Electrophoresis

ELECTROPHORESIS
Edited by Kiumars Ghowsi

ELECTROPHORESIS


Electrophoresis
http://dx.doi.org/10.5772/2905

Contributors
Kiumars Ghowsi, Hosein Ghowsi, Constantina P. Kapnissi-Christodoulou, Tomasz Piasecki,
Aymen Ben Azouz, Brett Paull, Mirek Macka, Dermot Brabazon, Elsa Lamy, Ana R. Costa, Clia
M. Antunes, Rui Vitorino, Francisco Amado, Ying Zhou, Vibeke Simonsen, Dario G. Pighin,
Luca Gonzlez-Arenzana, Rosa Lpez, Pilar Santamara, Isabel Lpez-Alfaro, Malgorzata K.
Sulkowska, Marcelo G.M. Chacur, Theopoline Omagano Itenge, Flavia Simone Munin, Maria
Regina de Aquino-Silva, Maria Luiza Barcellos Schwantes, Vera Maria Fonseca de Almeida-Val,
Arno Rudi Schwantes, Yoshimi Sato

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Electrophoresis, Edited by Kiumars Ghowsi
p. cm.
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Contents

Preface IX
Chapter 1 New Looks at Capillary Zone Electrophoresis (CZE)
and Micellar Electrokinetic Capillary Chromatography
(MECC) and Optimization of MECC 1
Kiumars Ghowsi and Hosein Ghowsi
Chapter 2 Method Development by Use of Capillary
Electrophoresis and Applications in Pharmaceutical,
Biological and Natural Samples 17
Constantina P. Kapnissi-Christodoulou
Chapter 3 Numerical Modelling of Light Propagation
for Development of Capillary Electrophoretic
and Photochemical Detection Systems 41
Tomasz Piasecki, Aymen Ben Azouz, Brett Paull,
Mirek Macka and Dermot Brabazon
Chapter 4 Protein Electrophoresis in Saliva Study 63
Elsa Lamy, Ana R. Costa, Clia M. Antunes,
Rui Vitorino and Francisco Amado
Chapter 5 Electrophoresis of Myocardial Cells 85
Ying Zhou
Chapter 6 Isozymes: Application for Population Genetics 97
Vibeke Simonsen
Chapter 7 Electrophoresis as a Useful Tool in
Studying the Quality of Meat Products 117
Dario G. Pighin
Chapter 8 Application of the Different Electrophoresis
Techniques to the Detection and Identification
of Lactic Acid Bacteria in Wines 137
Luca Gonzlez-Arenzana, Rosa Lpez,
Pilar Santamara and Isabel Lpez-Alfaro
VI Contents

Chapter 9 Isoenzyme Analyses Tools
Used Long Time in Forest Science 157
Malgorzata K. Sulkowska
Chapter 10 Seminal Plasma Proteins as Potential Markers
of Relative Fertility in Zebu Bulls (Bos taurus indicus) 173
Marcelo G.M. Chacur
Chapter 11 Identification of Polymorphism in the Keratin Genes (KAP3.2,
KAP6.1, KAP7, KAP8) and Microsatellite BfMS in Merino
Sheep Using Polymerase Chain Reaction-Single Strand
Conformational Polymorphism (PCR-SSCP) Analysi 193
Theopoline Omagano Itenge
Chapter 12 Temporal Expression of Isozymes, Alozymes
and Metabolic Markers at the Early Ontogeny of Prochilodus
argenteus (Characidae Prochilodontidae) from So
Francisco Basin, Trs Marias, Minas Gerais, Brazil 221
Flavia Simone Munin, Maria Regina de Aquino-Silva,
Maria Luiza Barcellos Schwantes, Vera Maria Fonseca
de Almeida-Val, Arno Rudi Schwantes and Yoshimi Sato

Preface

Electrophoresis experiment was first carried out by Tiselius in 1930. In his thesis titled,
The Moving Boundary Method to Study the Electrophoresis of Proteins, Tiselius
utilized the electric charge carried by the macromolecules to achieve some pioneering
separation of blood plasma proteins in free solution on a photographic film.
Electrophoresis is defined as the transport of electrically charged particles in a direct
current electric field. Electrophoretic separation is based on differential rates of
migration in the bulk of the liquid phase and is not concerned with reactions occurring
at the electrodes. In the early days, electrophoresis was carried out either in free
solution or in the supporting media such as paper, cellulose acetate, starch, agarose,
and polyacry lamide gel. In between 1950 to 1970, an enumeration of techniques and
instrumentation for Electrophoresis were developed.
Gel electrophoresis has been rarely used for the separation and identification of small
charged molecules of molecular weight less than about 1000 Dalton. In addition, the
major draw back of gel electrophoresis is lack of complete automation.
To overcome the low efficiency and reduce thermal effects, Hjerten carried out
electrophoresis in narrow diameter tubes of 300 m internal diameter the first time in
1967. This was the birth of open tubular capillary electrophoresis. However, in the
following decade, capillary electrophoresis did not draw enough attention from
researchers. Only until 1981 when Jorgenson and Lukacs demonstrated the use of
narrow capillaries to produce high efficiency for the separation of dansy and
fluorescamine derivatives of amino acids, dipeptides and simple amines, high
performance capillary electrophoresis was born and a new era of capillary
electrophoresis has begun. After the introduction of commercial CE instrument in late
1988 that allowed the full automation of CE analysis to be possible, more and more
research publications and industrial applications have made capillary electrophoresis
one of the dominant technologies in the separation field. In 1985 Terabe et. al added a
new dimension to capillary electrophoresis. They added micelles to the aqueous
electrolyte and were able to separate neutral molecules such as benzene and phenol.
Using this technique it is possible to separate various drugs which are neutral or even
charged. One that can separate enantiomers using this technique is called micellar
electrokinetic capillary chromatography (MECC).
X Preface

The present book contains few fundamentals on capillary electrophoresis and diverse
application of electrophoresis in general. We hope this collection will entertain the
readers who are interested in fundamental as well as applications of electrophoresis in
general.

Dr. Kiumars Ghowsi
Majlessi Branch, Islamic Azad University,
Iran

Chapter 1
New Looks at Capillary Zone
Electrophoresis (CZE) and Micellar
Electrokinetic Capillary Chromatography
(MECC) and Optimization of MECC
Kiumars Ghowsi and Hosein Ghowsi
Additional information is available at the end of the chapter
http://dx.doi.org/10.5772/45760
1. Introduction
The origin of theoretical plates for electrophoresis in general sense has been obtained from
the work of Giddings. Giddings has derived the following equation for the number of
theoretical plates.

2
ext
N
RT
A
=
O
(1)
where coefficient =unity when molecular diffusion acts alone and exceed unity when other
process contributes. R=gas constant and T=temperature and
ext
=chemical potential
conventionally substution of
ext
ZFV A = into eqn.(1) gives for the number of theoretical
plates.

2
ZFV
N
RT
= (2)
For an ideal process, in which =1 and T=298K,this equation reduces
N 20ZV = (3)
where we have used the Faraday constant F=96500 coulombs/mol. This voltage drops V in
the range of 100-10000 v with charge number Z=1 capable of yielding 2000-200000
theoretical plates, a range comparable to that found in chromatographic system. Jorgenson
and Lukacs, also in their land mark paper provided a theory for capillary zone

Electrophoresis 2
electrophoresis (CZE) in which they proposed two fundamental equations for resolution
and migration time. Resolution and number of theoretical plates are the focus of this work.
This is how number of theoretical plates are derived for capillary zone electrophoresis and
used in resolution equation by Jorgenson and Lukacs by starting with Gidding number of
theoretical plates.

2
ZFV
N
RT
= (4)
V=potential across capillary is substituted by EX where E=electric field and X=length of the
capillary. Z=charge number for ion is assumed to be 1, hence

2
FFX
N
RT
= (5)
[ ( ) ]
eo ep AB
X t v v = + (6)
Eqn.6 where
)
1
( [( ) ( ) ]
2
AB
ep ep A ep B
v v v = + is substituted by X in eq.(5). t is the time it takes the
analyte travels across the capillary and
eo
v is the electroosmotic velocity ( )
ep A
v and ( )
ep B
v
are the electrophoretic velocity of two analytes A and B with close electrophoretic velocities.
By substitution of equation(6) into equation (5) one obtains
[( ) [ ( ) ]]
2
eo ep AB
E
N Et v v
RT
= + (7)
There is mistake occurs in Jorgenson and Lukacs work, t in eqn. (7) is replaced by eqn.(8),
this is the case when electroomosis is absent.

ep
X
t
v
= (8)
By making this mistake substitution eqn (8) , what has been obtained by Jorgenson and
Lukacs for N , eqn.(2) is
[( )[( ) ( ) ]]
2
eo ep AB
ep
F X
N E v v
RT v
= + (9)
E, X, is the V, voltage across the capillary and
ep
v the electrophoretic velocity of the analyte
and
eo
v the electroosmotic flow in the capillary are substituted in eqn.(9) by
ep
E and
eo
E
where
ep
and
eo
are the electrophoretic and electroosmotic mobilities and E is the electric
field. By this substitution the following eqn.(10) is obtained for N
New Looks at Capillary Zone Electrophoresis (CZE) and
Micellar Electrokinetic Capillary Chromatography (MECC) and Optimization of MECC 3

( )
( )[ ]
2 ( )
eo ep AB
ep AB
FV
N
RT

+
= (10)
By using Einstain relation eqns.(11) and (12) we substitute for
ep
from eqn.(12)

ep
D RT
F
= (11)

ep
FD
RT
= (12)
Where D is the Diffusion constant of the analyte by this substitution and simplification
eqn.13 is obtained
[ ( ) ]
2
eo ep AB
V
N
D
= + (13)
This is the equation for the number of theoretical plates the Jorgenson and Lukacs use to
obtain the resolution equation. N is driven by mistake. The resolution equation is given by
them

( ) ( )
.
( ) 4
ep A ep B
s
eo ep AB
N
R

=
+
(14)
substituting eqn.(13) for N into eqn.(14), the resolution can be expressed as

1/ 2
(( ) ( ) )
.
( ( ) ) 4 2
ep A ep B
s
eo ep AB
V
R
D

=
+
(15)
The above eqn.(15) is the same equation that was obtained by Jorgenson and Lukacs.
This equation is incorrect. In the present work equation for resolution which is cosidered
correct equation is proposed.
2. Theory
According to Giddings theory the evaluation of the ultimate capabilities of zone
electrophoresis is possible. To calculate the number of theoretical plates and separable zone
achieveable in ideal zone electrophoresis, the electrostatic force exerted on a mole of charged
particles on electric field of strength E is
Force ZFE f = = (16)
where z=net charge of a single particle in proton units and F=Faraday constant the negative
chemical potential drop across the separation path.

Electrophoresis 4

ext
fX ' A = (17)
In which X is the distance where f, force applied in capillary eletrophoresis. For
conventional mode of capillary zone electrophoresis, electroosmotic and electrophoretic
velocities are in opposite direction. This conventional mode is similar to tread mill and
electric stairs where the moveing object has two movements one walking and the other one
movement of the stairs.
There X in eqn. (17) is not the length of capillary, it is effective distance where the electric
force which is applied is greater than the length of capillary is called

ext
fX ' A = (18)
Then by substituting eqn.(18) into eqn.(1) , the eqn.(19) for the number of theoretical plates
results.

2
fX
N
RT
'
= (19)
X=effective length which looks like a treadmill, the solute is like a person which can run for
miles on the mill but actually he has stayed stationary, f=FE force is Faraday constant times
electric field. By substituting force in eqn. (19) one gets

2
F
N EX
RT
' = (20)
By definition, the effective distance the analyte travels under the force of electric field, X
divided by the relation times, t, is equivalent to electrophoretic ( )
ep AB
v velocity , ( )
ep AB
v , as
the following
( )
ep AB
X
v
t
'
= (21)
Where
)
1
( [( ) ( ) ]
2
AB
ep ep A ep B
v v v = +
By the help of eqn.(21) X can be substituted into eqn.(20). Now eqn.(22) can be rewritten to
include electrophoretic velocity.
( )( )
2
ep AB
F
N v Et
RT
= (22)
Replacing the electrophoretic velocity variable with the product of electrophoretic mobility
and electric field
ep ep
v E = yields the following expression:

2
[( )[( ) ]
2
ep AB
F
N E t
RT
= (23)
In order to observe the dependence, efficiency has on capillary length and electroosmosis, a
substitution for the time variable in eqn.(21) is made. Net displaccment of the analyte or
capillary length, X, is related to the retention time, t as shown
[ ) ( ) ]
eo ep AB
N t v v = + (24)
Rearragement of eqn.(24) given eqn.(25)

( )
eo ep AB
X
t
v v
=
+
(25)
substituting eqn.(25) into eqn.(20) yields the expression,

( )
[ ]
2 ( )
ep AB
eo ep AB
v
F
N XE
RT v v
=
+
(26)
By making additional substitution for electrophoretic and electroosmotic velocity produced,
( )
AB ep AB
v E = and
eo eo
v E = , a final equation for efficiency is

( )
( )( )
2 ( )
ep AB
eo ep AB
F
N XE
RT

=
+
(27)
plate height is the ratio of effective length X to efficiency N is

X
H
N
'
= (28)
Substituting equation into eqn.(28) yields an expression for plate height.

2RT
H
FE
= (29)
This interesting result shows that the theoretical plate height is independent of
electroosmotic flow when it is based on the effective distance the analyte travels rather than
the capillary length. Instead, plate height has a simple inverse relation with the electric field
strength.
Fig .1 Shows the invers relation between theoretical plate height and electric field strength.
Electric field strength is the voltage across two ends of capillary divided by the length of
capillary.
Equation for resolution: Based width resolution is the quantitative measure of ability to
separate two analytes. For two adjacent peaks with similar elution times, peak showed be
nearly identical:

A B AB
W W W ~ ~ (30)

Electrophoresis 6

Figure 1. Theoretical plate height versus electric field for CZE.
where
1
( )
2
AB A B
W W W = + .
Assuming eqn.(30) is true, resolution for species A and B expressed in terms of their
retention times and the peak base width for either species.

( ) ( )
R B R A
s
AB
t t
R
W
= (31)
The conventional expression for separation can be written with parameters related to either
species A or B, shown here using the retention time and peak base width for species B.

2
( )
16[ ]
R B
AB
t
N
W
= (32)
By combining eqns. (31) and (32) an equation for chromatography is produced that
expresses resolution in terms of efficiency and retention time
4
.

( ) ( )
[ ]
4 ( )
R B R A
s
R AB
t t N
R
t
= (33)
The resolution time variable
R
t and the efficiency N are eliminated by inserting eqns.(25)
and (26) into eqn.(33) for the analyte B:

1/ 2
( ) ( ) ( )
[( )( ) ] [ ]
32 ( ) ( )
ep AB ep A ep B
eo ep AB eo ep AB
v v v
F
R XE
RT v v v v
=
+ +
(34)
when electrophoretic velocity,
ep
v , is replaced with
ep
E and applied patential, V is
substituted for XE one obtains the following equation for the resolution:

1/ 2
3/ 2
( ) ( ) ( )
[ ] [ ]
32
( ( ) )
ep AB ep A ep B
s
eo ep AB
FV
R
RT

=
+
(35)
In order to make a comparison between new resolution eqn.(35) obtained in present work and
Jorgenson Lukacs equation for resolution eqn.(15) could be transformed to the following
equation by using Einstein relation relating diffustion constant to mobility
D RT
F
= and

1/ 2
1/ 2
( ) ( )
[ ] [ ]
32 ( )
( ( ) )
ep A ep B
s
ep AB
eo ep AB
FV
R
RT

=
+
(36)
Jorgenson and Lukacs equation (eqn.36) and new derived Ghowsi equation(eqn.38) for
resolution are given as

1/ 2
3/ 2
( ) ( )
[ ] [ ]
32 ( )
( ( ) )
ep A ep B
s
ep AB
eo ep AB
FV
R
RT

=
+
(37)
This interesting observation that for the absence of electrosmotic flow in Jorgenson and
Lukacs equation the resolution eqn.36 is converted to

1/ 2
( ) ( )
[ ] [ ]
32 ( )
ep A ep B
s
ep
FV
R
RT

= (38)
The other resolution equation obtained in present work Ghowsis eqn.(35) with presence of
electroosmotic flow
s
R is converted to

1/ 2
( ) ( )
[ ] [ ]
32 ( )
ep A ep B
s
ep AB
FV
R
RT

= (39)
It is interesting that only for this case when electroosmosis is absent the resolution equation
of Jorgenson and Lukacs eqn.(15) with the help of Einstein relation is equal to Ghowsis
derived equation eqn.(38) at present for capillary zone electrophoresis.

Electrophoresis 8
3. Micellar electrokinetic capillary chromatography (MECC)
By converting figure of Merits in MECC to electrochemical parameters
5
and pursuing similar
procedure we applied to capillary electrophoresis and using effective length solute travels rather
than length of capillary and then converting the resolution equation in terms of chromatography
parameters new equation for resolution could be found which is published in another paper
7
.
4. Optimization of micellar electrokinetic capillary chromatography
(MECC) as a nano separation technique using three dimensional and two
dimensional plottings of characteristic equations
Feyman with the lecture of plenty of room at the bottom at an American Physical Society at
Caltech on December 29, 1959 considered the possibility of direct manipulation of individual
atoms as a more powerful forms of synthetic chemistry than those used at the time . In
conventional chromatography there are two phases involved one is the stationary phase and one
is the mobile phase
6
. Terabe et al proposed Micellar Electrokinetic Capillary Chromatography
8
,
MECC, which has the smallest pseudo stationary phase within nano range called micelle.
The very high strength of separation comes from these nano sized materials. That is why we
call this technique MECC.
5. Nano separation technique
There are several work which have been done to final the optimum conditions of this Nano
Separation Technique
5,8-10
.
In all optimization characteristic equation is the focus.
What is the characteristic equation?
In column chromatography the resolution equation is given as
6

1/ 2
1
. .
4 1
s
N k
R
k
o
o
'
=
' +
(40)
Where k is the capacity factor, is the selectivity and N is the number of theoretical plates.
Terabe
8
et al proposal MECC for the first time and they proposed the resolution equation for
MECC:

1/ 2
1 / 1
. . .
4 1 1 /
o mc
s
o mc
t t N k
R
k t t
o
o
'
=
' + +
(41)
Where N, and k were already defined , a new term is appearing in equation (41) for
resolution is
1 /
1 /
o mc
o mc
t t
t t
+
. In this term k is the capacity factor and
o
t and
mc
t are retention
times of the aqueous and micellar phases respectively. The characteristic equation for MECC
to optimize is from equation(41):

1 /
( , / ) .
1 1 ( / )
o mc
o mc
o mc
t t k
f k t t
k t t k
'
' =
' ' + +
(42)
This characteristic equation has two variables k and the ratio /
o mc
t t . In a recent work a
new model for the MECC using a model based on effective length solute migrated as a
similar to tread mill case were proposed
7
. Based an this model

1/ 2 1/ 2 3/ 2
1 ( / ) 1 1
[( ) / 4] ( )[ ][ ] .
4 1 1 ( / )
o mc
s ep B pseudo
o mc
t t k
R D RT N
k t t k
o
o
'
=
' ' + +
(43)
Variables of this equations were defined in the previous work
7
. The characteristic equation
of
s
R for equation (43) is the last two terms.

3/ 2
1 /
( , / ) ( ) .( )
1 1 ( / )
o mc
o mc
o mc
t t k
f k t t
k t t k
'
' =
' ' + +
(44)
In a comparison between the characteristic equation (42) obtained by Terabe
8
and the
characteristic equation obtained based on the new model
7
,equation(44) , the difference is the
power of ( )
1
k
k
'
' +
term where in equation (42) the power of this term is 1 and in equation (44)
the power of this term is 3/2. The rest of these two equations (42) and (44) are the same.
There are two other characteristic equations need to be obtained. For the first time J. P. Foley
obtained an equation for a compromise between resolution and the migration time. He
introduced /
s R
R t .
R
t is given in Terabes work
8
as

1
[ ]
1 ( / )
R o
o mc
k
t t
t t k
' +
=
' +
(45)
Two characteristic equations for /
s R
R t are obtained one is for Terabe
s
R equation (41) and
the other one is for
s
R in equation (43). The two characteristic equations for /
s R
R t are
given as below correspondingly.

2
( , / ) .(1 )
(1 )
o
o mc
mc
t k
f k t t
t
k
'
' =
' +
(46)

5/ 2
( , / ) .(1 )
(1 )
o
o mc
mc
t k
f k t t
t
k
'
' =
' +
(47)
6. Two dimensional and tree dimensional plots of characteristic equations
In present work by the help of modern technology of computer and three dimensional
software of Dplot direct access to the plot of characteristic equations (42) and (44) are possible.

Electrophoresis 10
Figure 2 shows the three dimensional plot of characteristic equation (42) obtained by Terabe
8

et al.

Figure 2. Three dimensional plot of Terabes characteristic equation for resolution of MECC.
In this work ( , / )
o mc
f k t t ' is a function of two independent variables k and /
o mc
t t .k is
changing between 1 to 10 and /
o mc
t t changes between 0.1 to 1. In figure 2 the surface of the
three dimensional plot shows maximums. The greatest maximum occurs at
max max
( , / ) 0.51
o mc
f k t t ' = .
These maximums are local maximums.
In figure 3 the characteristic equation (44) based an proposal thread mill model
7
is plotted. This
plot shows no local maximums but there is a single maximum occurs at / 0
o mc
t t = ,k=10
where ( , / ) 2.25
o mc
f k t t ' = . Similar information could be obtained for equation (42) from level
curves plotted by Dplot software presented at this work fig.4. This two dimensional plot is the
image of three dimensional plot on the surface of the plan / 0.1
o mc
t t = , k=1.
Similar information could be obtained for equation (44) from level curves plotted by Dplot
software presented at this work fig.5. This two dimensional plot is the image of three
dimensional plot on the surface of the plane / 0.1
o mc
t t = ,k=1.
Foleys characteristic equation / 0.1
o mc
t t = (46) has also been plotted three dimensionally
figure 6 and two dimensionaly level curves figure 7. The information obtained from either
these two plots is as following. Maximum ( , / ) 0.225
o mc
f k t t ' = occurs at / 0.1
o mc
t t = ,k=1
and minimum ( , / ) 0
o mc
f k t t ' = occurs at / 1
o mc
t t = ,k=1.

Figure 3. Three dimensional plot of Ghowsis characteristic equation for resolution of MECC.

Figure 4. Two dimensional plot, level curves, of Terabes characteristic equation for resolution of MECC.

Electrophoresis 12

Figure 5. Two dimensional plot , level curves , of Ghowsis characteristic equation for resolution of MECC.

Figure 6. Three dimensional plot, Foleys characteristic equation for resolution per unit time /
s R
R t .

Figure 7. Two dimensional plot, level curves, of Foleys characteristic equation for resolution per unit
time /
s R
R t .
Ghowsis characteristic equation (47) has also been plotted three dimensionally figure 8 and
two dimensional level curves figure 9. The information obtained from either these two plots
is the following: Maximum ( , / ) 0.16
o mc
f k t t ' = occurs at / 0.1
o mc
t t = , k=2 and minimum
( , / ) 0
o mc
f k t t ' = occurs at / 1
o mc
t t = ,k=1.

Electrophoresis 14

Figure 8. Tree dimensional plot of Ghowsis characteristic equation for resolution per unit time /
s R
R t .

Figure 9. Two dimensional plot level curves of Ghowsis characteristic equation for resolution per unit
time /
s R
R t .
7. Conclusion
The fundamental eqn.12 for the number of theoretical plates has been used to obtain
resolution equation by Jorgenson eqn.14. The number of theoretical plates equation in
previous work by Jorgenson is wrong, automatically makes the resolution equation
wrong.
With the consideration that length X in eqn. 17 is the distance where force is applied in
capillary electrophoresis, number of theoretical plates which depends on eqn.17 is
discussed.
Another equation is the result obtained in eqn.28 which indicates the new result based on
effective length. In comparison with eqn.12 Jorgenson equation different eqn.26 has been
used to obtain eqn.34 which is the new resolution equation. Similar procedure could be
applied to obtain the number of theoretical plates for effective length for MECC and the
new resolution equation could be obtained too. This work for MECC was shown in
reference 7.
In present work eight graph are presented as characteristic equations of MECC. Four of
them are three dimensional and four of them are two dimensionals. It is very interesting
observation that the Terabes et al characteristic equation shows local maximums in three
dimensional plots but the three dimensional plot of characteristic equation obtained by us
based on thead mill model shows no local maximums on the surface of three dimensional
plot.
Author details
Kiumars Ghowsi
Department of Chemistry Majlesi Branch, Islamic Azad University, Isfahan, I.R. Iran
Hosein Ghowsi
Department of Mathematics, Payame Noor University, Tehran, I.R. Iran
8. References
[1] J.C. Gidding, Sep.Sci.,4,181 (1969).
[2] J. Jorgenson and K.D. Lukacs , Anal. Chem., 53, 1298 (1981).
[3] C. D. Dunn, M. G. Hankins and K.Ghowsi , Sep.Sci. Tecnol. , 29,2419 (1994).
[4] W. R. Jones and J. Jandik, J. Chromatogr., 546, 445 (1991).
[5] K. Ghowsi, J. P. Foley and R. G. Gale , Anal. Chem.,62, 2714 (1990).
[6] D. A. Skoog, Principles of Instrumental Analysis, Saunder college Publishing
Philadelphia, edn.5 (1992).
[7] K. Ghowsi and H. Ghowsi, Asian J. Chem., 23,3084 (2011).
[8] S. Terabe and T. Ando, Anal. Chem., 57,834 (1985).

Electrophoresis 16
[9] J. P. Foley, Anal. Chem. 62,1302 (1990).
[10] K. Ghowsi and H. Ghowsi submitted to Asian. J. Chem. Accepted.
Chapter 2
Method Development by Use of
Capillary Electrophoresis and Applications in
Pharmaceutical, Biological and Natural Samples
http://dx.doi.org/10.5772/45834
1. Introduction
Electrophoretic methods compose a family of related techniques that use narrow-bore fused-
silica capillaries to perform high efficiency separations of both small and large molecules.
These methods are commonly known as capillary electrophoretic methods. Capillary
electrophoresis (CE) has, over the years, demonstrated its powerful separation ability in the
area of chiral and achiral analysis. This is contributed to the advantages it offers when
compared to chromatographic techniques: (1) low consumption of samples and solvents; (2)
high separation efficiency and resolution; (3) versatility [1,2].
Two of the most important modes of CE, which will be discussed in this chapter, are
capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC).
CZE is the simplest and the most widely used mode of CE [1]. The separation mechanism of
the analytes is based on their difference in charge-to-size ratios and their difference in
electrophoretic mobilities, which, in turn, result in different velocities. However, due to the
fact that neutral species do not possess an electrophoretic mobility, they cannot be separated
by use of this mode.
In order to circumvent this problem, new modes of CE have been suggested as alternatives.
MEKC, which combines the best features of both electrophoresis and chromatography, is
considered an alternative mode because it can be used for the separation of charged as well
as neutral compounds. It involves the introduction of a surfactant at a concentration above
the critical micellar concentration (CMC), at which micelles are formed. MEKC was first
introduced by Terabe et al. in 1984 [3]. Although it is a form of CE, its separation principle is
more similar to HPLC than to CE. In this mode, analytes are separated according to their
partitioning between the mobile and stationary phase and, when charged, their
electrophoretic mobility. The driving force for the partitioning of analytes is hydrophobicity.

Electrophoresis 18
In addition, hydrogen bonding, dipole-dipole, and dispersive interactions can contribute to
the solute partitioning between the two phases [4-6].
CE was originally considered as a powerful analytical tool for the analysis of biological
macromolecules. It has though, over the years, been extensively used for the separation of
other compounds, such as chiral drugs, food additives, pesticides, inorganic ions, organic
acids, and others. In this chapter, the ability of CE, and particularly CZE and MEKC, to be
used for the qualitative and quantitative determination of compounds in pharmaceutical,
biological and natural samples is investigated. In each approach, a number of studies are
reported and discussed. These studies involve establishment of optimum separation
conditions, method validation, optimization of sample-preparation procedure and
application for the determination of the analytes under study in real samples. The first part
of this chapter involves the determination of polyphenolic compounds using CZE with UV-
Vis detector in red and white wines, while the second part involves the determination of
pharmaceutical compounds in biological samples, such as blood and urine, using the
hyphenated technique CE-MS (mass spectrometry). The third and final part emphasizes the
importance of MEKC in chiral analysis since it has been known that usually only one
enantiomer is active, while the other may be less active, inactive or has adverse effects.
2. Determination of polyphenolic compounds in natural samples
Polyphenolic compounds exist in a variety of natural products, such as fruits, vegetables,
beverages (tea, wine and juices), honey, cacao and herbs. They attract a lot of interest due to
their beneficial implication in human health. They have been widely studied due to their
antioxidant capacity and their association with several pathological conditions, such as
hypertension, cardiovascular disease, dementia, and even cancer [7-9]. Therefore, due to
their health significance, numerous analytical methods have, over the last decades, been
developed for their separation, identification and quantitation in natural products [10-13].
According to literature, the simplest CE method, CZE, proved to be the best method for the
determination of polyphenolic compounds in wine samples [14-17]. In such studies, and in
each case, when the optimum CZE method was applied to different red and white wines, it
was established that red wines have higher levels of polyphenolic compounds than white
wines and that the polyphenolic composition varies among different wines.
2.1. Method development and validation
In this part of the chapter, a representative study performed recently in Cypriot wines is
briefly described [17]. The influence of several experimental parameters is initially
illustrated in order to obtain improved selectivity and resolution for the separation of seven
flavonoids, which constitute the most important group of polyphenols, and trans-rasveratrol
that are usually present in wine. This is accomplished by use of CZE and by examining
different sample preparation procedures. Due to the low concentrations of flavonoids in
wine and the high complexity of wine matrices, preconcentration methods are required,
which can simplify the electropherograms. The optimized CZE and pre-treatment methods
proved to be effective in characterizing flavonoids in red and white wine samples.
Method Development by Use of Capillary Electrophoresis
and Applications in Pharmaceutical, Biological and Natural Samples 19
The effect of column temperature, and concentration and pH of background electrolyte
(BGE) were investigated. These parameters, along with the applied voltage, are the most
common parameters that are required to be examined in order to optimize a separation
in CZE. Figure 1 illustrates the influence of the pH on the resolution and the analysis time.

Figure 1. Effect of pH value on the separation of the eight polyphenols. (A) pH 9; (B) pH 9.3; (C) pH 9.6.
Conditions: BGE 50 mM borate, 10 mM phosphate and 20 mM SDS; pressure injection, 30 mbar for 3
sec; applied voltage, 25 kV; temperature, 25 C; fused-silica capillary, 64 cm (55.5 cm effective length) x
50 m i.d.; detection, 205 nm. Peak identification: trans-resveratrol (1), epicatechin (2), catechin (3),
naringenin (4), kaempferol (5), apigenin (6), myricetin (7), quercetin (8) [17].

Electrophoresis 20
The last two increased with increasing the pH, possibly due to an increase in the negative
charge, which resulted in a greater affinity and a higher complexation between borate and
phenols. Taking into consideration the migration times, the peak efficiency and the
sufficient resolution, the following parameters provided a baseline separation of all
polyphenolic compounds: BGE containing 50 mM borate and 10 mM phosphate at pH 9.6
and column temperature of 25 C (Figure 1C). The use of alkaline borate-based BGEs, in
CZE, resulted in a sufficient separation of polyphenols due to the complex-formation ability
of borate. In addition, an increase in the borate concentration from 25 to 50 mM and an
increase in the pH value from 9 to 10 resulted in an increase in the migration times of all
analytes, while the resolution was significantly improved. At pH 10 though, the analysis
time was very long (~ 50 min) and joule heating effects, such as high current generation and
peak broadering, were observed. An increase in pH increased the negative charge of the
analytes, which, in turn, favored a greater affinity for the buffer and a higher complexation
between borate and phenols [18].
The method was then validated by the terms of linearity, precision and LOD. Linearities for
the eight analytes were very good, and precision, which was based on the relative standard
deviation, was below 1%, indicating an excellent reproducibility. In addition, LODs, which
were calculated as three times the standard deviation via the slope of the calibration curve,
were between 0.03 and 5.05 g/mL for all eight polyphenolic compounds.
2.2. Application
The qualitative and quantitative analysis of analytes in real samples is often difficult due to
interruptions caused by different interfering substances found in the sample matrix.
Therefore, a sample-preparation procedure is a necessary step prior to the electrophoretic
analysis, in order to isolate the analytes under study from real samples. Different
preconcentration methods have been used over the years, including solid-phase extraction
(SPE) with C-18, silica, or other cartridges [14,16,19] and liquid-liquid extraction (LLE) with
different organic solvents [10,20,21].
In the study performed in Cypriot wines, the sample preparation procedure was optimized
in order to determine the one that was simple, fast and reliable [17]. Therefore, three LLE-
procedures (C,D,E), a SPE-procedure (F), a procedure that involve evaporation and
reconstitution of wine sample (B) and a direct injection of wine sample after dilution and
filtration (A) were compared and the most effective method was applied to Cypriot wines.
The electropherograms obtained by use of each sample preparation procedure are
illustrated in Figure 2. When no extraction was performed, the electropherograms were
complex, while SPE was found to be ineffective for the isolation of polyphenolic compounds
from wine samples. LLE with diethyl ether, followed by evaporation of organic layer by
nitrogen stream and reconstitution in ethanol proved to be the optimum sample pre-
treatment method. When the optimum method was applied to Cypriot wine samples, the
quantification of polyphenolic compounds was successfully achieved. It was observed that
epicatechin and catechin exist in all wine samples in comparable concentrations, whereas
myricetin and quercetin exist only in two of the three wine samples. Polyphenolic
composition varies among different wines, because it depends on several factors, such as the
type of grapes used, the vivification process used, the type of yeast that participates in the
fermentation, weather variations and other biological effects [22].

Figure 2. Electropherograms of the wine samples obtained using six different sample preparation
procedures under optimum conditions. Conditions: BGE 50 mM borate, 10 mM phosphate and 20 mM
SDS (pH 9.6); pressure injection, 30 mbar for 3 sec; applied voltage, 25 kV; temperature, 25 C; fused-
silica capillary, 64 cm (55.5 cm effective length) x 50 m i.d.; detection, 205 nm. Peak identification:
epicatechin (2), catechin (3), myricetin (7), quercetin (8) [17].
Another important observation was that in white wine, the only flavonoid that was detected
was catechin at a concentration of 7.3 g/mL. This was not surprising since the majority of
flavonoids in wine come from the extraction derived from grapes solids. White wine is
made by pressing the juice away from the grapes solids, and then, by allowing it to ferment.
So, red wines have higher levels of polyphenolic compounds [23].
3. Determination of pharmaceutical compounds in biological samples
Quantification of drugs in biological fluids, like plasma, has an important role in drug
discovery and development. There are two main aspects that are taken into account in order

Electrophoresis 22
to make the identification of drugs in biological fluids possible. The first aspect is the
development of an accurate analytical method, with high sensitivity, capable to identify
desirable compounds in concentrations comparable to that in biological fluids. The second
one is the exploration of the optimum extraction method that can effectively extract the drug
from the biological matrix.
Over the years, CE coupled to electrospray ionization-mass spectrometry (ESI-MS) has been
utilized as a bioanalytical tool for the analysis of drug compounds in biological samples [24-
29]. Even though the most common detector in CE is the UV detector due to its easy
manageability and low cost, it has the drawback of low sensitivity due to the short optical
path length. An alternative to this is the use of MS. The coupling of CE with MS is a well-
established technique, which combines the high efficiency and resolution that are provided
by CE and the detection sensitivity and selectivity and the identification potential that are
provided by MS [25,30].
In recent years, a large number of publications have been provided on the general
developments and biological applications of CE-ESI-MS [24-29]. Zheng et al. developed a
CZE-ESI-MS method for monitoring the antiepileptic drug lamotrigine in human plasma
[27]. The optimum conditions were obtained by varying a big number of BGE, sheath liquid
and MS spray chamber parameters. In each case, both the CZE separation, as well as the MS
detection sensitivity, were evaluated, and the parameter that provided a reasonable
compromise between resolution and detection sensitivity was chosen as the optimum. The
developed method was then applied to assay blank samples spiked with lamotrigine in
order to set up the calibration curve and estimate the limit of detection (LOD). Both linearity
of calibration curve and LOD (0.05 g/mL) were good, and the optimum method was
applied to 14 human plasma samples collected from a lamotrigine-treated subject over a
period of 96 h after oral administration of 50 mg lamotrigine.
In a 2011 study, Elhamili et al. analyzed the anticancer drug Imatinib by use of CE coupled
to ESI time-of-flight MS in human plasma [29]. The CE separation and ESI parameters were
initially investigated and optimized in regard to peak efficiency, peak intensity and
electrospray stability. The LOD and limit of quantitation (LOQ) were evaluated by injections
of standard solutions of the drug compound, and they were determined to 5 and 20 ng/mL,
respectively. In addition, the extraction recovery of Imatinib from human plasma using a
common liquid-liquid extraction (LLE) method and a new strong cation exchange (SCX)
solid-phase extraction (SPE) column was investigated and compared. The highest extraction
recoveries were obtained by using the latter method. The SCX-SPE extraction followed by
CE-ESI-TOF-MS analysis in patient plasma samples demonstrated good repeatability,
linearity and sensitivity for possible therapeutic monitoring of Imatinib level. The authors,
in this manuscript, also conclude that this method could be applied for the analysis,
quantification, and clinical assessment of other drug compounds and their metabolites.
3.1. Method development
The performance and usefulness of CE-MS is also demonstrated here by providing a more
in-depth analysis of a research work that was performed in a blood sample obtained from a
patient with Alzheimers Disease (AD) [24]. In this study, a CZE-ESI-MS method was
developed for the analysis of the acetylcholinesterase inhibitor rivastigmine, using
neostigmine bromide as an internal standard, which is highly recommended in order to
avoid problems that are related to sample injection [31]. Rivastigmine is a pseudo-
irreversible carbamate inhibitor of acetylcholinesterase, and it is clinically used for the
symptomatic treatment of mild to moderate AD [32].
In a previous paper, MEKC coupled to a diode-array detector was used for the simultaneous
separation of nine acetylcholinesterase inhibitors, including rivastigmine [33]. This method
was validated and successfully applied to a real blood sample that was obtained from a patient
who was not under any of this medication. The sample was spiked with rivastigmine in order
to establish the ability of the method to separate the drug from other components that might
exist in the blood sample. In this study, the blood sample was not directly injected into the
capillary, because some components that exist in the sample can be absorbed to the capillary
wall and deteriorate the performance of the column [34]. The blood sample was therefore
diluted ten folds with the BGE [12.5 mM Na2HPO4 / 12.5 mM Na2B4O7 / 20 mM SDS (pH 10)],
and it was then spiked with 25 g/mL of rivastigmine. However, due to the low sensitivity
obtained by CE with on-column UV detection, the identification of rivastigmine in biological
fluids using CE remained a challenge. In order for the technique to be used for the quantitation
of an acetylcholinesterase inhibitor in body fluids, the sensitivity, and consequently the LOD
had to be improved. The increased interest in exploring CE-MS and its potential to serve as an
alternative method allowed further investigation for the determination of rivastigmine and
related drugs in complex biological matrices.
When the CZE-UV method was compared with the CZE-MS, the first demonstrated a
shorter analysis time of approximately 2 min due to the shorter effective length, while the
S/N for the peak of rivastigmine at the SIM mode was estimated to be eight times bigger
than with UV detection. This, in turn, indicated the high specificity and selectivity of the
ESI-MS detector [24]. In the CZE-ESI-MS study, several electrophoretic and ESI-MS
parameters were also examined, which were classified in three categories: the BGE
parameters, such as the concentration, the pH and the use of organic modifier, sheath liquid
parameters, such as the composition, the methanol (MeOH) content and the flow rate, and
finally some spray chamber parameters, such as the temperature and the flow rate of the
drying gas and the nebulizer gas pressure. The effect of each parameter on the S/N, and
consequently the LOD, was examined and the optimum one was chosen for further
optimization.
In the case of BGE parameters, it was observed that ammonium acetate provided the most
reproducible migration times, a concentration of 40 mM ammonium acetate resulted in the
highest S/N, while a higher concentration decreased the ratio, probably due to the Joule
heating effect that increases the level of noise (Figures 3a & 4a). When the pH was
examined, it was concluded that at pH 9, where rivastigmine starts to have a negative
charge (pKa=8.6), both the analysis time and resolution increased, and a higher S/N was
obtained (Figures 3b & 4b).

Electrophoresis 24

Figure 3. Effect of (a) ionic strength and (b) pH of the BGE on the separation of rivastigmine (2) and I.S
(1). Conditions: BGE: ammonium acetate, sheath liquid 1 % acetic acid in water:MeOH (50:50 v/v) at a
flow rate of 10 L/min, analyte and I.S. concentrations 0.3 mg/mL. Drying gas flow rate 6 L/min and
temperature 200 C, nebulizer gas pressure 20 psi [24].

Figure 4. Effect of (a) ionic strength of the BGE and (b) pH of the BGE upon S/N ratio. Conditions: BGE:
ammonium acetate, sheath liquid 1 % acetic acid in water:MeOH (50:50 v/v) at a flow rate of 10 L/min,
analyte and I.S. concentrations 0.3 mg/mL. Drying gas flow rate 6 L/min and temperature 200 C,
nebulizer gas pressure 20 psi [24].
As far as the sheath liquid parameters are concerned, it was observed that its composition
and its flow rate affected the ESI-MS sensitivity significantly. This was not a surprising
observation since the sheath liquid plays an important role in the CE-MS system. The sheath
liquid is used as the make-up liquid that can solve the flow-rate incompatibility problems
between CE and MS [35]. These problems are encountered because the flow rate through the
CE column is very low (nL/min), and it cannot support a stable electrospray, whose flow
rate is typically a few L/min. In addition, the sheath liquid is used for establishing an
electrical connection at the cathode end of the CE capillary, and it provides the suitable
solvent conditions for the electrospray, which does not depend on the CE BGE [36].
When different sheath liquids were evaluated, the one that was able to support the
formation of positively charged ions, and consequently provide the highest S/N, was acetic
acid (1%) (Figure 5a). The influence of methanol as an organic modifier in the sheath liquid
was also examined, because the use of such solvents allows an easier protonation of the
analytes, which results in a higher signal [28]. By varying the percentage of methanol, it was
concluded that 50% was the optimum since the noise level was the lowest (Figure 5b).
Finally, the flow rate of the sheath liquid was set at 10 L/min (Figure 5c). Other values
were either too low to establish an electric contact that is required to achieve separation, or
they affected the spray stability negatively, which, in turn, lead to higher noise levels.

Figure 5. Effect of (a) sheath liquid composition, (b) sheath liquid organic modifier and (c) sheath liquid
flow rate upon S/N ratio. Conditions: BGE: ammonium acetate 40 mM, at pH 9.0; analyte and I.S.
concentrations 0.3 mg/mL. Drying gas flow rate 6 L/min and temperature 200 C, nebulizer gas pressure
20 psi [24].

Electrophoresis 26
The spray chamber parameters, which are the last parameters examined in this study, have
an important effect on the response of the MS system. One of these parameters involves the
drying gas, which is used for accelerating the buffer desolvation, increasing the MS
sensitivity, and eliminating any undesirable ions from entering into the MS system. It was
observed that the drying gas flow rate has an effect on the stability of the electrospray, and
consequently, the levels of the noise. The flow rate was set at 6 L/min, because at this flow
rate an increased number of ions come closer to the liquid-gas interface, and this increases
the desolvation velocity [37] (Figure 6a). In addition, other flow rates that were examined in
this study either caused an unstable electrospray or lowered the S/N. The drying gas
temperature was varied from 150 C to 350 C, and the highest S/N was obtained at 200 C,
which was considered as the optimum (Figure 6b). The nebulizer gas pressure was the last
parameter examined in this category, and based on the stability of the electrospray and the
S/N, 20 psi was selected as the optimum. At 20 psi, the electrospray is more efficient,
probably due to an improved ion evaporation process because smaller initial droplets are
obtained with higher nebulizer gas pressure (Figure 6c).

Figure 6. Effect of (a) drying gas flow rate, (b) drying gas temperature and (c) nebulizer gas pressure
upon S/N ratio. Conditions: BGE: ammonium acetate 40 mM at pH 9.0, sheath liquid 1 % acetic acid in
water:MeOH (50:50 v/v) at a flow rate of 10 L/min, analyte and I.S. concentrations 0.3 mg/mL [24].
3.2. Method validation
All the parameters mentioned above are the common parameters that need to be examined
in a method development process that involves a CE-MS system. These parameters affect
the analysis time, resolution, response of the analyte under study, noise level, and
sensitivity of the system. All these are important if the developed method is expected to be
applied to biological samples for the detection and the quantification of drug and other
compounds.
When the optimum conditions for the analysis of rivastigmine were determined, the method
was validated in terms of linearity, precision, stability, recovery, LOD and LOQ. Two
calibration curves were constructed, in human plasma and in standard solutions, and
linearity was good in both cases. The precision, which was evaluated based on migration
times and peak areas, was excellent, and particularly in the case where the peak area of the
internal standard was also taken into consideration. The LOD and the LOQ were
determined based on the standard deviation of the peak area and the slope of the calibration
curve. The LOD and the LOQ were calculated as 3 and 10 times the above correlation,
respectively. In the plasma sample, the LOD and the LOQ were found to be 2.8 ng/mL and
8.4 ng/mL, respectively, while in standard solutions they were 1.6 ng/mL and 5.0 ng/mL,
respectively. These values are considered satisfactory for the accurate and precise
quantification of rivastigmine in AD patients treated with the particular drug compound,
and this is based on clinical studies that were performed in such patients [38,39].
3.3. Application
Biological matrices are among the most difficult samples to analyze because of the big
number of components they contain that they may be adsorbed onto the capillary wall or
interfere in the detection and/or separation process. Therefore, before plasma analysis, it is
important and necessary to perform a sample preparation procedure. In addition to this, the
concentration of most of the analytes in biological samples is low; so, a preconcentration
step before the detection and quantitation is required. In many cases, different sample pre-
treatment methods are used and compared in order to determine the most effective one, in
regard to analyte recovery, difficulty, time and reproducibility. In this study, one LLE and
two different SPE procedures were examined. In the case of SPE, two different SPE
cartridges were used, a C18 cartridge and an Oasis HLB cartridge. LLE proved to be
inefficient for rivastigmine assay, and it was time consuming because the extraction step
was followed by additional steps that involved evaporation and reconstitution of the
residue in an organic solvent. When the two SPE methods were compared, the C18-SPE
cartridge proved to be the optimum, because the S/N was three times higher (S/N=154) than
when Oasis HLB cartridge was used (S/N=52), and it provided better recoveries.
The optimum CZE-ESI-MS parameters and the optimum sample preparation procedure
were finally applied for the determination of rivastigmine in a plasma sample obtained from
an AD patient following rivastigmine patch administration (dose of 9.5 mg/mL
rivastigmine/24-h). Figure 7 demonstrates the SIM electropherograms of C18-SPE extract of

Electrophoresis 28
plasma sample collected 2.0 hours post-application, at m/z 223 and 251, for I.S. and
rivastigmine, respectively. The mean ( S.D.) plasma concentration obtained for
rivastigmine was 14.6 ( 1.7) ng/mL.

Figure 7. Electropherograms of C18-SPE extracts of plasma from an AD patient following rivastigmine
patch administration in a dose of 9.5 mg/mL / 24-h in the SIM-mode at (a) m/z 223 (I.S.) and (b) m/z 251
(rivastigmine). Conditions: BGE: 40 mM ammonium acetate at pH 9, sheath liquid 1 % acetic acid in
water:MeOH (50:50 v/v) at a flow rate of 10 L/min, analyte and I.S. concentrations 0.3 mg/mL. Drying
gas flow rate 6 L/min and temperature 200 C, nebulizer gas pressure 20 psi [24].
Based on the studies mentioned above, the CZE-ESI-MS method proved to be a promising
technique in drug and pharmaceutical analysis. The development of such a method has
several advantages over HPLC-MS and GC-MS. The most important ones are the reduction
of the reagents cost, the low injection volume requirements, and the avoidance of disposing
large volumes of organic waste. In particular, when the study described here is compared to
previous studies, where HPLC-MS and GC-MS were used for the analysis of rivastigmine,
the required injection volume of plasma for a single analysis is reduced from microliters [40-
43] to nanoliters.
4. Determination of enantiomers in pharmaceutical samples
In the last three decades, there has been a growing interest in the separation, detection and
quantification of enantiomers in pharmaceutical, clinical, environmental and food analysis.
It has been known that usually only one enantiomer is active while the other may be less
active, inactive or has adverse effects. Among the separation techniques, HPLC [44-48] GC
[44,45,49] and CE [50-55] are most often applied in chiral analysis. Temperature and
derivatization are major problems encountered in GC, and poor separation efficiency is
observed in HPLC. CE has proven to be a powerful separation technique in the area of chiral
analysis, since it has the major advantage of low consumption of samples and solvents.
The most common modes of chiral CE are electrokinetic chromatography (EKC) in the
presence of a chiral selector, MEKC, capillary electrochromatography (CEC), where the chiral
selector can be either used as a coating (OT-CEC), a packing (P-CEC) or a monolithic material
(M-CEC) in the capillary, and others [55-63]. The prerequisite for separation of enantiomers in
CE, as in every chromatographic system, is the formation of either stable diastereoisomers by
the use of a chiral derivatization agent or reversible diastereoisomeric complexes with the
addition of a chiral substance, (chiral selector). In the first case, the two enantiomers are
separated based on their different physicochemical properties, while in the second case, they
are separated based on their different mobilities. In general, the three point rule, illustrated
by Easson and Stedman [64], describes the interactions that are necessary for chiral
discrimination. A minimum of three simultaneous interactions have to occur between the
chiral selector and one of the enantiomers so that chiral separation is achieved. The other
enantiomer, due to spatial restrictions, should have at least two types of interactions, which
can be hydrophobic interactions between the hydrophobic core of the polymer and the analyte,
electrostatic interactions between the polar head group of the polymer and the analyte, dipole-
dipole forces, such as hydrogen bonding between the polar group of the chiral selector and the
analyte, and secondary interactions, such as - interactions, ion-dipole bonds, and Van der
Waals forces. This difference in the number and type of interactions between the enantiomers
and the chiral selector generates a mobility difference between the enantiomer-chiral selector
complexes, which is necessary for the achievement of a chiral separation.
A big number of chiral selectors have been widely used, over the years, in CE for improved
chiral separations of various classes of analytes. These chiral selectors include cyclodextrins,
polymeric surfactants, cyclofructans, macrocyclic antibiotics, crown ethers, and others.
Cyclodextrins are molecules with large ring-like structures composed of -(1,4)-linked D-
(+)-glucopyranose units. Native cyclodextrins are cyclic oligosaccharides consisting of six (-
CD), seven (-CD) and eight (-CD) glucopyranose units. The chiral recognition ability of
cyclodextrins can be improved by their derivatization with different functional groups, such

Electrophoresis 30
as methyl-, sulfate-, acetyl- and prolyl-, and with the modification of the hydroxyl groups,
which are present on the rim of the CD. The mechanism of enantiomeric discrimination is
the inclusion of the hydrophobic group of the analyte into the cavity and interactions of the
hydroxyl groups of the C2 and C3 at the upper rim of the CD, such as hydrogen bonds and
dipole-dipole interactions.
Navarro et al. [65] developed a CZE method for the analysis of lansoprazole enantiomers in
three different pharmaceutical preparations (Davur, Alter and Cinfa). -CD was used as a
chiral selector and sodium sulphite was used as an additive. Recoveries of 91-102% of the
label content were obtained, and this demonstrated the potential of the method for the
routine quality control of lansoprazole enantiomers in pharmaceutical formulations.
Chai et al. [66] used the chiral selector hydroxypropyl--cyclodextrin in order to separate the
antifungal drug iodiconazole and the structurally related triadimenol analogues. This chiral
selector provided the best results in regard to resolution due to its large cavity and the
hydrogen bonding between the analytes and the cyclodextrin. The mechanism for the chiral
discrimination of hydroxypropyl cyclodextrins possibly involves the development of
secondary interactions between the chiral analyte and the hydroxypropyl groups on the
cyclodextrin rim after the inclusion of the analyte into the cavity. The degree of substitution
and the type of the hydroxyalkyl group on the cyclodextrin rim, which influences the depth
of the cavity, can therefore change the enantiorecognition ability of the cyclodextrin [67,68].
4.1. Method development and validation
The use of CE, and particularly MEKC, in chiral analysis is demonstrated further here by
providing a more in-depth analysis of a research work that was performed in a pharmaceutical
formulation that contained one of the enantiomers of Huperzine A [55]. Huperzine A is
considered to be a potent, highly specific and reversible inhibitor of acetylcholinesterase with
high efficiency and low toxicity. The mechanism of complexation of Huperzine A with
acetylcholinesterase is similar to that of other pharmaceutical drugs that are used for the
treatment of AD [69]. The (-)-enantiomer of Huperzine A is three times more biologically
active than the synthetically racemic mixture, and only this form behaves as a potential
acetylcholinesterase inhibitor. Therefore, the development of an analytical method for the
enantiomeric separation of the synthetic Huperzine A is of greatest importance.
It is important here to mention that the type of the chiral selector used in this study was the
polymeric surfactant. The use of polymeric surfactants in both chiral and achiral CE has
attracted considerable attention. In 1994, Wang and Warner [70] were the first to report the
use of a polymeric surfactant added to the BGE in MEKC. Polymeric surfactants offer
several distinct advantages over conventional micelles [63,71-73]. Firstly, polymerization of
the surfactant eliminates the dynamic equilibrium due to the formation of covalent bonds
between the surfactant aggregates. This, in turn, enhances stability and improves resolution.
Secondly, polymeric surfactants can be used at low concentrations because they do not
depend on the CMC. This usually provides higher efficiencies and rapid analysis. They
have, over the years, been extensively used in a BGE [74-80], in a polyelectrolyte multilayer
coating [63,74,81-83], and in a CE-MS system [84-86].
In this study, the optimal conditions, in regard to resolution, efficiency and analysis time,
were initially established by varying different electrophoretic parameters. The BGE type,
concentration and pH are usually the first parameters to be examined in a method
development procedure. Sodium acetate at acidic and neutral pHs, where the analyte
exhibits cationic behavior, was chosen as the optimum. BGEs with basic pHs did not exhibit
any enantiomeric discrimination, and the analysis time was very long. The optimum pH
was 5.0 because it provided slightly better peak shapes, and the optimum concentration was
50 mM because it provided higher resolution (Figure 8). The very low peak efficiency, which
needs to be improved, is clearly illustrated in this figure.

Figure 8. Effect of BGE concentration on the separation of the enantiomers of Huperzine A: (A) 20 mM,
(B) 35 mM and (C) 50 mM. Separation conditions: BGE: sodium acetate (pH 5.0), 0.075% (w/v) poly-LL-
SULV; pressure injection, 30 mbar for 3 s; applied voltage, 20 kV; temperature, 25 C; fused-silica
capillary, 64 cm (55.5 cm effective length) x 50 m i.d.; detection, 230 nm [55].

Electrophoresis 32
As far as the chiral selector is concerned, different polymeric surfactants were examined,
such as poly(sodium N-undecanoyl-L-leucinate) (poly-L-SUL), poly(sodium N-undecanoyl-
LL-leucyl-leucinate) (poly-LL-SULL), poly(sodium N-undecanoyl-LL-leucyl-valinate) (poly-
LL-SULV), poly(sodium N-undecanoyl-L-valinate) (poly-L-SUV), poly(sodium N-
undecanoyl-L-valyl-glycinate) (poly-L-SUVG), poly(sodium N-undecanoyl-LL-alanyl-
valinate) (poly-LL-SUAV), poly(sodium N-undecanoyl-LL-leucyl-alanate) (poly-LL-SULA),
and poly(sodium N-undecanoyl-LL-valyl-valinate) (poly-LL-SUVV). The polymeric
surfacant poly-LL-SULV, which has shown the best chiral discrimination ability for a
number of pharmaceutical compounds [80], was the first to be examined in different
concentrations. The concentration of 0.075% w/v was chosen as the optimum, based on
analysis time, efficiency and resolution. This concentration though did not provide baseline
resolution.
Another parameter examined in order to improve peak efficiency and resolution was the
addition of modifiers. None of the organic solvents at different concentrations were able to
improve the separation. An alternative to this was the addition of a salt, such as D- and L-
alanine tert-butyl ester hydrochloride (D- and L-AlaC4Cl). poly-LL-SULV became insoluble
when the salt was added into the BGE. Therefore, the other polymeric surfactants
mentioned above were examined at different concentrations. In each case, D- and L-AlaC4Cl
were used individually as additives, the electropherograms were obtained, and resolution
and efficiency were estimated. Based on this, the combination of poly-LL-SUAV at a
concentration of 0.20% (w/v) with L-AlaC4Cl provided the best results.
However, the use of L-AlaC4Cl did not provide satisfactory reproducibility of the migration
time and efficiency. This is probably due to the hydrolysis of the salt in an aqueous BGE
solution. An alternative involved the use of tert-butanol, one of the hydrolysis products, at
different concentrations. Figure 9 clearly demonstrates the improved peak efficiency, in
comparison with Figure 8. Each electropherogram was obtained at a different concentration
of tert-butanol. A concentration of 10% (v/v) was the optimum, because it provided the
highest resolution (1.45) and the highest peak efficiency (Figure 10).
The validation of the method demonstrated good linearities and very low relative standard
deviation values, indicating excellent run-to-run and day-to-day reproducibilities. In addition,
the LOD and LOQ were determined to be 4.17 g/mL and 13.92 g/mL, respectively.
4.2. Application
As previously shown, after method development and validation, the optimum separation
conditions are applied to a real sample. In this case, the optimum parameters were applied
to a pharmaceutical formulation in order to detect and quantitate the acetylcholinesterase
inhibitor (-)-Huperzine A. The extraction procedure followed for extracting Huperzine A
from the pharmaceutical formulation proved to be effective because the enantiomer
determined in the sample was in a relatively good agreement with the amount that was
stated on the bottle. Therefore, the developed MEKC-UV method is able to control the
purity of (-)-Huperzine A in pharmaceutical formulations.

Figure 9. Effect of the concentration of tert-butanol on the separation of the enantiomers of Huperzine
A: (A) 5%, (B) 7.5%, (C) 10% and (D) 12% (v/v). Separation conditions: BGE: 50 mM sodium acetate (pH
5.0), 0.2% (w/v) poly-LL-SUAV; pressure injection, 30 mbar for 3 s; applied voltage, 20 kV; temperature,
25 C; fused-silica capillary, 64 cm (55.5 cm effective length) x 50 m i.d.; detection, 230 nm [55].

Electrophoresis 34

Figure 10. Effect of the concentration of tert-butanol on the efficiency. Separation conditions: Same as
Fig. 4. BGE: 50 mM sodium acetate (pH 5.0), 0.2% (w/v) poly-LL-SUAV; pressure injection, 30 mbar for 3
s; applied voltage, 20 kV; temperature, 25 C; fused-silica capillary, 64 cm (55.5 cm effective length) x 50
m i.d.; detection, 230 nm [55].
5. Concluding remarks
Analysis of chiral and achiral analytes in natural, pharmaceutical and biological samples can
be extremely difficult. Co-migration may occur, which can cause problems in detection, and
the electropherograms obtained can be very complex. In addition, the analytes of interest are
usually present in the matrices at very low concentrations. Therefore, all the analytical steps,
including method development, detection and sample preparation, which is an essential
stage in any analysis process, have to be optimized in order to obtain the desirable
sensitivity, resolution, robustness and analysis time.
Among the separation techniques that have so far been used for pharmaceutical, clinical and
food analysis, CE has been established as a powerful analytical tool, which has rapidly been
developed and matured since its introduction. CE and its related techniques offer a number
of advantages, including low consumption of sample and solvents, high separation
efficiency, rapid method development, fast migration times, versatility, and simple
instrumentation. Another important aspect involves its ability to separate small and large
molecules, charged and neutral species, inorganic and organic molecules, synthetic and
natural compounds, along with proteins and peptides.
The coupling of CE to MS provides nowadays a promising alternative to UV detection. The
combination of high sensitivity, high selectivity, and high specificity provided by MS with
high resolution, and high efficiency provided by CE makes it an attractive technique in
different fields, such as clinical, forensic, pharmaceutical, and others. However, chiral
analysis by use of CE-MS still needs some improvement, in regard to resolution and peak
capacity. In addition, contamination of the ionization source induced by the chiral selector
added in the BGE is still considered a main problem, even though different procedures
have, in recent years, been developed in order to overcome this limitation [87].
Author details
Department of Chemistry, University of Cyprus, Nicosia, Cyprus
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Chapter 3
Numerical Modelling of Light Propagation
for Development of Capillary Electrophoretic
and Photochemical Detection Systems
Tomasz Piasecki, Aymen Ben Azouz, Brett Paull,
Mirek Macka and Dermot Brabazon
http://dx.doi.org/10.5772/45764
1. Introduction
Capillary electrophoresis (CE) has been used for over 30 years as an efficient separation
technique [1]. These separations are typically preformed using capillaries with internal
diameter ranging from 2 m to 200m and as the diameter increases above 100m, a
reduction in separation performance is observed [2]. One of the main factors limiting the
overall performance of CE separations is Joule heating associated with the passing of the
electric current through resistive medium. The high surface-to-volume ratio in a smaller
capillary diameter allows for efficient heat dissipation which is beneficial for compound
separation, however, simultaneously this has a negative impact on the detection
performance due to the corresponding reduction in analyte volume available for detection
[3].
Historically when capillaries were first used for CE, they were coated with polyimide and
presented excellent robustness but posed problems with optical detection as polyimide is
highly absorbing below 550nm preventing optical detection in that spectral range. Such
capillaries were striped of the coating to enable the optical detection in UV and low
wavelength visible range. However, capillaries striped of their coating are brittle and can
easily be damaged making usage often impractical. The introduction of the
polytetrafluoroethylene (PTFE) coated capillaries allowed for optical detection within the
UV range as well as across the entire visible spectrum. Although the PTFE coated
capillaries are not as durable as the polyimide coated capillaries, they are significantly
more robust than coatless ones and allow for easier deployment during typical daily
laboratory routine.

Electrophoresis 42
Absorbance photometry is a detection technique used to determine the concentration of
target species in a liquid sample based on interaction between the probe light and species
[4]. Typically it is a measurement of the light intensity with and without a sample placed in
the light path. A scheme of light intensity measurement with the sample located for
detection is presented in Figure 1. The sample transmittance, T, is defined as the ratio of the
initial light intensity, I0, to the recorded light intensity, I1 (Eq. 1). I0 should be measured with
the sample holding cuvette empty. This allows for reflections and potential absorption by
the cuvette material to be taken into account during sample measurement. Sample
absorbance, A, is measured as the negative log of the transmittance (Eq. 2). The cuvette
length l is known, as well as species molar absorptivity coefficient , which is a specific
characteristic of every species. Light attenuation along the light path is governed by Beer-
Lamberts law where c is molar concentration (Eq. 3) [5]. The method of calculating the
actual optical path length is presented elsewhere [6].

Figure 1. Schematic of light intensity with sample located for photometric detection
I =
I
1
I
0
(1)
A = log I = log
I
1
I
0
(2)
I(l) = I
0
c
-ucI
= I
0
c
-A
(3)
Despite excellent performance in analyte separation, CE techniques still constitute a
minority of the commercial applications due to detection limitations imposed by popular
absorbance photometric detectors. Various developments in CE have occurred such as the
development of UV-transparent capillaries which facilitated CE by making it possible to
employ the commercially available detectors. In the past different approaches have also
been employed to alter the geometry of the detection system in order to improve the
detection performance for CE. These include the application of rectangular capillaries to
reduce refraction effects on the cylindrical boundaries [2], multi-reflection cells for increased
signal intensity [7], and development of Z-shaped flow cells with increased optical
pathlength for analyte detection [8]. Some detectors were built employing these methods,
for Development of Capillary Electrophoretic and Photochemical Detection Systems 43
but to date the majority of the commercially available absorbance based detectors work on
the basis of a single passage of light through the sample contained in a capillary.
To gain a better understanding of light propagation through capillaries and how to improve
detection levels, the modelling of the light propagation through capillaries has been
previously undertaken. Much of this work was limited to two-dimensional projections and
two-layer models (no coating present) taking into account capillary material and inner
cavity [9]. Previous numerical simulations of light propagation through capillaries have
been reported as a tool for flow-cell design and have been limited to prediction of light path
for these specific cases only [10]. The numerical problem of ray-tracing through a capillary is
an excellent example where advantage can be taken of the ability to perform numerous
calculations quickly on a computer to allow for ray paths, ray path overlaps, and resultant
light intensities to be calculated.
This book chapter presents a theoretical study on the light propagation through coated
capillaries, focusing on PTFE-coated capillaries. These models can be used to increase the
performance of absorbance photometric detection and for associated photochemical applications.
2. General description of the model
The Light propagation and light intensity distribution models were developed using
LabVIEW 2011 graphical programming environment. Graphic-related work, such as
generation of multi-colour maps and reading pixel colours were conducted with Adobe
Photoshop CS 3 ver. 10.0.1. Dimension and angle measurements taken from photos were
performed using Image J 1.43u software.
Whenever light is incident on the boundary of two transparent dielectrics part of it is
reflected and part is transmitted, see Figure 2. The angle of incidence is related with the angle
of transmittance by Snells Law (Eq. 4). In the case of cylindrical symmetry, as in capillaries, it
is possible that the exiting ray will not be parallel with the incident, see Figure 3.

Figure 2. Light incident on boundary of two different dielectrics.

Electrophoresis 44

Figure 3. Schematic of light transmission through, (a) a flat transparent material and (b) a cylindrical
capillary, showing curvature affects on angle of incidence and transmittance.
n
sin 0
= n
t
sin 0
t
(4)
where ni is the refractive index of the incident ray transmission medium, i is the angle
between incident wavevector and the normal to surface, nt is the refractive index of the
material through with the transmitted ray passes and t is the angle between transmitted
wavevector and normal to the surface. Light attenuation inside the absorbing medium is
governed by Beer-Lambert law (Eq. 5).
I = I
0
c
-ucI
(5)
where I is the initial light intensity, I0 is the light intensity behind the sample, is the molar
absorptivity coefficient, c is concentration of the compound and l is length is the absorption
path length.
The numerical modelling software was developed to calculate the light ray path through
multi-layered cylinders, and the light intensity distribution map through the cylinder
cross-sectional area. Light propagation within multimode optical fibres occurs by the
phenomena of total internal reflection, where the values of refractive indices and fibre
diameter remain within the limits of geometrical optics. The size of the capillary used in
this work was comparable with the size of multimode optical fibres. It was assumed that
capillary body, coating and bore were perfectly cylindrical and concentric. Only the right
half of the capillary cross-section is displayed in the developed model, as the diameter
acts as the modelled axis of symmetry and no light ray could propagate through from left
to right side. In general such occurrence is possible, but only for higher values of
refractive indices approximately twice those of glass and PTFE which were used in this
work.
The programmed model calculated the light ray path equations in the Cartesian coordinate
system. Separate linear functions to describe each of the light ray path segments were used
(for example between air/tube, tube/tube or tube/liquid).

Figure 4. Schematic of reference lines and points, and light ray path calculated by each sub-routine for
light passing through a three layer system in which the light ray path is passing through a coated
hollow capillary.
Each light path segment was calculated in a separate subroutine calculating the light path in
each zone. Incident light was in the form of rays parallel to y-axis, see Figure 4. The first
subroutine calculated the coordinates of the light incident from infinity on the air/coating
boundary under desired angle. This point of incidence of the ray a1 on outermost circle c1
was assigned as p1. An extended radius r1 going from (0, 0) through p1 was drawn for
calculation of angle of incidence i1 and in turn angle of transmission t1 was calculated
from Eq .4, for the use inputted values of refractive indices ni and nt. A line a2 through p1
was drawn representing the refracted ray in the coating with t1 as the angle between a2 and
r1, ending the first program subroutine, see Figure 4A. The next subroutine began with
calculation of point p2 (where line a2 crossed boundary c2) and the drawing of extended
radius r2 from (0, 0) through p2. Angle of incidence i2 was calculated as the angle bounded
by r2 and a2, see Figure 4B. Angle of transmittance t2 was calculated from Eq. 4 and the line
a3 was drawn where t2 was and angle between r2 and a3, ending the second program
subroutine, see Figure 4B. This subroutine was iterated a further three times to calculate light
ray paths segments along lines a4, a5 and a6 after refraction on each encountered boundary.
These next subroutines of the light path formation are illustrated in Figure 4C to E and the
complete generated light path is presented in Figure 4F without reference lines. The entire
light path was represented as a sum of individual rays calculated separately according to
the symbolic algorithm:

Electrophoresis 46
r = (o
k
c
k
p
k
r
k
0
k
0
t
k
o
k+1
)
2n
k=1

where r is a light ray path, k is a step number; n is a number of layers; a, c, p, r, i and t are
as described earlier. The experiments on model validation and their results are presented
elsewhere [11].
3. Application of the developed model for optimisation of the capillary-
based optical detection system
The main aim of this part is to present quantitative information about light behaviour in
capillary base detection setups. Their highly complicated geometry and multi-layered
construction results in unusual optical properties can significantly affect the performance of
the detection system if not designed and manufactured carefully.
The light attenuation inside light absorbing medium is governed by Lambert-Beers law (Eq.
2). However in this form it is assumed that the light that is collected by a detector is
concurrent with the light that passed through the absorbing medium. In real situation there
is additional component that can seriously affect photometric measurements: the stray light.
It can be defined as the light that omits the sample but still is collected by the detector. In
such case (Eq. 2) should be written in another form (Eq.4):
A = log
I
A
-I
s
I
0
(6)
where: A is absorbance, IA is the light intensity of the attenuated light beam, IS is intensity of
the stray light, and I0 is the initial light intensity. In such case, with increase of the amount of
light attenuating medium IA goes down to zero and Eq. 6 becomes Eq. 7, effectively
imposing maximum possible absorbance:
A = log
I
S
I
0
= const. (7)
Figure 5 presents the maximum possible absorbance recorded depending on the percentage
amount of the stray light in the detection system. For the illustration purpose, the relation
between concentration and absorbance is set to one. The line labelled Ideal represents
situation with no stray light at all, giving completely linear response depending on the test
compound concentration. The other four curves labelled 10%, 1%, 0.1% and 0.01%
represent the same response with the respective percentage of the stray light present in the
system. This allows visualising the detection limits associated with the imperfectness of the
detection system. Also it is noteworthy to compare the deviation from the linearity that is
associated with the stray light.
Figure 6 presents the deviation from the ideal response depending on the amount of the
stray light. As it can be read from the graph on Figure 6, the deviation is small, and therefore
assumption of linearity is valid typically one absorbance unit before the maximum, e.g. for
0.1% of the stray light, the maximum measure absorbance is 3 AU, and the assumption of
linearity is valid from 0 to 2 AU.

Figure 5. A comparison of different amounts of stray light present in the detection system and how it
imposes detecion limits. Concentrations are scaled in arbitrary concentration units (ACU). Absorbance
scaled in absorbance units (AU).

Figure 6. A comparison of the deviation from the linear response depending on the amount of stray
light in the system. Concentration scaled in arbitrary concentration units (ACU). Absorbance scaled in
absorbance units (AU).
For numerous reasons it is convenient to employ the same capillary for detection after any
reactions or separations. The cylindrical geometry of the capillary combined with its multi-
layer structure creates a quite complex medium for the light to propagate through and
eventually can significantly alter the light path. Figure 7 presents a simplified image of

Electrophoresis 48
possible light refractions on different boundaries (air/coating, coating/fused silica body,
body/content). In this figure only light perpendicular to the capillary symmetry axis is
portrayed. In real situation light coming out from an uncollimated light source can be
incident under any angle. This situation can be compared with a capillary illuminated by a
laser.

Figure 7. Schematic of a capillary cross-section illuminated by a beam of parellel rays.
In order to prevent undesired light to reach the detector the light source and the detector
should be comparable with the capillary bore internal diameter. This can be achieved by
installing an aperture or by using an optical fibre to illuminate the capillary and collect the
light for detection. Such application of optical fibres has been reported already several years
ago [12], [13]. Application of any of these two approaches has an impact on the overall
performance of the detection system. Light going through an aperture undergoes diffraction
and light coming out of an optical fibre is divergent with the spread angle confined by
numerical aperture (NA) of the fibre itself. The NA of 0.22 gives cone of acceptance with
maximum possible angle of 25.4, being as well the maximum angle at which the light will
exit the fibre. Figure 8 present a comparison of the light intensity that can interact with
capillary bore content depending on the distance at which a 100m collecting optical fibre of
with NA of 0.22 for four different maximum incidence cones. Zero degrees is equivalent of an
ideally parallel beam, while 25 degrees is an equivalent of the light coming out of an optical
fibre. Light intensity loss is the effect of the capillary geometry only and the capillary coating,
capillary body and the theoretical solution filling the capillary bore are assumed to be
completely transparent. As it can be seen the light coming out of the optical fibre has the
angle of divergence of 25 degrees. This results that only over 22% of the delivered light
intensity can reach the capillary bore, interact with its content and be collected by similar
100m diameter optical fibre. That number decreases with distance as light after propagating
through the entire capillary becomes even more divergent, similarly to a ball lens.

Figure 8. Relation between the percentage of light passing though the capillary bore, comming from a
100 m fibre, and the distance between the collecting 100 m optical fibre and the capillary. These
results were calculated for incident beams with 0, 5, 10 and 25 degrees divergent cone of illumination.
The light intensity loss associated with the divergent illuminating beam is not the only
concern. At the same time due to the capillary geometry it is possible that the light exiting
the illuminating fibre will enter the capillary, propagate through omitting the capillary bore
completely and exit it in a manner that can still enter the collecting optical fibre. Such light
would contribute toward the stray light and reduce overall efficiency of the detection
system. Figure 9 illustrates the percentage amount of the stray light that can enter collecting
optical fibre under an angle below the numerical aperture depending on a distance between
the capillary and the end of the fibre.
For the collecting fibre perfectly touching the capillary the amount of stray light is zero.
However already at distance of 0.5mm this goes up to 40% if the capillary is illuminated
with an optical fibre as well. The observed maximum for 10 degrees divergent incident
beam (Figure 9 - green line) followed by drop of stray light to zero a 2mm from capillary is a
result of the capillary geometry light omitting the capillary bore after passing the coating
and exiting the capillary becomes divergent and eventually is not present within the central
region where the collecting optical fibre is located. However the value of 10 degrees
divergent incident beam is possible theoretically it is unlikely to occur in the real
experimental setup.

Electrophoresis 50

Figure 9. Percentage of stray light collected by a 100m optical fibre related to its distance from
capillary and for four different values of the incident beam divergence angle: 0, 5, 10 and 25 degrees.
The divergence value of 25 degrees as the divergence of the light exiting fused silica optical
fibre was selected for other comparison what diameter of the illuminating and collecting
optical fibre should be used to maximise the amount of light interacting with the capillary
content while minimising the amount of stray light. The values were modelled for 100m
internal diameter capillary. Two different approaches were tested:
- wide-area illumination and small area collection, attempting to collect only the light
passing through the capillary bore.
- narrow-area illumination attempting only to illuminate the capillary bore and collecting
as much light as possible
The results are summarised in Figure 10 and Figure 11. Figure 10 presents the collected light
intensity that passed through the capillary bore and could interact with its content
depending on the distance of the collecting fibre from the capillary.
Figure 11 presents percentage amount of stray light associated with the respective setting.
The first number represents the illuminating optical fibre diameter in micrometres, and the
second represents the collecting fibre diameter in micrometres as well.
The highest overall light intensity of the light able to interact with the capillary content
was achieved with 50m illuminating and 200m collecting fibres. However at the
position when maximum light intensity is collected the amount of stray light was
unsatisfactory (close to 20%). The large diameter of the collecting fibre, twice of the
internal capillary diameter, was large enough for the light to propagate around the
capillary bore and still enter the fibre under a sufficient angle to stay within the optical
fibre.

Figure 10. The amount of the initial light intensity that propagates through the capillary bore and
subsequently is collected compared for different combinations of diamteres of the illuminating fibre
(the first number) and the collecting fibre (the second number)

Figure 11. The amount of the stray light collected, compared for different combinations of diamteres of
the illuminating fibre (the first number) and the collecting fibre (the second number)
Reducing the diameter of the collecting fibre to 100m helped avoid stray light even when
the distance between the collecting fibre and the capillary was around 1mm. This result can
be very helpful as it allows for the high efficiency of the light collection combined with the
minimisation of the stray light due to imprecise assembly of the detection system.
Combination of 100m illuminating and 100m collecting fibres gave good results in terms
of the available light intensity, but even a slightest misalignment of the collecting optical

Electrophoresis 52
fibre (0.2mm distance between can result in large amount of stray light delivered to the
detector. Other combinations (100m and 200m illuminating fibres and 50m collecting
fibre) produced significantly lower available light intensity combined with high
vulnerability to stray light introduction due to non-perfect assembly of the optical system.
In order to design a capillary based photo detection system the following occurrences should
be considered: effect of stray light, amount of light intensity within the capillary bore,
location of illuminating and collecting optical fibres and diameters thereof, as well as their
angle of incidence. When considering all of these factors in conjunction, a complex hyper-
plane of possible solutions presents itself which cannot be simply understood or calculated. It
is therefore valuable to have a numerical model which can simultaneously take into account
these factors. Such a model can also be used for other applications. An example of this is
presented in the following sections for the application of photopolymerisation.
4. Optimisation of the photochemical reactions in capillaries
The main aim of this part is to present qualitative and semi-quantitative information about
light behaviour in photopolymerisation setups. The capillary geometry and its multi-layered
construction resulting in unusual and unexpected optical properties should be better
understood as it allows to improve the work efficiency with capillaries. As explained in later
part of this article many of described problems occur only in polytetrafluoroethylene (PTFE)
coated capillaries.
For photoinitiated polymerisation in UV-range polyimide coated capillaries cannot be used
because of polyimide high absorptivity below 550 nm. PTFE-coated capillaries were selected
as PTFE is transparent in UV region. During several experiments it was noted that position
of LED versus capillary play significant role in shape and quality of formed polymer. When
LED was placed too close to the capillary monolith was growing distance of several
millimetres under photomask. It was also noted that edges of formed polymer were uneven
and blurry. If LED tilted there was noticeable difference in growth of one end of monolith.
Observed problems in polymerisation setup are presented schematically on Figure 12. A
series of experiments was conducted to repeat mentioned observation and approach them
qualitatively and semi-quantitatively. The experimental conditions for photopolymerisation
reactions are presented elsewhere [14].
A series of experiments were conducted in order to evaluate the degree of monolith growth
under the masked region of a capillary where direct light should not enter. Observed results
in experiments with photopolymerisation using same mixture and light sources showed that
monolith can grow few millimetres under the mask. Such growth is considered undesired.
Table 1 and Figure 14 shows average of three results of photoinitiated polymerisation of
monolith with measured distance of the part formed under the photomask in a region
where direct light should not reach and therefore polymerisation would not be anticipated
or well controlled. There was a clear trend of increased length of monolith grown under the
mask as the LED was placed closer to the capillary. Also when the LED was tilted by an
angle of 45 growth was significantly larger that side compared to when the LED was held
perpendicularly. The light source was located directly over the edge of the photomask as
shown in Figure 13.

Figure 12. Schematic illustration of observed problems with monoliths obtained by photoinitiated
polymerisation: (a) ideal monolith, (b) monolith growing up to several milimeters under the photomask
in both direction when LED was too close to the capillary, (c) monolith growing on one end when the
LED was tilted.

Figure 13. (a) schematic of the LED placement versus the capillary for perpendicular illumination and
(b) for illumination under 45 angle. Red arrow marks distance from capillary to the LED, blue arrows
marks distance of the monolith growth.

LED perpendicular LED tilted by 45
LED distance from
capillary
Growth length
LED distance from
capillary
Growth length
0 cm 1.1 mm 0 cm 2.3 mm
1 cm 0.7 mm 1 cm 1.9 mm
2.5 cm 0.5 mm 2.5 cm 1.7 mm
4 cm 0 mm 4 cm 1.4 mm
Table 1. Monolith growth depending on light source position.

Electrophoresis 54

Figure 14. Relation between distance from the light source and monolith growth distance beyond the
photomask covering for the case of the LED shining perpendicularly to the capillary and at an angle of
45 to the capillary.
Previously described theoretical model for light propagation was used in order to
understand what is happening during photopolymerisation with light, solution and
monolith. Tilting of the LED and thus delivering significantly more light to one side of the
monolith suggest that capillary geometry plays significant role.
When the light is incident on a boundary of two dielectrics with different dielectric constant
(e.g. PTFE/fused silica, fused silica / polymerization mixture) a portion of it undergoes
reflection reducing the intensity of transmitted light. The intensity of the reflected and
transferred light is given by Fresnels equations (Eq. 8) separately for each polarisation
of light:
R
s
= j
sn(0
i
-0
t
)
sn(0
i
+0
t
)
[
2
R
p
= j
tun(0
i
-0
t
)
tun(0
i
+0
t
)
[
2
(8)
The uncollimated light is mixture of all possible polarisations, that is combination of linear
polarisations and can be separated for p polarisation (vector of the electric field is parallel to
plane of incidence) and s polarisation (vector of the electric field is perpendicular to plane of
incidence). Graph on Figure 15 shows percentage of light being reflected on boundary
PTFE/fused silica (light coming from medium with lower refractive index to medium with
higher one) versus angle of incidence for both polarisations calculated from Eq. 8.
The light that reflects multiple times on dielectric boundary quickly loses intensity. The
developed numerical model showed that incidence angles that are present in a capillary at
the fused silica/PTFE boundary illuminated from outside do not provide significant
0
0.5
1
1.5
2
2.5
3
0 1 2 3 4 5
M
o
n
o
l
i
t
h

g
r
o
w
t
h

u
n
d
e
r

t
h
e

p
h
o
t
o
m
a
s
k

[
m
m
]
Distance from LED to capillary [cm]
LED perpendicular
LED tilted by 45
reflectance and light is quickly transmitted outside of the capillary. Figure 16 provide
information with upper limits of the light intensity that can be reflected by the capillary
assuming total lack of absorption at this stage.

Figure 15. Theoretical reflectance of s and p polarised light incident on boundary PTFE/fused silica
versus the angle of incidence; calculated from Eq. 8.

Figure 16. Schematic of the light propagation by multiple reflections inside PTFE-coated fused silica
capillary with upper limit of the initial intensity I0 (no more than). Dark blue PTFE, light blue fused
silica, white polymerisation mixture, red light path

Electrophoresis 56
Figure 16 shows schematic cross-section of capillary with sample light ray. After two
reflections (first on boundary fused silica/polymerisation mixture, second on boundary fused
silica/PTFE) not more than 0.16% of intensity from point A is delivered to point B. The real
value of the light intensity in point A is already lower than I0 due to reflection on boundary
air/PTFE and boundary PTFE/fused silica, but these effects are neglected and rounded to I0 is
this discussion. After three reflections intensity drops to not more than 0.06% of initial, and
after four to not more than 0.02%. Dimensions of the capillary and refractive indices of PTFE,
fused silica and polymerisation mixture are enforcing angles in further reflections and
transmittances after around 300 m from initial point of illumination total delivered light
intensity is below 0.2% of the initial light intensity I0 delivered to the capillary is available.
This calculations are based on assumption that all materials (air, PTFE, fused silica and
polymerisation mixture) are completely transparent and do not absorb any light. Because
their transmission coefficients are below 1 total amount of light available will be significantly
lower, making impossible to penetrate distances observed in the experiments.
The size of a standard capillary is comparable with size of multimode optical fibres, where
geometrical optics is sufficient to explain observations and calculate results. Capillaries and
coating have form of coaxial cylinders. The basic principle of optical (multimode)
waveguide is total internal reflection (TIR). This phenomenon is occurring when dielectric
with refractive index n1 (e.g. fused silica (FS)) is covered with dielectric with refractive index
n2 < n1 (e.g. PTFE, nPTFE < nFS). The light wave incoming from medium with higher refractive
index to boundary can undergo TIR provided angle of incidence is high enough. The graph
on Figure 17 shows dependence of intensity of reflected light versus angle of incidence (for
fused silica/PTFE boundary).
In order to observe total internal reflection for light incident on fused silica/PTFE boundary,
angle of incidence must be higher than 64.8, otherwise some light will be transmitted
through the boundary resulting with loss of the light intensity. The developed numerical
model showed that no angle higher than 44.7 is available in any part of the capillary
meaning that no light introduced to capillary from source placed above the capillary can
achieve angle sufficient to reflect totally within the fused silica.
Capillaries are made of fused silica and have very similar diameters to optical fibres. PTFE
has lower refractive index than fused silica. Initially a hypothesis about the capillary acting
as an optical waveguide was discussed to explain occurrences of monolith growth under the
photomask. There were two major contradictions: light coming from outside of cone of
acceptance (i.e. from source located above the capillary) for optical fibre it cannot be
transmitted over longer distance. The value of the highest possible angle of incidence on
boundary fused silica/PTFE for light interacting with capillary content obtained from the
developed model is significantly lower than required for the TIR. Graph on Figure 17 shows
that for angles of incidence below 45, reflectance is very low, and light is mostly
transmitted through the boundary.
The last possibility was reflection on boundary PTFE/air the highest possible ratio of
refractive indices. In order to observe TIR on this boundary the incidence angle of light must
not be lower than 49.25
1
. The highest incidence angle on fused silica/PTFE boundary
calculated using light propagation model gives angle of 44.57. Although amount of light
reflected on that boundary is around 10% it does not satisfy the condition to observe total
internal reflection and could not explain growth of few millimetres.

Figure 17. Theoretical reflectance on boundary fused silica/PTFE versus the angle of incidence.A
reflectance value of one (starting at 64.8) shows where total internal reflection occurs and no light is
transmitted through the boundary (from Eq. 8).
These attempts mentioned above to explain observed result were based on a static system
with constant time-independent properties. A capillary with on-going polymerisation
reaction is a dynamic system, which changes its physical and optical properties in time. A
hypothesis that a monolith forming inside capillary is changing optical properties of the
setup during the polymerisation was posed. To prove this hypothesis, new photographs of
capillary filled with polymerisation mixture and monolith were taken to show the
transmission of light when the LED was shining on the capillary. The capillary was installed
vertically above the digital microscope. In order to prevent any undesired light, a black
cardboard separated the microscope objective from the rest of the setup and photos were
taken in total darkness. Any possible openings near capillary wall were covered with a
sealant. Schematic of that setup is shown on Figure 18.
It can be clearly seen in the Figure 19a that light was transmitted through the fused silica. The
distance from the light source to the microscope was 20 mm to prevent any other discussed
method than waveguiding to propagate light toward the end of capillary. To confirm that the

1
Calculated from Eq. 22, refractive index of PTFE nPTFE=1.32 and refractive index of air nair=1

Electrophoresis 58
observed effect has nothing to do with collimation of the light the experiment was repeated
using 532 nm green laser as light source. Result is shown on Figure 20. Black spots are effect
of destructive interference of laser beam with itself after multiple reflections inside fused
silica. LED light is non-collimated thus the interference effect was not observed.

Figure 18. Schematic of experimental setup for observing light waveguiding inside the capillary with
monolith.

Figure 19. (a) Capillary with monolith and (b) empty capillary.
Image collected by digital microscope using setup from Figure 18.

Figure 20. Image observed when 532 nm green laser is used as light source (setup same as in Figure 18).
The monolith inside the capillary has a very irregular surface. Moreover the refractive
index of a polymer is higher than of the fused silica. The incident light was scattered on the
monolith surface (polymerisation mixture/monolith boundary) and due to the morphology
of the monolith surface it was scattered in all directions (Figure 21). This type of reflection
is called diffuse reflection or diffuse scattering. In this situation light can be reflected under
an angle sufficient to undergo total internal reflection on boundary fused silica/PTFE.
These angles are not available for light that is not a subject to diffuse reflection. The
refractive index of polymethacrylic polymer is higher than fused silica and ranges from
1.472 to 1.506 [15]. Light reflected diffusively in one part of monolith can propagate
through the monolith, cross the boundary monolith/fused silica, and then remain in the
TIR regime in regions where no monolith is present, effectively turning capillary into an
optical waveguide.
Also diffuse scattering allows photons entering fused silica under angles higher than for
those coming directly from light source. Wherever total internal reflection is occurring, on
opposite side of boundary an evanescent field appears strong enough to initiate
photopolymerisation. Figure 22 shows a schematic of that principle.

Figure 21. Schmatic of the diffuse scattering of incident light on the porous surface of the monolith that
has formed inside the capillary.

Electrophoresis 60

Figure 22. Schematic showing formation of the evanescent field (yellow arrow) outside dielectric inside
which light undergoes total internal reflection. Dark blue PTFE, light blue fused silica, transparent
polymerisation mixture.
5. Conclusions
The developed numerical model was used for investigation of the capillary optical
properties during the photopolymerisation reaction. One of the important goals of this work
was determining the relation between alignment of the light source and the shape and size
of produced polymerised monoliths. Previous observations suggested that light
transmission through the capillary fused silica body occurs in a similar manner as in an
optical fibre. The numerical model was required to determine the conditions necessary for
total internal reflection of the light used to initiate the polymerisation reaction, which in
consequence would lead to waveguiding of the light through the capillary and enabled
evanescent polymerisation in an obscured region under the photomask.
From the modelling and experimental results presented above, it can be seen that in cases
where light is incident outside of the cone of acceptance, it cannot be confined by total
internal reflection and thus does not propagate for significant distance. The presence of the
photopolymerised monolith within the capillary, provides a surface for diffuse reflection,
which in turn presents angles of internal reflection which would not otherwise be present.
This enables the capillary to act as an optical waveguide.
The minimum required angle to observe total internal reflection without monolith was not
present in the system. This result is concurrent with basic knowledge of the optical fibres:
the light must be introduces into the waveguide under a sufficient angle to remain within
total internal reflection regime. If the initial angle of incidence is higher than required the
beam will eventually escape the waveguide. This hypothesis was tested using an empty
capillary and a capillary filled with polymerisation mixture, which was illuminated
perpendicularly. A photodetector was used to record the light emitted though the capillary
cross-section. The results in both cases showed that there was no propagation of light along
the capillary.
During this investigation the dynamics of the polymerisation process which are affected by
the system optical properties was studied. The polymerisation process and the
corresponding formation of a monolith inside the capillary would be expected to affect the
optical properties of the whole system. An organic monolith is a highly porous, amorphic
structure, that when illuminated reflects a significant amount of light. The presence of the
monolith formed during the reaction changes the optical properties of the capillary that may
no longer be regarded as a uniform cylindrical structure with constant refractive indices
through the cross-section.
The monolith inside the capillary bore provided a surface for diffuse light reflection. The
monoliths surface morphology allowed for presence of angles that are normally unavailable
inside the capillary system, but with monolith waveguiding of the light becomes possible.
The light can be transmitted along the capillary for significant distances (measured distance
was 20 mm) utilising the total internal reflection mechanism. When light is entering the
capillary walls as in Figure 22 and undergoes total internal reflection within fused silica, an
evanescent field will appear within the coating and the internal boundary of the capillary
bore which is strong enough to initiate the reaction of photopolymerisation. Increased
distance between the capillary and the light source significantly reduces the amount of light
reflected from the monolith surface and thus the amount of light that can propagate through
the capillary.
Author details
Tomasz Piasecki, Aymen Ben Azouz and Dermot Brabazon
School of Mechanical Engineering and Manufacturing, Dublin City University, Dublin, Ireland
Irish Separation Science Cluster, Dublin, Ireland
Brett Paull and Mirek Macka
School of Chemistry, University of Tasmania, Hobart, Australia
6. References
[1] Capillary and microchip electrophoresis: Challenging the common conceptions. Breadmore, M.
C. 2012, Journal of Chromatography A, Vol. 1221, pp. 42 55.
[2] Rectangular Capillaries for Capillary Zone Electrophoresis. Tsuda, T., Sweedler, J. V., Zare, R.
N. 1990, Analytical Chemistry, Vol. 62, pp. 2149-2152.
[3] Capillary Electrophoresis. Kuhr, W. G. 1990, Analytical Chemistry, Vol. 62, pp. 403R-414R.

Electrophoresis 62
[4] Enhancement of detection sensitivity for indirect photometric detection of anions and cations in
capillary electrophoresis. Johns, C., Macka, M., Haddad, P. R. 2003, Electrophoresis, Vol.
24, pp. 2150-2167.
[5] Skoog, D. A., Holler, F. J., Crouch, S. R. Principles of instrumental analysis. Belmont :
Thomson Books/Cole, 2007. ISBN 13: 978-0-495-12570-9.
[6] Practical method for evaluation of linearity and effective pathlength of on-capillary photometric
detectors in capillary electrophoresis. Johns, C., Macka, M., Haddad, P. R., King, M., Paull,
B. 2001, Journal of Chromatography A, Vol. 927, pp. 237-241.
[7] Nanoliter-Scale Multireflection Cell for Absorption Detection in Capillary Electrophoresis.
Wang T., Aiken, J. H., Huie, C. W., Hartwick, R. A. 1991, Analytical Chemistry, Vol. 63,
pp. 1372-1376.
[8] Optical Improvements of a Z-Shaped Cell for High-Sensitivity UV Absorbance Detection in
Capillary Electrophoresis. Moring, S., E., Reel, R. T. 1993, Analytical Chemistry, Vol. 65,
pp. 3454-3459.
[9] On-column capillary flow cell utilizing optical wave-guide for chromatographic applications.
Bruno, A. E., Gassmann, E., Pericles, N., Anton, K. 1989, Analytical Chemistry, Vol. 61,
pp. 876-883.
[10] The pigtailing approach to optical detection in capillary electrophoresis. Bruno, A. E., Maystre,
F., Krattiger, B., Nussbaum, P., Gassmann, E. 1994, Trends in Analytical Chemistry, Vol.
13, pp. 190-198.
[11] Numerical model for light propagation and light intensity distribution inside coated fused silica
capillaries. Piasecki, T., Macka, M., Paull, B., Brabazon, D. 2011, Optics and Lasers in
Enginnering, Vol. 49, pp. 924-931.
[12] Fiber-optic-based UVvisible absorbance detector for capillary electrophoresis, utilizing focusing
optical elements. Lindberg, P., Hanning, A., Lindberg, T., Roeraade, J. 1998, Journal of
Chromatography A, Vol. 809, pp. 181-189.
[13] Capillary Electrophoresis Detector Using a Light Emitting Diode and Optical Fibres. Butler, P.
A. G., Mills, B., Hauser, P. 1997, Analyst, Vol. 122, pp. 949-953.
[14] UV-LED photopolymerised monoliths. Abele, S., Nie, F. Q., Foret, F., Paull, B., Macka, M.
2008, Analyst, Vol. 133, pp. 864-866.
[15] Brandrup, J., Immergut, E. H. Polymer Handbook. Ney York : John Wiley & Sons, 1975.
Chapter 4
Protein Electrophoresis in Saliva Study
Elsa Lamy, Ana R. Costa, Clia M. Antunes,
http://dx.doi.org/10.5772/48586
1. Introduction
Saliva started for been less studied than other body fluids, but in the last years it has being
receiving an increased attention. Until now, more than 2000 different proteins and peptides
have been identified in whole saliva and salivary glandular secretions [1]. From these, more
than 90% derive from the secretion of the three pairs of major salivary glands (parotid,
submandibular and sublingual glands). The remaining 10% derives from minor salivary
glands and from extra-glandular sources, namely gingival crevicular fluid, mucosal
transudations, bacteria and bacterial products, viruses and fungi, desquamated epithelial
cells, and food debris [2].
Saliva secretion is mainly under autonomic nervous system regulation. Sympathetic and
parasympathetic stimulation have different effects on the flow rate and composition of
saliva secreted. Whereas parasympathetic stimulation results in the production of a high
volume of saliva with low protein concentration, stimulation of the sympathetic branch of
the autonomic nervous system is responsible for the secretion of a small amount of saliva
with increased protein concentration. Besides this distinctive characteristic, and inversely to
what is observed for the majority of body systems, the effects of parasympathetic and
sympathetic innervations are not antagonic but rather exert relatively independent effects in
which the activity of one branch may synergistically augment the effect of the other [3,4].
Despite the thought of an exclusive nervous regulation, recent in vivo animal experiments
indicate a short-term endocrine regulation of salivary glandular activities as well [5-9].
The primordial function of saliva is to aid in the moistening and preprocessing of food,
aiding in deglutition. Besides this, other important functions exist for saliva, which can
generally be grouped in digestive (and ingestive) and protection [10]. For digestive (and
ingestive) purposes, saliva contains enzymes, including proteases, lipases and
glycohydrolases, which initiate partial break-down of food components. Among these

Electrophoresis 64
enzymes, alpha-amylase is by far the enzyme present in higher amounts. There are also
salivary proteins involved in food perception, such as: salivary PRPs (proline-rich proteins),
which bind dietary polyphenols (mainly tannins) and are involved in astringency
perception [11]; carbonic anhydrase VI, suggested to influence bitter taste sensitivity [12];
and alpha-amylase, involved in sweet taste sensitivity [13]. Additionally, recent studies
suggest changes in saliva composition induced by taste [14-16], reinforcing the potential of
saliva in food perception and ingestive choices. Concerning protection role, several different
salivary proteins have been identified, namely mucins, acidic PRPs, statherins, among many
others. For example, salivary proteins adsorbed to the enamel surface form the enamel
pellicle, which helps to protect teeth [17]. It is also relevant to point the presence of proteins
with more than one function, and the sharing of the same function by different families of
proteins. This functional redundancy may help to ensure that a given function is always
present under a broader range of physiological conditions [18, 19]. An in-depth analysis of
saliva proteome, including the posttranslational modifications can therefore provide a
valuable resource for saliva function research.
The high potential of saliva as a source of biomarkers was one of the main responsible for
the great interest in this fluid. Several analytes are present in saliva in amounts that relates
to blood, with the great advantage of being collected using simple and non-invasive
methods. Proteomic techniques such as two-dimensional electrophoresis (2-DE), 2D-liquid
chromatography/mass spectrometry (2D-LC/MS), matrix-assisted laser desorption
ionization-time of flight mass spectrometry (MALDI-TOF/MS), and surface enhanced laser
desorption/ionization time-of-flight mass spectrometry (SELDI-TOF/MS), have been used in
saliva studies. Based on those techniques, potential salivary biomarkers for diseases such as
Sjgren syndrome [20], diabetes mellitus [21] and some different cancers [22] have been
suggested. 2-DE has been one technique of choice for the global analysis and initial profiling
of salivary proteins, being used as a first step for protein separation, followed by MS or
tandem MS (MS/MS) [23].
Whereas the salivary proteins from humans have deserved substantial attention, both in
terms of identification and characterization, as well as functional properties, animal saliva
has been much less studied. However, interest for the latter is being increasing, due to the
convenience on the use of animal models for diverse pathological and physiological
conditions and due to the potential of this fluid for disease diagnostic and for understanding
behavioral and physiological processes, important in animal production. Moreover, a
multidisciplinary approach that integrates knowledge about salivary proteins in the animal
kingdom (most important in mammals) and draws comparisons to possible functions in
humans would be valuable.
The following sections will give an overview about the use of electrophoresis in saliva
studies. Methodological issues and the major advantages and limitations for the use of this
technique in human and animal saliva studies will be presented. We will finish the chapter
by presenting alternatives to electrophoresis for the study of salivary proteome.

Protein Electrophoresis in Saliva Study 65
2. Applications of electrophoresis for saliva proteome characterization
Even before the advent of proteomics, electrophoresis was frequently used for salivary
protein separation. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
[24], PAGE in non-denaturing conditions [25], isoelectric focusing [26], two-dimensional
electrophoresis (2-DE) [27], capillary electrophoresis (CE) [28,29] and free flow
electrophoresis [30] have all been used in saliva studies, with different purposes.
One-dimensional gel electrophoresis under denaturing conditions has several advantages:
virtually all proteins are soluble in SDS, allowing their separation; it covers a relatively high
range of molecular masses (from 10000 to 300000 Da) and allows the possibility of extremely
acidic and basic proteins to be visualized [31]. Moreover, SDS-PAGE has the advantage of
having a low sensitivity to salt concentration. However, by separating proteins only based
in their molecular masses, only limited information is obtained, since each of the bands
present in the gels is frequently constituted by different proteins. Limitation in the number
of proteins separated also occur using IEF, which separates proteins only based in their
charges.
2-DE takes advantages of the two different properties of proteins (molecular masses and
isoelectric points), allowing the separation and visualization, in a gel matrix, of a
considerable number of different proteins. This technique, which originates from the work
of OFarrell and Klose in the 1970s [32,33] became very useful for the study of complex
protein mixtures, such as saliva. Besides the very high separation capability of 2-DE,
ensuring well-resolved protein maps with more than 2000 protein spots, this technique has
also the advantage of mapping posttranslational modifications. Coupled to mass
spectrometry, for protein identification, 2-DE has been considerably used in several different
samples, including saliva [27,34]. Using this approach, the protein spots observed in 2-DE
gels are subsequently digested using a protease (usually trypsin), with the resultant digest
products analyzed by mass spectrometry. 2-DE can be used to compare expression levels of
proteins in related samples, such as those from altered experimental conditions, allowing
the response of classes of proteins to be determined. This approach has been suceffuly used
in a number of saliva studies [e.g. 35].
Salivary peptides and proteins have been analyzed by a variety of CE approaches (reviewed
in [36]. The term CE, although often used as shorthand for capillary zone electrophoresis,
refers to a family of related techniques, all based on the performance of the separations in
narrow-bore capillaries across which an electric field is applied. The types of CE that have
the greatest potential in proteomics (commonly used together with mass spectrometry) are
capillary zone electrophoresis and capillary isoelectric focusing [37]. The use of CE to
analyze saliva proteome, have been described and allowed the profiling and identification
of several salivary proteins [38]. Recently this technique was successfully used for the
identification of salivary profiles in cancer diagnosis [29].
The existence of different electrophoretic methodologies, allowing separations based on
different protein characteristics is of great utility in proteomics, contributing for the

Electrophoresis 66
resolution of complex samples such as saliva. The electrophoretic methods described are
complementary of each other and since all have advantages and limitations, the choice for
each one will depend on the objectives of the study and on the salivary proteins of interest.
3. Methodological issues related to saliva proteome analysis
The concept of salivary proteome is related to the creation of a salivary protein catalogue,
where information that can be further used for several different purposes (e.g diagnostics,
physiological status) can be placed [39]. In this context, it is important that the results
obtained from different laboratories can be compared. Accurate examination of salivary
components requires optimal collection, processing and storage conditions. Moreover, most
of the studies aim comparisons among pathological/physiological conditions, and as such it
is essential that the differences obtained are not due to external factors. To avoid changes in
the protein and peptide composition from salivary secretions, standardized salivary
sampling protocols, processing and storage conditions need to be applied [23,39,40].
3.1. Sample collection, processing and storage
One of the particularities of saliva is its capacity of rapidly adapt to different conditions.
This is related to the fact of salivary secretion being mainly regulated by the two branches of
the autonomic nervous system (both sympathetic and parasympathetic), with only a minor
regulation from hormonal origin. Such type of regulation results in variations in
composition according to the stimulus. Circadian rhythm [41], gender [42], drugs [43],
exercise [44], among others, are factors that change salivary flow rate and saliva
composition. Additionally to variations in the composition from each salivary gland cell
type, the contribution of each individual salivary gland to the total fluid is not the same. For
example, minor salivary glands and submandibular glands have an important contribution
at rest, whereas in response to strong stimuli during feeding is the parotid contribution that
becomes dominant [45]. Concerning daytime, the flow rate of resting and stimulated saliva
is higher in the afternoon than in the morning, with the peak occurring in the middle of the
afternoon. The salivary protein concentration also follows this diurnal pattern [2]. Eating is
another strong stimulus for the secretion of saliva, and as such the interval between feeding
and saliva collection influences salivary flow rate, viscosity and protein composition.
The points referred above are important for the selection of the collection method: with or
without stimulation. For unstimulated saliva collection the draining method is usually
choose. Accordingly, saliva is allowed to drip off the lower lip to a tube maintained on ice.
On the other hand, stimulated saliva is frequently obtained after parafilm mastication, or
after sour taste stimulation [46].
Advantages and disadvantages exist for both approaches. Unstimulated saliva collection is
several times preferred, as stimulated saliva contains a diluted concentration of several
proteins, which may be of interest. However, it is difficult to have saliva completely free of
stimulation: due to the high range of stimulus influencing salivary secretion, even small

variations in collection conditions, among different saliva donors (e,g light intensity,
temperature, emotional status, or others) may be sufficient to induce differences in results [39].
Stimulated saliva collection is generally used to have higher volumes of sample. In some
pathological conditions (e.g Sjgrens syndrome, xerostomia) or post-radiation, stimulation
of salivary secretion may be the only way of obtaining adequate amounts of saliva samples
for analysis. However, difficulties may exist to make uniform the intensity and duration of
the stimulus and the secretion of certain proteins may be affected by the duration of
stimulation. For example, with prolonged stimulation of salivary flow, certain glycoproteins
may be incompletely glycosylated [39].
Another issue in regard with saliva sampling is the origin of the collected secretions, i.e.
whether it is glandular or whole saliva. Glandular fluid can be obtained through the use of
adapted collection devices, for both parotid and submandibular/sublingual secretions [47].
Through this, changes in salivary proteins by exogenous enzymes are avoided, since the
fluid is collected before it reaches the mouth.
Most of the studies on saliva in general have been obtained from human saliva. Concerning
animal saliva less is known and, although methodologies of collection, processing and
storage are based on the ones reported for humans, in most cases it is difficult to obtain
unstimulated saliva from animals. Only in domestic ruminants, which produce considerably
high volumes of saliva daily, in a continuous flow, it is possible to obtain whole saliva
samples by simply collecting the mouth flow into a large beaker [48]. Moreover, this method
only works with domesticated animals that are used to be handled by man.
For all the animals that do not produce large amounts of saliva, or for specific investigation
purposes, stimulation may be necessary. Under controlled experimental conditions, the
collection of stimulated saliva can be initiated either mechanically or chemically. Cotton
rolls (e.g. Sallivetes) are currently used for animal saliva collection for being both practical
and efficient in getting this fluid. Animals chew for some time, being saliva production
stimulated by mastication. The cotton roll moistened by the saliva is further centrifuged to
release the fluid. There is the possibility of some salivary proteins such as mucins and/or other
potential biomarkers irreversibly adsorb to the cotton roll, resulting in their loss. However, to
our knowledge, studies to elucidate this aspect are lacking. Chemical (pharmacological)
stimulation is sometimes used for saliva collection in animals with low amounts of saliva. For
example, in small rodents, it is very difficult to collect sufficient amounts for analysis without
stimulation and the use of parasympathetic agonists (e.g. pilocarpine) appears to be effective
[49]. This type of stimulant is referred to increase the volume production without changing the
proportion of the proteins secreted. Others types of chemical stimulants may be used. For
example, sympathetic agonists, such as isoproterenol, induce the synthesis and secretion of
granular proteins from salivary glands, thereby increasing salivary protein concentration and
changing the salivary protein profile [50].
While whole saliva can be collected using the already mentioned cotton-rolls, or through
direct aspiration from the mouth [49], glandular saliva collection can be achieved using

Electrophoresis 68
catheters inserted into the ducts of the gland of interest. Although this method is invasive,
the level of invasion is minimal, and once the collection period is finished, the catheter can
easily be removed without causing any damage to the animal. This method was used to
study sheep and goat parotid salivary proteomes [34,51]. Collection of glandular saliva has
the advantage of accessing the secretion product of certain glands, as well as obtaining a
clear saliva sample with virtually no interfering compounds, such as food debris or
microorganisms. Glandular collection is most important for studies that aim on unraveling
the effects of particular factors on the secretion from individual glands [e.g. effects of dietary
constituents on parotid saliva [35]. However, this approach, if not carried properly, might
have the disadvantage of faulty catheter insertion which causes leakage of plasma proteins
into the salivary secretions due to disruption of epithelial integrity.
Wherever in humans or animals, the collection method of choice, as well as the origin of
fluid selected, will affect the outcomes and thus should depend on the objectives of the
study and on the specific group of proteins of interest.
In complex protein mixtures, such as saliva, sample preparation and fractionation constitute
one of the most crucial processes for proteome study. None of the currently available
proteomic techniques allow the analysis of the entire proteome in a single step. In body
fluids such as saliva, proteins have different physicochemical characteristics and a wide
concentration dynamic range and, as such, fractionation is essential. In this context,
electrophoresis may also be useful for that purpose. Preparative isoelectric focusing using
free flow electrophoresis is an example of the methods available for sample fractionation
and which has been used in saliva [30]. Before 2-DE separation, free flow electrophoresis has
the advantage of generating different fractions, which can be independently run. For
example, a first separation using isoelectric free flow electrophoresis results in fractions with
different pI ranges. Each of the resulting fractions can be further separated according to
charge, by using narrow pH ranges, in the first dimension allowing a more detailed picture
of the protein profile [30].
Saliva contains proteins present in high levels, and numerous low abundant proteins, for
which analysis may be of interest. The observation of the latter in electrophoretic gels is
obscured by the presence of the high-abundant proteins. When salivary proteome is used
for disease diagnostic purposes, the analysis of low abundant proteins is almost always
necessary, since most of potential salivary biomarkers are present in relative low amounts.
The major components of saliva are mucins, proline-rich glycoproteins, amylase and some
antimicrobial proteins that include agglutinin, lisozyme, lactoferrin, immunoglobulins,
histatins and defensins [27]. The protein alpha-amylase contributes to almost 60% of total
salivary proteome [52] and its depletion allows the analysis of less abundant proteins.
Salivary alpha amylase depletion can be achieved through elution of samples from starch
columns to deplete this protein specifically [53].
One of the main concerns when working with protein sample is to avoid undesirable
alterations during the several steps that go from collection to final analysis. One
characteristic of saliva is that many salivary proteins enter post-translational modifications

(PTMs), namely glycosylation, phosphorylation, sulfation and proteolysis. From the
considerable amount of glycoproteins present in saliva, mucins represent an important
group, which is responsible for bacterial agglutination and lubrication of the oral cavity
tissues. Phosphoproteins also exist in a considerable amount of salivary proteins, with
several diverse roles [54]. Such modifications start in the acinar cells of salivary glands, and
continue when saliva enters the mouth, mainly due to the presence of host- and bacteria-
derived enzymes, what results in additional protein modifications [55].
Since these forms of PTMs are responsible for many functions of this fluid, a particular
attention should be directed to saliva collection processing and storage, in order to minimize
proteolysis, de-glycosylation and de-phosphorylation. Different research groups have
employed different methods for avoiding proteolysis, de-glycosylation and de-
phosphorylation. The addition of 0,2% trifluoroacetic acid to saliva after collection has been
one protocol used [56]. In a recent study, it was observed that the addition of protease
inhibitors to saliva may allow its storage at 4C for approximately two weeks, without
significant degradation [53]. Nonetheless, some authors report that not even an inhibitor
cocktail can prevent all protein degradation [57]. Additionally, it was suggested to be possible
to keep the samples at room temperature (for a period of about two weeks), without
considerable changes in salivary proteome occurred, only by adding ethanol, [53].
Nevertheless, working on ice for no longer than one hour and subsequent storage of samples
at -80C has been considered a safe and practical handling protocol [57-59]. Nonetheless, long
time storage, as well as freeze-thaw cycles can induce protein precipitation, in particular from
low molecular mass components [57]. In any case, little research has been directed on ways of
minimizing degradative processes, and this is clearly needed [39].
3.2. Staining procedures and PTMs in-gel analysis
Protein visualization is necessary for quantitative and qualitative analysis. In electrophoresis
this is achieved through reversible or irreversible binding of a colored organic or inorganic
chemical to the protein. An ideal staining procedure would be the one with a very low
detection limit, an optimal signal to noise ratio, a wide dynamic range and a wide linear
relationship between the quantity of protein and the staining intensity, and compatible to
mass spectrometry [60]. However, such an ideal stain does not exist.
Silver, Coomassie Brilliant Blue (CBB) and fluorescent staining are the most frequently used
methodologies. Silver staining presents a high sensitivity, making possible the visualization
of proteins present in amounts as low as 1 ng [61]. Silver staining techniques are based upon
saturating gels with silver ions, washing the less tightly bound metal ions out of the gel
matrix and reducing the protein-bound metal ions to form metallic silver. In 2-DE, Silver
staining is regularly used due to its potential for the visualization of low intensity protein
spots. However, it presents a narrow dynamic range and the tendency of the dye to stain
differently based on amino acid composition and PTMs. Moreover, by detecting low levels
of protein, each of the stained spots may have not sufficient amounts o protein for
subsequent analysis by mass spectrometry.

Electrophoresis 70
CBB is a disulfonated triphenylmethane textile dye. CBB staining presents a linear dynamic
range and a moderately sensitivity. As such, CBB dyes are suitable for protein quantitative
analysis, which is necessary in proteomics analysis. Moreover, this staining technique is
compatible with mass spectrometry. Two modifications of CBB exist: R-250 and G-250. In
acidic solutions the dye sticks to the amino groups of the proteins by electrostatic and
hydrophobic interactions. Inversely to silver stain, CBB is not so extremely sensitive, thus
being necessary to load higher amounts of proteins in the gel. Consequently, CBB stained
spots contains considerable amounts of proteins, suitable for mass spectrometry analysis.
Saliva samples present particularities that should be considered for silver and CBB staining.
Parotid saliva contains a considerable amount of proline-rich proteins (PRPs), which are
difficult to stain with silver since they present low amounts of amino acids containing
sulfur, necessary for the binding of silver ions [61]. On the other hand, when stained with
CBB R-250 they usually present a violet-pink stain, which allows differentiating them from
all the other salivary proteins that stain blue. A destain protocol of 10% acetic acid, instead
of the common 10% acetic acid/ 10% methanol is generally used for this purpose [62].
The fluorescent dyes were more recently developed, presenting a high sensitivity and a
linear dynamic range, and in this way, being advantageous relatively to common silver
staining. For example, SYPRO staining technique has been used in several salivary
proteomic studies [e.g. 63]. This is a novel, ruthenium-based fluorescent dye. SYPRO Red
and Orange, bind to the detergent coat surrounding proteins in SDS denaturing gels, thus,
staining in such gels is not strongly selective for particular polypeptides [64]. SYPRO stains
are compatible with mass spectrometry and can be used in combination with other staining
techniques, for detection of PTMs, such as glycosylations or phosphorylations.
DIGE (Difference Gel Electrophoresis) technology represented an improvement in 2-DE
based proteomics, allowing more accurate comparisons among samples. Its convenience is
also true for saliva samples [65]. DIGE is based on the modification of proteins, before
electrophoresis, by attaching a fluorescent labeling. Cyanine-bases dyes (Cy2, Cy3 and Cy5)
are used with this purpose. These dyes label the amine group of protein lysines specifically
and covalently to form an amide. A different cyanine-based dye is added to each individual
sample. The dyes are designed to have the same molecular mass and charge to ensure that
proteins common to both samples have the same relative 2-DE mobility. The samples are
mixed and resolved in a single 2-DE gel. The proteins from the different dyes are visualized
by alternatively illuminating the gel at different wavelengths. With this technique, it is
possible to avoid some inter-gel variation.
PTMs, such as glycosylation and phosphorylation, can be accessed in electrophoretic gels
through staining procedures. As it was referred before, several different salivary proteins
suffer PTMs, which confer their characteristic functions. Consequently, their visualization in
electrophoretic gels is valuable. Detection of phosphoproteins can be achieved using
different procedures. Recently, Pro-Q Diamond was developed for phosphoproteins
staining. This affords wide specificity and high sensitivity. Phosphoserine,
phosphothreonine and phosphotyrosine containing proteins are detected [66].

O- and N- glycosylated proteins are abundant in saliva. Some of the most well studied
salivary glycoproteins are mucins (MUC5B and Muc7) and proline-rich glycoproteins. The
most classical procedure for glycoproteins staining is periodic-acid Schiff (PAS) with the
protocol that allows detection of glycoproteins in gels being adapted from protocols of
histochemistry. However, it does not present a high sensitivity, resulting in the need of high
levels of protein load [67]. Another possible method is the more recently developed stain
Pro-Q Emerald that allows glycoprotein detection, presenting approximately 50-fold more
sensitivity than PAS [68] very useful when amount of sample is limited.
3.3. Protein identification
Proteome studies may be performed with two main different but somewhat complementary
purposes: to characterize a particular sample (e.g organism, cell, fluid), or to compare
samples from different experimental conditions. Independently of which of these two is the
focus, protein identification is a central aspect. In proteomic studies, electrophoresis is
frequently coupled to mass spectrometry technologies. Proteins selected in the gels are
excised and subjected to enzymatic in-gel digestion. In this process it is important that each
of the excised spots (or bands) present the amount of protein sufficient for MS analysis. So,
the choice of the staining methodology should take this into consideration, as well as the
need for the use of dyes compatible with MS (referred in previous section).
After proteins being identified, MS identifications need further to be validated, by Western
blotting, through the use of antibodies to the proteins of interest. This is only possible for
proteins for which commercial antibodies are available. There are some particularities of
saliva samples that may be considered for Western blotting. Using this technique, only the
protein spots (or bands) that react with the antibody are visualized. When comparison
among different samples is to be made, it is important to have the same load of protein in all
lanes or 2-DE gels. Different loads may result in erroneous results. The existence of internal
controls, i.e., proteins for which the levels are proportional to the amount of total protein
loaded in the gels, is important and is commonly used to circumvent putative differences in
protein loads [e.g 69]. The simultaneous use of a primary antibody for such internal control
and the use of a primary antibody for the protein of interest may allow adjustments: by
comparing the intensity of these internal controls, the relative amount of protein run for
each sample can thus be estimated. For saliva studies such strategy is not possible, since it is
not yet known one salivary protein which relative amount to total protein content remains
constant. One way to circumvent this limitation is through the staining of the membrane,
allowing the relative evaluation of protein content, before incubation with the primary
antibody [70]. With this procedure, it is possible to visualize the several lanes and
consequently the expression of the protein of interest may be compared as a percentage of
the total band intensity. The reversible Ponceau red staining [71] is the standard procedure,
despite its low sensitivity (detection limit in the range of 1 g/spot).
Although in human saliva, protein identification can be generally performed with success,
for animal saliva this is not always the case. One of the major limitations in animal

Electrophoresis 72
proteomic studies is the lack of complete and annotated genome and protein sequences for a
great number of species, making salivary protein identification challenging. As consequence
there is the need for search in other related species databases, at the cost of eventually
producing high number of false positive results [72]. Moreover, the inexistence of
commercial antibodies for most of animal proteins makes validation of identifications more
difficult. Nevertheless, recent advances in sequencing the genomes of various domestic
animals (cattle, pigs and sheep) are increasing the ability to identify salivary proteins in
these animal species.
4. Advantages and limitations of electrophoresis for saliva proteome
analysis
Electrophoresis has been widely used in saliva characterization. Despite the advances in the
knowledge of salivary protein composition that SDS-PAGE and IEF allowed, the
development of 2-DE and its application to the study of saliva contributed for a great
advance in the comprehension of this body fluid. 2-DE has played a major role in the birth
and developments of proteomics, although it is no longer the exclusive separation tool used
in this field. Nevertheless, 2-DE continues to be essential in proteomics studies. Apart from
the great advantage arising from its efficiency in resolving a high number of proteins in
complex samples such as saliva and allowing the visualization of PTMs, 2-DE method has
also the advantage of being a method relatively inexpensive (at least comparatively to most
of the other techniques used in proteomics).
Although the great advantages of electrophoresis for the study of salivary protein
composition, saliva presents several particularities that need to be consider, and that will
limit the use of this technique. One of these particularities is the considerable ionic content
that saliva presents, which is important to account for when IEF or 2-DE is to be used in
protein separation; since separation according to electrical charge will occur, the existence of
charged compounds, or salts, in the sample may cause interference. Lowering of ions and
salt content may be achieved using ultracentrifugation membranes [e.g. 51], or through
protein acid precipitation (e.g. 10% TCA/90% acetone), in which the precipitate is
subsequently re-suspended in a buffer. Both of these procedures will also increase the
protein concentration, which may constitute an advantage since saliva samples may
sometimes be too diluted (for example, after parasympathetic stimulation). In any case some
protein loss will inevitably occur.
Another characteristic of saliva that limits its study by electrophoresis is the considerable
amount of mucins present. These are glycoproteins with high molecular masses, which
present particular physychochemical characteristics that difficult analysis using
electrophoretic separation. Moreover, the presence of mucins also limits the study of other
salivary proteins, since mucins form complexes with several proteins, interfering in the
analysis of the latter. Human salivary glands secrete two types mucins: oligomeric mucin
(MG1) with molecular mass above 1 MDa and monomeric mucin (MG2) with molecular
mass of 200250 kDa, which together represent about 26% of total proteins from saliva [73].

The high molecular mass of these molecules impede them to migrate through the
polyacrylamide matrix, resulting in their deposition in the top of the gel [74]. The exact mucin
content is dependent on the proportion of contribution from the different major salivary
glands for the total saliva. As such, the type of stimulation will influence the amount of
salivary mucins present. Stimuli that increase sublingual and submandibular saliva result in
higher amount of these proteins than when parotid glands are the major contributors.
The presence of high amounts of mucins confers a high viscosity to saliva, what has practical
drawbacks in sample preparation for analysis, such as difficulties in sample pipetting. Usage
of denaturing conditions, such as buffers containing 4-6M guanidine hydrochloride (GdmCl),
or the reducing agent dithiothreitol (DTT) can diminish the viscosity of mucous salivary
secretions [75]. However, this effect is achieved at expense of effects in the structure of
proteins, and should be avoided in studies where the maintenance of such structure is
necessary (for example when studying salivary complexes). The centrifugation of saliva
samples, which is one of the first approach usually performed in saliva preparation, also aids
in the removing of mucins. However, mucins and other glycoproteins are frequently involved
in protein complexes with other salivary proteins, namely amylase, statherin and PRPs,
resulting in particular protein losses, when the pellet is discharged.
Another limitation for the study of saliva using electrophoresis, which is common to the
majority of body fluids, is the high diversity in the levels of the different protein species. As
it was mentioned in section 3.1., a few salivary proteins are present in high amounts,
whereas many others appear in low levels. Depletion methods are needed, in order to
visualize the low abundant proteins, which may be of particular importance. If considering
the important application of saliva as source of disease biomarkers, and knowing that many
of those potential biomarkers are not secreted by the salivary glands, it is essential to have
access to the low abundant proteins, such as metabolic enzymes with antimicrobial activity
(e.g. lysozyme and lactoperoxidase).
Finally, saliva contains several low-molecular mass components that have important
functions, namely important bactericidal activity (e-g- histatins, defensins) [76]. The fact that
electrophoresis does not allow the separation of these compounds constitutes another
limitation in the application of electrophoresis to saliva characterization.
5. Alternatives to electrophoresis for saliva proteome analysis
Through this chapter the use of electrophoresis in saliva proteome analysis has been
emphasized. 2-DE and protein MS represent an integrated technology by which several
thousand proteins can be separated, quantified and identified. And, as has been being
referred, this approach has been considerably used in saliva proteome studies. However,
although the advantages of 2-DE, it does not allow the study of the complete proteome.
Moreover, as it was stated before, 2-DE have drawbacks which include poor gel-to-gel
reproducibility, and the requirement of relatively large amounts of sample, as well as
extensive labor and a considerable time required. As such, other proteomic techniques are
valuable for the study of saliva proteome.

Electrophoresis 74
With the introduction of high-throughput LC coupled to tandem MS (MS/MS), the study of
complex systems moved towards a bottom-up proteomics analysis, where complex protein
samples are digested and the generated peptides, which are separated by high pressure
liquid chromatography (HPLC), are introduced into a mass spectrometer for fragmentation
and sequencing to identify and quantify the parent proteins. However, LC-MS analysis of
highly complex proteomic samples remains a challenging endeavor [77]. With this
approach, tryptic cleavage generates multiple peptides per protein so that proteomic
samples typically consist of hundreds of thousands of peptides. To date, no separation
method is capable of resolving so many components in a single analytical dimension prior
to the MS analysis. Thus, many research efforts have focused on the development of a more
sensitive multidimensional liquid chromatography (MudPIT) with higher peptide
separation power [21,22]. Initially, large scale shotgun proteomics was defined as an ion
exchange chromatography (specifically strong cation exchange-SCX) coupled to reverse
phase (RP) and mass spectrometry [78]. Nowadays, alternative configurations to SCX with
RP have also been investigated and include the use of anion exchange chromatography and
RP, affinity chromatography (AC) and RP, isoelectric focusing (IEF) and RP, capillary
electrophoresis (CE) [79,80]. Recently, a RPRP system was proposed for proteomics where
the first RP column uses a pH of 10 and the second RP column uses a pH of 2.6 [81,82]. This
later approach yielded higher proteome coverage when compared with the SCX-RP
approach [81,83].
These methodologies have been applied to saliva proteome characterization aiming the
extension of salivary proteins catalogue and further comparison within other biological
fluids. For instance, the use of a classical MudPit approach through combination of SCX-RP,
allowed the identification of more than 100 proteins [84]. In other experiments a shotgun
approach using only LC-MS/MS resulted in the identification of more than 300 proteins [85].
Different chromatographic combinations have been succeeded [30,80,86] which in
conjugation to instrumentation advances conducted to the identification of more than 3000
different components in saliva. Other approaches were also performed aiming the reduction
of saliva sample complexity through the utilization of combinatorial chemistry derived
hexapeptide libraries (Proteominer from BioRad) which lead to the identification of more
2300 different proteins [1]. This has been later used for PTM characterization mainly N-
linked salivary glycoproteins and their glycosylation sites [87]. Other methodologies aimed
at characterizing salivary glycoproteins consist in enrichment procedures based on affinity
chromatography with lectins [88] acting as a first dimension. In fact, saliva is a rich source of
both N- and O-linked glycoproteins, which play an important role in the maintenance of
oral health and protection of teeth [68]. In line with the characterization of the most
abundant PTMs in salivary proteins, namely phosphorylation, other systems were also
developed. For instance, Salih et al [89] developed an enrichment procedure based on
chemical derivatization using dithiothreitol (DTT) leading to the identification of 65
phosphoproteins. In a different approach, combining hexapeptide libraries, immobilized
metal ion affinity chromatography, SCX and RP, 217 unique phosphopeptides sites were
positively identified representing 85 distinct phosphoproteins [54].

As stated above, and similarly to other bodily fluids, saliva contains several protein species
of low molecular weight which comprise around 40-50% of the total secreted protein content
[90]. Albeit particular functions can be attributed to the major peptide classes, several
questions about their precise role in oral cavity remain unclear. A strategy based on acidic
precipitation or passing the saliva supernatant through a defined cut-off filters and LC-
MS/MS have been widely adopted to perform the characterization of this low molecular
weight fraction [91-107]. Behind the identification of several fragments deriving from those
major peptide classes, several PTMs were also assigned. For instance, novel N- and O-
glycosylation sites were identified in PRPs [108] as well as S-Glutathionyl, S-cysteinyl and S-
S 2-mer recently identified in cystatin B [109].
More than identification of proteins, many studies aim the evaluation of protein expression
under different purposes and pathophysiological conditions. In fact, quantitative proteome
profiling is key for comparative analysis of proteins from normal and diseased patients, as
similar proteins may be present in both states but at significantly different concentrations.
Without quantitative information, the value of these differentially abundant proteins as
biomarkers may be overlooked. In the pursuit of these goals, gel-free approaches using
stable-isotope tagging or label free (based on spectral counting) have been used for
comparative analysis involving salivary samples. In these approaches, the typical flowchart
starts by protein digestion being, in case of isotope labeling, derivatized with respective
isotope, mixed and analysed simultaneously. Depending on the approach, up to 8 different
samples (iTRAQ-8plex, Absciex) can be compared at same time. As an example, Streckfus et
al. [110] evaluated the salivary protein expression in patients with breast cancer using a
iTRAQ approach identifying 55 proteins that were common to both cancer stages in
comparison to each other and healthy controls while there were 20 proteins unique to Stage
IIa and 28 proteins that were unique to Stage IIb. In case of label free, eluted peptides are
aligned in terms of retention time and comparative analysis will be based on spectral
counting [111]. For instance, Ambatipudi et al. [112] by MudPit and label free evaluated
the aging effect in the abundance of human female parotid salivary proteins where
extensive age associated changes in the abundance of many of salivary proteins were noted,
especially for proteins associated with host defense mechanisms.
6. Concluding remarks
The proteome of human saliva received considerable attention in the last years. However,
the improvement of the methods to study and characterize salivary proteins and/or the
changes in its profile is still an issue since the identification of potential biomarkers for
several pathological and physiological conditions is yet to be established. Moreover, a
complete knowledge on the importance of each salivary protein in oral environment is not
completely understood. Additionally, the growing interest in animal saliva, both due to
their value as models for humans, as well as for veterinarian and production purposes,
justifies the efforts to develop new protocols adapted to the particularities of these samples.

Electrophoresis 76
Despite the existence of limitations, electrophoresis continues to be an essential tool in the
study of salivary proteome. It constitutes the bases for the separation of several different
components, allowing a summary characterization and also providing a purification step
prior the application of more selective and commonly more expensive methods.
Nevertheless, enhanced methodologies for sample fractionation and processing might be
useful to circumvent some of the limitations for the study of this fluid by electrophoresis.
Profiling such a fluid that rapidly changes according stimulus and where some of its
constituents interact with each other is challenging. Improved approaches will be necessary
to cope with the challenges in understanding of these interactions, their functions and health
consequences the so called interactome which will be the future in saliva characterization
and biomarker identification.
Author details
Elsa Lamy
*

ICAAM Institute of Mediterranean Agricultural and Environmental Sciences,
University of vora, vora, Portugal
QOPNA, Mass Spectrometry Center, Department of Chemistry, University of Aveiro,
Aveiro, Portugal
Ana R. Costa
Department of Chemistry, University of vora, vora, Portugal
Clia M. Antunes
Department of Chemistry, University of vora, vora, Portugal
Center for Neuroscience and Cell Biology, University of Coimbra, Portugal
QOPNA, Mass Spectrometry Center, Department of Chemistry,
University of Aveiro, Aveiro, Portugal
Acknowledgement
Authors acknowledge the financial support from FCT - Fundao para a Cincia e a
Tecnologia Science and Technology Foundation (Lisbon, Portugal) of the Ministry of Science,
Technology and Higher Education (Post-doctoral grant SFRH/BPD/63240/2009 Elsa Lamy) and
by FEDER Funds through the Operational Programme for Competitiveness Factors -
COMPETE and National Funds through FCT - Foundation for Science and Technology under
the Strategic Projects PEst16C/AGR/UI0115/2011 and PEst-C/QUI/UI0062/2011.

* Corresponding Author

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Electrophoresis 82
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Chapter 5
Electrophoresis of Myocardial Cells
Ying Zhou
http://dx.doi.org/10.5772/53508
1. Introduction
Mammals are composed of a large number of surface charged cells. The cell structure and
functions in different tissues and organs are different. Through the method of cell
electrophoresis the information of the cell surface structure can be obtained and is valuable
for the function study of cells, tissues and organs. Many studies have been reported on the
fluidic electric phenomena of cells (Ertan & Rampling, 2003; Aki et al., 2010; Brown et al.,
1985; Pimenta & de Souza, 1982), but quite limited on the separation of myocardial cell
electric phenomena possibly due to the short of myocardial cell electrophoresis
technology to conduct the experiments. The methods only observing through separation
and investigation of myocardial cell surface complex sugars and plasma membrane
phospholipid composition on the cell contraction within the ion flow (inward ionic
current) may not actually take the cell membrane and membrane structure as a whole but
insularly highlight the single component in achieving cardiac function. Integration is not
equal to the simple sum of the single components. The heart is the vital organs of humans
and animals, its interfacial electric phenomena (such as electrocardiogram) and its
response to the pacemaker have revealed the hints on the close relationship between the
myocardial cell membrane structure and function of the heart (Podrid et al., 1995). For this
reason, electric phenomenon of the myocardial cells was systematically studied. Although
electrophoresis does not directly measure the cell surface charge density and zeta
potential, it can help to find a trace to elucidate the mentioned relationship by exploring
the classic knowledge of colloidal particles and to set up a electric double layer model for
insight into the structural characteristics of the myocardial cell surface and their
variations. It opens a way to realize the mechanism in respect of the cardiac function. We
found that the myocardial cell is rich and complex internal and external membrane
structure, and the distribution and variation of the adsorbed ions on cell surfaces are
completely different from the colloidal particles, these should be the basis for completion
of the cardiac function.

Electrophoresis 86
2. Experimental
2.1. Cell preparation
Ventricular myocardial cells were isolated from adult Sprague-Dawley rats (23 months old,
weight 225300 g) using standardized enzymatic techniques (Zhou et al., 2000). Freshly
isolated single cells were stored in Tyrodes solution containing (in mM) 137 NaCl, 5.4 KCl,
1.2 MgCl2, 1 NaH2PO4, 1 CaCl2, 20 glucose, and 20 HEPES (pH 7.4). (Fig. 1)

Figure 1. Photo of isolated myocardial cells under low power lens.
2.2. Preparation of cell suspensions
Cell suspensions were prepared in isotonic solutions composed of an aqueous glucose
solution and 60%, 30%, 15%, 10%, 7.5%, 5% and 2.5% (v/v) of a stock solution of 145 NaCl
and 2.97 CaCl2 (referred to in Table 1). To adjust the transmembrane potential, which is
determined primarily by the K
+
transmembrane equilibrium potential (Fozzard et al., 1991),
K
+
was added at a required concentration depending on the potential needed which can be
calculated using the Nernst equation: ln
Kin
K
Kout
c RT
E
F c

, where EK is the K
+

transmembrane equilibrium potential, R is the universal gas constant, T the absolute
temperature, F the Faraday, and cKin and cKout are the concentration of K
+
inside and outside
the cell, respectively (Liu, 2005). Aqueous Dextran 40 (free of ion, transmembrane potential
not known) was from Shijiazhuang No.4 Pharmaceutical (China). A proper amount of

Electrophoresis of Myocardial Cells 87
hydroxypropyl methyl cellulose (HPMC) was added into all suspensions to adjust the
viscosity of the final suspensions at 3.8 mPas (24C, Table.1).
2.3. Removal of sialic acid from myocardial cells
Myocardial cells were suspended, at 0.5%(v/v), in the Tyrodes solution containing
0.25U/mL neuraminidase (Sigma Chemical, St. Louis, Mo) at pH 6.0, continually shaken at
37C for 90 minutes, and then washed with the Tyrodes solution (Post, 1992) .
2.4. Procedure of electrophoresis
The prepared myocardial cells were washed three times with the suspending medium and
then suspended in the same medium at 0.1% (v/v) measured at 24C. The used media were
collected in Table 1 where the No. 10-14 solutions were used for the suspension of enzyme-
treated myocardial cells while the No. 1-9 for non-treated cells. The suspended cells were
loaded on a cell electrophoresis system (Beijing Warder Biomedicine Instrument Company,
China) and electrophoresed, within 2-3 minutes, at 2-7V/cm depending on the ionic strength
of the suspension. Only the perfect cells were recorded. Each sample solution was
determined in parallel for 12 times. All the measurements were finished within eight hours
after the myocardial cells were isolated.

Table 1. The suspensions used in this study.

Electrophoresis 88
2.5. Note
2.5.1. Cell separation should be to reduce the loss of the charged matter on cell surface
a. Single collagenase (collagenase II, 1mg/ml) can be used as the first choice for myocardial
cell separation enzyme; b. enzymatic digestion time of myocardial tissue control in about 15
minutes (not more than 20 minutes), c. the same experimental animals age and enzyme
digestion time should be consistent.
2.5.2.
Select the electrophoresis tank stationary layer as the level of observation and measurement
of the electrophoretic velocity, to ensure the accuracy of the measured values.
2.5.3.
When the high ionic strength suspensions were studied, the electrophoresis voltage is better
selected an proper high value in compromising with the electrophoresis time (making the
cells migrate at <10m s-1).
2.5.4.
The experiment should be completed within 6-8 hours after the cell separation, to ensure the
normal activity of the cells.
3. Data processing and interpretation
Myocardial cells showed different features under an electric field depending on the nature
of the suspensions used. Although the cells may contract at high ionic strength or may not at
low ionic strength, the impact of the contraction on the electrophoretic speed did not affect
the analysis and judgment of experimental results measured from different conditions.
Commonly, myocardial cells show negative charges in the electric field. But their
electrophoretic speed may have obvious difference even in same a suspension, suggesting
that the composition of the charged layer on myocardial cell membrane is variable. In fact,
cells were possibly affected by enzyme action (time-dependent), mechanical damage and so
on in the preparation. However, this also did not impact on the judgment of variation
tendency of the cell mobility.
In the case that the type and proportion of adions on cell surface were maintained, the cell
electrophoretic mobility (EPM) showed a zigzag increase (Fig. 2A). At an ionic concentration
from 9.7 to 33.2mM, no electromigration of some cells were observed at some conditions,
mostly at pH 5.0 and 6.0, rare at pH 7.35 (Table 2). However, reversed electromigration has
not yet been observed. The variation of the mobility was largely dependent of the
suspension pH. The mobility was found to be in common greater at low pH (2.5-4.5) than at
high pH (4.5-7.35) with the minimum at pH 5.0 (Fig. 2C). The mobility commonly decreased
as pH increased but changed to fast increase when pH value was above 7.0. The curves of

the EPM against pH values under the conditions of the same transmembrane potential and
different ionic strength was basically the same as (Fig. 3).

Figure 2. A: Zigzag increase of EPM measured in the suspensions of 5 mM K
+
, pH 7.35 and ionic
strength 8.8-156mM (P<0.01). B: an obvious zigzag increase of EPM in the suspension of 5mM K
+
at
156mM ionic strength and pH 2.5-7.35 (P<0.01). C: increasing tendency of EPM in the suspensions of
ionic strength 151-160mM, pH 7.35 and K
+
strength 3-12mM (P<0.01).
The zigzag increase of cell mobility was also measured when the transmembrane potential
or K
+
increased (from 3 to 12mM K
+
, Fig. 2B). This suggests that the electromigration of
myocardial cells may be imposed largely by the transmembrane potential. Such a zigzag
variation of mobility was found in different conditions with somewhat similar features
except for some point as shown in Figure 4. Myocardial cells tended to die at the ionic
strength < 6.7mM and pH < 5.0 or at the ionic strength <151mM and pH < 2.5. The death
could largely be avoided when the cells were suspended in Dextran solution at pH5.0.

Electrophoresis 90
Table 2. Cells with zero mobility found in various experimental groups

Figure 3. Plot of EPM against ionic strength measured in the suspensions of 5 mM K
+
and 8.8-156mM
ionic strength at pH values as shown in the figure.


Figure 4. Plot of EPM against the concentration of K
+
from suspensions with ionic strength of 151-
160mM at pH values as shown in the figure.
At zero transmembrane potential (140 mM K
+
), the variation tendency of cell mobility along
with the pH change was still the same but the range changed to 3.09 - 6.00, the highest and
the lowest value appeared at the pH value of 6.5 and 6.0. At a transmembrane potential of
approximately - 83.9mV (5 mM K
+
) and in 153mM ionic strength suspension, the cell
mobility generally increased with an exception at pH7.35 (Fig. 5).
After the surface sialic acid was cut off, the mobility of myocardial cells decreased at pH 5.0
as expected (Fig. 6, the first three bars). The decreasing extent was found to depend on the
ionic strength. Unexpectedly, the mobility increased when they were suspended in dextran
medium (Fig. 6, the 5th bars) or at transmembrane potential of 0 (Fig. 6, the 4th bars).
In conclusion, the zigzag mobility variation (over a range from 0 to 8.67) of myocardial cells
was observed for the first time, and the variation was found to depend not only on the
suspensions pH and ionic strength but also on K
+
or transmembrane potential.
4. Electric double layer on myocardial cell surface
4.1. Characteristics of myocardial cell mobility
This aberrant change could have four characteristics: First, the cell mobility changes in
parallel to the ionic strength and becomes large as the charged layer shrinks. Second, the
undulant mobility repeats in a certain range as the surface negative electric field is enhanced

Electrophoresis 92
or weakened. Third, the cell mobility rises abnormally as a component of the comprehensive
surface negative electric field decreases to its minimum or is equal zero (at pH 2.5 or zero
volt of transmembrane potential), Fourth, zero mobility appeared several times, especially
under the conditions of relatively low ionic strength where the mobility should originally be
slow (diffusion layer shrinks).

Figure 5. Plot of EMP against pH from the suspension of 140 mM K
+
(transmembrane potential was
zero). EMP at pH 7.35 decreased compared with that at 5mM K
+
(transmembrane potential was -
83.9mV) and 153mM ionic strength, and the others were increased (*: P<0.01).

Figure 6. Effects of neuraminidase treatment on EMP of myocardial cells. EPM were measured in
suspensions at pH 5.0 and ionic strengths of 12.4mM (Table 1, 11), 19.8mM (Table 1, 12) and
153mM(Table 1, 13), 140 mM K
+
(Table 1, 14) and Dextran 40 (Table 1, 10), respectively
(*: P<0.01).

4.2. Composition of the cell surface electric field
The fact that K
+
strength influenced on the cell EPM demonstrated that the transmembrane
potential participation should be considered to impose on the surface negative electric field
of myocardial cells, this conforms to the theoretic expectation. However, when the
transmembrane potential changes to zero, the characteristics of the EPM of myocardial cells
are still different from the common particles. After removing of the sialic acid on the surface
of cells, and at pH 5.0, which is lower than the isoelectric point of phospholipids (pI=6.5)
(Yamada et al., 1981) and changed the isoelectric point of membrane protein (Braun et al.,
2007; Popot & Engelman, 2000; Cho & Stahelin, 2005), or when the transmembrane potential
is zero (at 148mM ionic strengths), the EPM showed a strange rise, they may have very
complex contribution to the surface charges.
First, the cell mobility changes in parallel to the ionic strength and becomes large as the
charged layer shrinks. The role of transmembrane potential is complex, there should be a
complex negative electric field from intracell which concerns with the charged layer but has
an effect different from the charged layer. It enhances the half layer of the adsorbed ions and
reduces the diffusion layer at the same time or causes redistribution of the absorbed surface
ions, making the cell mobility changed zigzag. Second, the undulant mobility repeats in a
certain range as the surface negative electric field is enhanced or weakened. This is just an
effect of the complex negative electric field inside the cells: the adsorption layer of cell
surface varies as the charged layer does, causing part of the ions entering from the diffusion
layer. Third, the cell mobility rises abnormally as a component of the comprehensive surface
negative electric field decreases to its minimum or is equal zero (at pH 2.5 or zero volt of
transmembrane potential), implying that the effect of the complex intracellular negative
electric field is reduced as the charged layer shrinks significantly. Fourth, zero mobility
appeared several times, especially under the conditions of relatively low ionic strength
where the mobility should originally be slow (diffusion layer shrinks). The intracellular
complex negative electric field act thus in a limited space within the diffusion layer. The
zero mobility concerns only with a zero diffusion layer, not necessarily a zero value for cell
surface negative electric field and the adsorption layer. Therefore, the negative electric field
of myocardial cell surface is composed by the surface sialic acid, plasmalemma of
phospholipids, proteins and transmembrane potential (complex intracellular negative
electric).
Each component of negative electric field on the myocardial cell surface has different
influence on adions because its amount, isoelectric point and affect characteristics are not
the same. Sialic acid on the extracellular surface is about 50 nm away the plasma membrane
proteins, phospholipids and transmembrane potential, and in between the negative charges
sparsely distributes, including some penetrated adsorbed ions (Langer, 1978). More
specifically, in myocardial cells there are many of the same or similar membrane structures
besides the negative electric field (transmembrane potential). The surface area of these
structures is about 1/3 or more of the cell surface area. The special structure and composition
make the electric double layer of the myocardial cell surface different from the general

Electrophoresis 94
particles: There exist the intersection of the surfaces and inside/outside infiltration of matter,
and unique or even strange changes of the distribution characteristics of the cell surface
adsorbed ions and mobility. The so-called electric double layer structure is thus not sterling,
may be better called an aberrant electric double layer.
4.3. Analysis of mobility change
The one way declining of the cellular mobility after removal of only the extracellular surface
sialic acid, which is different from the zigzag mobility vibration, suggests the removal of
sialic acid has almost no impact on the complex intracellular negative electric field. At zero
transmembrane potential of zero, the mobility was not found to have an obvious
relationship with the phospholipid isoelectric point as the pH varied, implying that the pH
value may influence on all the membrane composition of sialic acid, protein, phospholipids,
and the intracellular complex negative electric field. The mobility is a result of multiple
integral ation. Thus the plasma membrane phospholipid may have only weak contribution
to the distribution change of adsorbed ions on a myocardial cell surface. After complete
removal of the effect of the transmembrane potential, the mobility almost allways increased
except for at pH7.35. This indicates the significant effect of the transmembrane potential
changes on the intracellular complex negative electric field: The more is the transmembrane
potential reduced, the weaker becomes effect of intracellular complex negative electric field
and the less are the ions entering from the diffusion layer into the adsorption layer. (In case
of pH7.35 there may be other charged components enhancing the effect). The fact that
removal of sialic acid and plasma membrane phospholipids and suppression of the
transmembrane potential could cause high mobility at high ionic strength, which should
also be an integral result, reveals that these factors have some effect on the intracellular
complex negative electric field.
By these imitated in vivo, broadly varied suspension conditions, the myocardial cells were
shown to have a complex aberrant electric double layer structure on the membrane surfaces.
The related surface electric field is a cellular character integrated from the complex
combinations of membrane sugars, phospholipids, proteins and transmembrane potential
(intracellular complex negative electric field). The ion layer is composed of a large
adsorption layer and a large cyclicly varied diffusion layer. This is a special structure,
making mobility and its related features completely different the general colloidal particles.
Interestingly, the contraction and Ca
2+
influx (slow inward calcium current) of a living
myocardial cell surface, which is chared only negatively, is not significantly affected after
removal of the negative sialic acid composition (Yee et al., 1991; Langer & Nudd, 1983). This
is due to the contribution of other powerful components in addition to the sialic acid. The
aberrant electric double layer structure, which is also distinguished from non-excitable cells,
is dependent on the variation of each complex membrane composition such as sugars,
membrane proteins, phospholipids and transmembrane potential (intracellular complex
negative electric field). The change may be the normal physiological performance but can
also be a result of pathology. The charged layer in combination with the intracellular
complex negative electric field connects the intra and extra cell environments as a whole

body. This point is very important for not only realizing the myocardial cell function
(contraction) but also for studying the cardiovascular disease mechanisms.
5. Challenges and prospects of myocardial cells electrophoresis
Animal and human cells are a complex system. According to the basic essence of systems
biology research--structure determines function we believe that the study of cell electric
phenomena is a key point to crack the hard shells of some diseases and related mechanism.
This may unroll all the true information on finding the formation and changing mechanism
and inside/outside cellular connecting routes of the cell surface electric double layer, which
should be different from the ordinary colloidal particles and on the integration of the double
layers characters into the overall cellular structure and natural biological behaviors. In fact,
the occurrence of some diseases is not only caused by one abnormal gene or protein by the
unusual results of the electric double layer structure of the cell membrane surface.
Therefore, the electric double layer structure of cell membranes or surfaces should be a
coherent part in biological researches, and the related methods and materials selected
should be able to imitate body conditions as much as possible during performing the
investigation of cell electric phenomena. It is also critical to establish cell model capable of
re-diplaying the cell functions.
Author details
Ying Zhou
PLA 309 Hospital, Beijing, P.R. China
Acknowledgement
I thank Professor Chen Yi for guidance and help.
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Electrophoresis 96
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Yamada, K.; Sasaki, T. & Sakagami, T. (1981) Measurement of isoelectric points of
phospholipid exchange proteins by gel isoelectric focusing. The Tohoku Journal of
Experimental Medicine. 135, 37-42. ISSN: 1349-3329
Braun, R. J.; Kinkl, N.; Beer, M. & Ueffing, M. (2007) Two-dimensional electrophoresis of
membrane proteins. Anal Bioanal Chem, 9, 1033-45. ISSN: 1618-2642
Popot, J. L. & Engelman, D. M. (2000) Helical membrane protein folding, stability, and
evolution. Annu Rev Biochem, 69, 881-922. ISSN: 0066-4154
Cho, W. & Stahelin, R. V. (2005) Membrane-protein interactions in cell signaling and
membrane trafficking. Annu Rev Biophys Biomol Struct. 34, 119151. ISSN: 1056-8700
Langer, G. A. (1978) The structure and function of the myocardial cell surface. Am J Physiol.
1978, 4,H461-468.
Yee, H. F. Jr.; Kuwata, J. H. & Langer, G. A. (1991) Effects of neuraminidase on cellular
calcium and contraction in cultured cardiac myocytes. J Mol Cell Cardiol, 23,175-185.
ISSN: 0022-2828
Langer, G. A. & Nudd, L. M. (1983) Effects of cations, phospholipases, and neuraminidase
on calcium binding to "gas-dissected" membranes from cultured cardiac cells. Circ Res,
53, 482-90. ISSN: 0009-7330.
Chapter 6
Isozymes: Application for Population Genetics
Vibeke Simonsen
http://dx.doi.org/10.5772/45875
1. Introduction
Electrophoresis as such has been applied for many purposes, among these, analysis of
isozymes or isoenzymes. As the designation indicates, this is a group of enzymes having the
same catalytic ability, i.e. they catalyse the same chemical reaction. Due to the fact that
enzymes are proteins and built from amino acids, they possess an electric change - so
isozymes are enzymes with the same catalytic ability, but different electric charge. The
charge will cause the enzyme to migrate in an electric field, the migration rate may be faster
or slower, depending on the carrying media (see section 3 on electrophoresis) and the power
applied. In order to detect the enzyme, the substrate for the particular enzyme and needed
co-factors are added, so that the enzymatic reaction can take place. An array of enzymatic
detection methods based on for instance histochemical or fluorogenic procedures are known
(Manchenko 1994). The revealed pattern is called a zymogram (Fig.1).

Figure 1. Zymogram of the enzyme glucosephosphateisomerase from 15 individual sprat eggs, dimeric
enzyme with three allozymes. For further interpretation, see section 5
Proteins are gene products, which mean that they are an easy way to get information on
genetic variation. If more zones of a particular enzyme are revealed in the same zymogram,
then the enzyme consists of isozymes. If one zone is interpreted as being the product of one
gene, it may reveal different alleles of the gene and, hence, this particular isozyme consists
of various allozymes as shown in Fig. 1. Interpretation of the zymograms leads to estimation
Allozyme 1
Allozyme 2
Allozyme 3

Electrophoresis 98
of allelic frequencies, which is the fundament of population genetics under the assumption
that the genes are inherited in accordance with the Mendelian laws. Another important
feature of enzymes is that they may consist of one, two, three or more polypeptides. As the
enzymes perform the same reaction needed in the metabolism of the organism and that
metabolism more or less is universal, the molecular structure is universal. That means that if
an enzyme is a dimeric enzyme, built by two polypeptides, this structure will be found
regardless of plant species, insect species or any other organism studied, see section 5.1 on
interpretation.
1.1. Procedures needed for studying isozymes
When studying isozymes, five major procedures must be considered: Sampling and storage,
preparation of the samples for electrophoresis, electrophoretic procedure, detection
procedure for the isozymes, and the interpretation of the zymograms.
2. Samples
As mentioned above, one of the key observations in population genetics is the detection of
alleles used for estimation of allelic frequencies. In order to achieve a reliable estimate of
allelic frequencies, many individuals must be analysed, at least 30 in order to minimise the
variance of the estimate of the allelic frequencies (see e.g. Hedrick 1983). When studying
vulnerable species, for instance species with low population size, the method for sample
collection has to be considered. Two methods for sample collection are considered:
None destructive method where the individual can continue life
Destructive method which will cause the death of the individual
2.1. None destructive methods
None destructive methods require that the samples used for detecting isozymes are
removed from the individuals at site of collection. In the case of plants, the samples may
consist of leaves or seeds collected from individual plants. For animals the samples may
consist of a small amount of blood that can be removed without killing the animal, and with
special equipment it may also be possible to remove small biopsies or hair samples. If hair is
pulled out, the small tissue sample on the tip of the hair can be used for analysis of
isozymes.
2.2. Destructive methods
As mentioned, destructive methods lead to the organism being killed. In this case, it has to
be considered how to utilise the samples in an optimal way in order to reduce further
collection. When dealing with plants, in the most cases the destructive method can be
avoided. However, when using seeds, this will result in a reduction in the next generation,
so collecting seeds is a partially destructive method. However, seedlings as samples for

Isozymes: Application for Population Genetics 99
analysis of isozymes are often a good choice due to the soft material compared to the fully
grown plant. Many small invertebrates have a size that only allows one analysis, e.g.
collembolans, but other invertebrates, e.g. earthworms, can support several analyses -
however, the collecting method will be destructive. Many fish species when caught will die
immediately, so removing biopsies will not save the fish. Analysing fish eggs is a
destructive method which will affect the next generation. However, many fish species
produce a huge number of eggs, so the actual damage may be small, depending on the
vulnerability of the species.
2.3. Transportation
Depending on the species, the samples may either be frozen or kept in a cold box with ice
when transporting the samples back to the laboratory. It is crucial that the samples are kept
cold in order to avoid destruction of the proteins and, hence, the enzymes. Furthermore, re-
freezing the samples will also destroy the proteins, so the number of times for
freezing/thawing the samples has to be minimised. Leaves or seeds can easily be transported
in a cool bag with cooling blocks if the transportation can be done in one day in the
temperate climate zone. If the material is frozen, transportation in a cool bag or a thick foam
box with cooling blocks may be sufficient to keep the samples cold, so the activity of the
enzymes is not reduced significantly. Frozen fingerlings of Indian major carps were
transported this way from Bangladesh to Denmark and later analysed for isozymes with
success, see Fig. 2. Transportation time was about 24 hours. For longer storage, the tissue
samples have to be kept either at -80C or in liquid nitrogen. For many organisms, storage of
none treated samples are better for storage than solutions of the samples, e.g. a small tissue
sample in a plastic bag will do better than a solution of the tissue during storage, see
preparation of samples, section 2.4.

Figure 2. Zymogram of the enzyme glucosephosphateisomerase from Indian major carps, from left to
right: 5 hybrids between mrigal and rohu, 5 hybrids between catla and rohu, 5 hybrids between catla
and mrigal, 5 rohu, 5 mrigal and 5 catla
2.4. Preparation of samples
Preparation of samples for the electrophoretic procedure may be done in various ways,
depending on the organism and the electrophoretic procedure chosen. The simplest way is

Electrophoresis 100
to use a drilling machine with a glass or plastic rod. A small about of a buffer solution, often
the gel buffer, is added and the sample with the buffer is treated with the drilling machine.
When dealing with very tough samples, a small amount of sand can be added. After this
procedure, the samples are centrifuged and the supernatant is used for the application
procedure. It is important that the samples are kept cold during the process in order to
minimise the loss of enzyme activity. More advanced milling machines are available and
they give a better grinding of the material. Depending on the organism, it may be an
advantage to add various chemicals to the grinding buffer, and especially for plants an array
of chemicals are suggested (Soltis & Soltis 1990). However, seedlings seem to do just as well
with a buffer as grinding solution. Sonication may also be a method for squeezing the
material as is treatment with liquid nitrogen.
Depending on the application method, it may be necessary to add sucrose or other
chemicals that increase the density of the sample solution. This is often used when the
samples are loaded on the surface of the gel, either on a small piece of filter paper (e.g.
Whatman No. 3MM) or as a droplet (1-2 L). Various application templates may be used.
It must be mentioned that fish eggs, which are stored in 40% sucrose (Paaver, pers.
comm.), can be picked up with a forceps after the sucrose with the eggs is spread on a
piece of filter paper, thereby removing the liquid. A single egg can be picked up and
placed on a piece of filter paper (size 4mm x 6mm) soaked with 5 L 40% sucrose, another
piece of filter paper also soaked with 40% sucrose is placed on the top, and the whole
sandwich is squeezed with a spatula. One piece of paper may be used immediately and
the other piece may be stored in an Eppendorf tube (0.5 mL) at -80C and used later for
electrophoresis.
3. Electrophoresis
Electrophoresis is the migration of electrically charged molecules in an electric field. In order
to establish an electric field which can be handled, a supporting media is needed. The media
may be paper, agar or agarose, starch, cellulose acetate or polyacrylamide. Each of these
media has advantages and disadvantages, so one has to select a media which suits to the
organism that has to be analysed. Among the media mentioned, I have no experience with
paper or cellulose acetate.
The electrophoresis may be performed either vertically or horizontally. Regardless of
orientation which you are using, you must ensure that the gel is kept cold during the
procedure. The electrophoretic procedure develops heat, and the heat may destroy the
activity of the enzyme. In general, the set-up consists of buffer trays which carry the
electrodes providing the electric field and provides contact to the carrying media and the
gel. When using a horizontal set-up, the contacts may be filter paper, cloths or sponges,
which establish the electric field across the gel. It must be mentioned that some enzymes are
sensitive to the material from which the contact is made, but ususally filter paper, Whatman
quality, will not harm the enzymes. A horizontal set-up is shown in Fig. 3.


Figure 3. Drawing of horizontal set-up
3.1. Electrophoresis performed with agar or agarose as supporting media
The two methods described in this paragraph are horizontal set ups. Years back, certain
purified agars were adequate for doing electrophoresis of e.g. haemoglobin, but
nowadays various agaroses are better suited for the electrophoretic procedure. The pore
size formed by the polymers is dependent on the concentration of agar/agarose. Due to
the fact that there has been a high demand for high quality agarose for separating DNA
fragments, many varieties of agarose have been developed, among these also products
suitable for proteins.
3.1.1. Electrophoresis performed with agar or agarose on microscope slides
A simple set-up is to use microscope slides for support of the media. The agar/agarose
solution (1%) is cooked in water bath or in microwave oven with the buffer used for
electrophoresis and the cleaned slides are coated with 2 mL agar/agarose solution, using a
pipette. The samples are applied in a slit (one slit, one sample) made by filter paper placed
vertically on the slide, see Fig. 4, or holes punched out by suction with a capillary pipette.
Depending on the method used, one to four samples may be applied to one microscope slide
and the electrophoresis is performed immediately after the application. An array of buffers
may be used, for suggestions, see Richardson et al. (1986).
Buffer tray
with electrodes
Gel with support
and placed on a
cooling plate

Electrophoresis 102

Figure 4. How to make a slit on an agar/agarose gel. The blue quadrangle at the left figure is filter
paper, the blue zone shows water (buffer) migrating up in the filter paper, the blue zone on the right
figure illustrates the slit
3.1.2. Electrophoresis performed with agar or agarose on glass plates
A different approach is to use glass plates 100 mm x 100 mm x 1 mm and Gel Bond film for
agarose. The film is rolled on the glass plate with 10% glycerol as glue. Strips of Dymo
tape are placed on another glass plate, so that when the two glass plates are placed together
by using Bulldog Clamps, the space between them is 0.2 mm. Thickness of the gel can be
increased by adding more Dymo tape strips. The plate with the Dymo tapes is siliconated.
The sandwich with a syringe needle between the glass plates is placed in a hot cabinet
(70C) for 30 minutes. When warm, the hot agar/agarose solution, cooked in the same way
as mentioned in section 3.1.1, is added using a syringe (also hot) with a gentle pressure on
the syringe. When the moulding form is nearly filled, the syringe with the needle is
removed. The gels are left between the two glass plates and may be stored this way for a
week in the refrigerator in a box with moist, but not soaking wet, paper towel.
Prior to applying the samples, the sandwich is opened and the gel on the Gel Bond is placed
on a cooling plate. Gel is removed from the Gel Bond film, creating 1 cm rim around the gel,
see Fig. 5. Application may be done either in punched holes, by a sample application strip
made from plastic with slits or the sample applicator developed for the Pharmacia
Phastsystem. It may be advantageous to dry the area, where the samples will be applied,
with a piece of filter paper, but this operation must be done very gently. On this size of gel, 8
to 16 samples may be applied. Furthermore, as the application takes place on the top of the
gel, it may be advantageous to have 10% sucrose in the sample grinding buffer. As these

Microscopeslide
with agarose
Slit
Filter paper
sucking
water

gels are thin, cooling from beneath is sufficient. However, there may be a risk of drying out
of the gel, especially the 0.2 mm gel, however, covering the surface with a small plastic film
may prevent this. The contact between the gel and the electrodes may be filter paper dipped
in the buffer trays containing adequate buffer and partially covering the gel. Another
possibility is to use two strips of Whatman No. 17 soaked in buffer and place the electrodes
on top of these, see Fig. 5.

Figure 5. Set up with filter paper as buffer container
The running time depends on the enzyme studied, but as a rule a running time of about -
1 hour is sufficient. Various dyes, e.g. bromphenol blue, may be applied to see how far the
front is running, and the closer to the edge the better. How much voltage and current to
apply depends on the buffer and the thickness of the gel. The concentration of agar/agarose
will also have an impact on the electrophoretic procedure.
3.2. Starch gel electrophoresis
Horizontal starch gel electrophoresis is a well-described method that also provides buffers
for gels and trays, see Ferguson (1980) and Murphy et al. (1996). This procedure uses potato
starch hydrolysed, in other words, the starch molecules form a structure with pores. The
pore size depends, more or less, on the starch gel concentration.
Various moulding forms can be made, and the ones used in our lab have been constructed
by a plexiglass frame and Mylar film glued together with high vacuum grease. The size of
Supporting film
Gel
Wicks

Electrophoresis 104
the frame is 250 mm x 190 mm x 6 mm outer size, leaving a form for the gel of 210 mm x
150 mm x 6 mm, and the width of the frame is 20 mm. The size of the gel allows a horizontal
slicing yielding three slices that can be stained individually, i. e. three enzymes can be
detected in one gel. The concentration of starch used for this purpose was 12 %. It is possible
to use thicker gels and they will allow more slices of the gel for detecting enzymes, but then
the cooling has to be increased. A higher starch concentration also is possible, but the higher
concentration, the slower the migration. The cooling set up is a cooling plate underneath the
gel, as shown in Fig. 3, and a tray with crushed ice on the top of the gel, so you have a kind
of cooling sandwich with the gel in the middle. The cooling is strongly needed, as the rather
thick gel will developed a high amount of heat which will destroy the activity of the
enzymes needed for detection.
The preparation of a starch gel consists of cooking the gel, evaporation of the gel and gel
settling. Two thirds of the buffer used for the gel is heated to cooking point using cooking
facilities as open fire, electric cooking plate or microwave oven. The starch is suspended
in the rest of the buffer, the suspension is added to the hot buffer and the whole solution
is swirled around for mixing. The hot solution is re-heated to boiling point two or three
times, allowed to settle for a few minutes and then evaporated. It is important to avoid air
in the gel solution, as it may damage the electrophoretic procedure. If evaporation is not
possible, cooking the solution several times may do the job. Upon evaporation, the
solution is poured into the gel frame, left for 15 minutes for settling, and then placed on
the cooling plate for another 15-30 minutes. Prior to being placed on the cooling plate, the
gel has to be covered with a plastic sheet to avoid loss of liquid. The gel has to be solid
before applying the samples.
A slit is cut in the gel for sample application. The samples are treated as described in section
2. The supernatant of the sample is sucked up in a piece of Whatman 3MM filter paper or
similar paper, size 4 mm x 6 mm, excess liquid is removed and the filter paper with the
sample is applied in the slit.
The gel should run for three hours at 80 mA if possible. The contact between the electrodes
in the buffer tray and the gel, see Fig. 3, is ensured with wicks made from Whatman 1. We
use 3 layers of the filter paper. Other media for contact may be used, but it has to be
considered whether the medium contains substances that interfere with the activity of the
enzyme.
An alternative method is to use a set up like the one shown in Fig. 5, but using wider
wicks also made from Whatman 17. The wicks are sucked in the tray buffer, two thirds of
the paper is placed on the gel, and the electrodes are placed on the outer edge of the filter
paper. If necessary, additional tray buffer is added during the electrophoresis when the
power is off.
Various dyes may be used to see how far the front has moved. After the electrophoresis, the
gel is cut into three slices with a string mounted in a U-frame. Guitar string E is very well

suited for this purpose. The adjustment of the string is done by guitar tuning pegs attached
to the frame. After slicing, the gel slices are stained, see section 4.1 on staining.
3.3. Polyacrylamide gel electrophoresis
Polyacrylamide gels are based on polymerisation of acrylamide monomers with a linking
reagent, e.g. N,N- methylenebisacrylamide, see Westermeier et al. (2001). The pore size is
controlled by the concentration of acrylamide (T) and the cross-linker (C), measured in per
cent. The concentration is estimated as
( ) 100 a b
T
V

100 b
C
V

where a = acrylamide in g, b = N,N- methylenebisacrylamide in g and V= the volume of the
solution in mL.
If C is kept constant and T is increased, the pore size decreases.
Polyacrylamide gel electrophoresis may be performed either horizontally, as the system
shown in Fig. 3, or vertically. Various equipment for electrophoresis can be purchased from
professional companies. As for starch gel electrophoresis, various buffers may be applied,
see e.g. Westermeier et al. (2001). Again, cooling may be important, depending on the
thickness of the gel and the sensibility of the enzyme.
3.3.1. Horizontal polyacrylamide gel electrophoresis
Many companies offer ready-made gels that may be advantageous to use, as the acrylamide
is toxic when unpolymerised. However, if the financial support is limited, the gels can easily
be prepared in the lab, but a casting frame is needed. The casting frame can be a glass-plate
covered with Gel Bond film (not needed if the glass plate is very thin) combined with a
siliconated glass plate with a U-frame. The two glass plates are clamped together with Bull
Dog clamps or similar equipment. A solution with the acrylamide, cross linker and catalysts,
needed for the polymerisation and dissolved in the gel buffer is filled into the form with a
syringe. A set up like one shown in Fig. 3 is applied for the electrophoresis. Application of
the samples can be performed as described for agarose gel electrophoresis, except that the
filter paper with the sample is placed on top of the gel. To improve the migration into the
gel, 10% sucrose is added to the sample grinding buffer, see section 2.4. A few mA are
applied for 5-15 minutes, then the filter paper is removed and a higher voltage is applied.
The running time depends on the thickness of the gel and the gel buffer. If adding
bromphenol blue or another dye that does not harm the sample to the grinding buffer, the
migration front can be followed.
Another way to apply the samples is to construct a U-frame with slot formers. These can be
made by gluing Dymo tape or other forms of tape to the glass plate, as shown in Fig. 6.

Electrophoresis 106

Figure 6. Template for horizontal polyacrylamide gel with slotformer
3.3.2. Vertical polyacrylamide gel electrophoresis
If a set-up for a vertical system is applied it may be an advantage first to make a running gel
and then a stacking gel on top of the running gel. The stacking gel is helpful for organising
the molecules according to size. By insertion of a comb in the stacking gel before the gel has
solidified, holes for applying the samples can be made. The holes are rinsed with the tray
buffer before the samples are applied. Then the holes are filled with the tray buffer and with
a syringe the sample is applied in the bottom of the hole. It is a good help to have a dye in
the sample supernatant, e.g. bromphenol blue, and also sucrose or another substance which
makes the sample heavier than water. Again it may be advantageous to apply a few mA for
a short while and then increase the voltage.
3.4. Isoelectric focusing in agarose
Isoelectric focusing can be performed either in agarose or polyacrylamide. Ampholytes are
added to the gel before it solidifies. The ampholytes are chemical substances that have a
high buffering capacity and low molecular weight. When power is applied to the gel, the
ampholytes create a pH gradient across the gel, which allows proteins to be organised
according to their pI value (isoelectric point (pI) where the protein is unchanged).

Figure 7. Zymogram of the enzyme esterase from 5 samples of plasma from minks, each allozyme
consists of two bands, which means that the five individuals four-banded phenotypes are
heterozygotes. For further explanation, see Simonsen et al. (1992)

Glass plate
U-frame
Slot former

The method using agarose as gel may be performed in a very simple way similar to the
method described in section 3.1.2. An outcome of the procedure is shown in Fig. 7.
4. Staining and data storage
After the electrophoresis, procedures for detecting the enzymes have to be applied, i.e. the
activity of the enzyme is detected. Furthermore, it is often important to be able to return to
the results for further analysis, so methods for storing the gels should be considered.
4.1. Staining
The staining procedure is based on the activity of the enzyme. In general, many proteins are
separated in a single analysis, as no attempt has been made to select specific proteins from
the sample supernatant, see section 2.4, and only few proteins are visible, e. g. haemoglobin,
the red dye in blood. Specific methods for detecting the proteins and, hence, the enzymes
are needed.
General histochemical methods for detecting proteins as staining with e.g. Coomasie Blue
are often used for visualization, but the methods are unspecific and not very sensitive. A
better method is to use the antigenic ability of the protein. If an antibody to the protein
exists, it is possible to detect the protein by first doing electrophoresis in the usual way.
Then cut the gel into pieces that are run in the second dimension in a gel with antibody. The
protein can then be revealed by special histochemical methods, for further information see
Axelsen et al. (1973).
The enzymes, which have catalytic abilities, are easy to detect. When electrophoresis is
done, the specific substrate for the enzyme is applied to the gel and the reaction will only
take place at the spot in the gel where the enzyme is located. More spots of a specific
enzyme are called isozymes, i.e. proteins with the same catalytic ability, but different electric
charge. If the spots can be interpreted as the result of a locus with more alleles, then the
spots are identified as allozymes.
There are three main principles for visualization of enzymes:
1. Transformation of the substrate into a visible product.
2. Transformation of the substrate into a product that can react with a dye.
3. Transformation of the substrate into a product that can be used as substrate for another
enzyme. This enzyme and a dye then have to be added to the solution, depending on
the reaction.
An example of the first principle is the use of 4-methyl-umbelliferyl (4-MUB)-substrates, e.g.
4-MUB-acetate. The enzyme esterase splits 4-MUB-acetate into 4-MUB and acetate, 4-MUB is
fluorescent and visible by UV-light.
An example of the second principle is the use of substrates that split off naphthol. By adding
diazo-compounds, the visualization takes place.

Electrophoresis 108
An example on the third principle is dehydrogenases that use either NAD or NADP as
coenzyme. This is depicted in Fig. 8.

Figure 8. Visualisation of enzymes able to reduce NAD or NADP, NAD is nicotinamide dinucleotide,
NADP is nicotinamide dinucleotide phosphate, NADH and NADPH are the respective reduced
substances of NAD and NADP, MTT is MTT-tetrazolium salt and PMS is phenazine-methosulfate, a
catalyst which transmits hydrogen ions
This method can be expanded with one step more by having an enzyme which can produce
a product on which a dehydrogenase can act. A frequently used helping enzyme is glucose-
6-phosphate dehydrogenase. For reactions where ammonia is released, glutamate
dehydrogenase works as a linking enzyme.
Many of the reactions are light sensitive, so they have to be developed in a dark cabinet and
mainly a hot cabinet will do the job.
Nowadays, it is possible to visualize about 100 different enzymes. For staining procedures,
see Harris and Hopkinson (1976), Tanksley and Orton (1983), Richardson et al. (1986),
Pasteur et al. (1989) and Manchenko (1994).
A couple of hints regarding staining procedure are that the reactivity of the enzyme may
increase with temperature and by using the optimal buffer for the specific enzyme in the
staining solution. The amount of enzyme in the sample solution may be very low and
especially very thin gels have a risk of rinsing off the enzyme. Applying the stain in an agar
overlay on the gel may improve the result.
Prior to performing electrophoresis, it is a good idea to check the sample solutions for
enzyme activity. This can be done by using the same glass plates as described in section
3.1.2. Pour 5-10 mL staining solution in 2% agar on a glass plate, apply a droplet of the
sample supernatant on the agar surface, place the glass plate in a hot cabinet for a while and
if the dye is developing there a good chance that electrophoresis of that particular enzyme
will work.
4.2. Gel storing
There are two methods for storing the gels; either a) dry the gels and save them or b) take a
photo.
All gels can be stored, but many of the stains are sensitive to light, so the stain may fade
away over time. The thin agarose gels are easy to dry. For the esterases shown in Fig. 7, once
stained for enzymes, the thin agarose gel is treated with 5% acetic acid for 10 minutes and
Substrate
Enzyme
NAD or NADP
NADH or NADPH MTT (Yellow)
Formazan (Blue)
PMS
Product

dried at room temperature. However, the 2 mm thick slides of starch gel are harder to dry.
Various methods have been tested and a drying oven has been developed. An easy way is to
dry the agar overlayer if the stain is applied in that way. A photo is even better, as it does
not take up that much storing space. This was a tedious job 20 years ago, when you had to
find the right film, develop it and to do the copying in the dark room. However, nowadays
it is much easier to take a photo. The best way to do this is to have light box providing
transmitted light and a stand for the camera. By using various softwares for photo editing,
you can enhance the bands on the gel, see Fig. 1 and 2. From the photo, it may possible to
estimate the relative amount of activity if you have a multi-banded zymogram by using e.g.
the software Image J (http://rsbweb.nih.gov/ij/).
5. Data analysis
To a wide extent, electrophoresis, applied to proteins, has been used concerning genetic
investigations from the sixties and to nineties. Several books and book chapters have been
written about this topic, e.g. Ferguson (1980), Richardson et al. (1986), Pasteur et al. (1989),
Whitmore (1990) and Murphy et al. (1990). However, now that the PCR-technique has been
developed, DNA is much more suited for genetic investigations. By analysing DNA, the genes
are studied directly and not after the translation of the genetic information into proteins.
Electrophoresis is a major part of the analytic methods used for both proteins and nucleic
acids, the interpretation and data analysis revealed for protein electrophoresis can be
applied for analysing the results achieved by studying nucleic acids and, hence, genes. A lot
of software for data analysis is available on the internet (e.g. GeneAlex written by Peakall &
Smouse (2006)). However, prior to carrying out advanced analysis of the data, it should
checked up, whether the genetic marker is inherited according to the Mendelian laws. When
dealing with proteins, it must also be considered whether the protein is codominant, i.e.
whether the heterozygote can be recognised. Most studied individuals are diploids, i.e. have
chromosomes in pairs, which means that an individual has two products (proteins) of each
gene, two alleles. If the two products are identical in construction, then the individual is
homozygous, but if the products are different, then the individual is a heterozygote. This
scoring of phenotypes interpreted as genotypes is the fundament for applying protein
electrophoresis for genetics.
5.1. Interpretation of zymograms
The outcome of a protein electrophoresis is called a zymogram, as mentioned in section 1. In
Fig. 1, the position of three different gene products is indicated as allozyme 1, allozyme 2
and allozyme 3. Fig. 9 depicts three zymograms with allozymes designated 1 and 2.
The first zymogram in Fig. 9 is determined by a locus with two codominant alleles and the
resulting protein is monomeric, which means that only one protein chain makes up the
active protein. The second zymogram is the result of a locus with two codominant alleles,
but the active protein is a dimer, i.e. two protein chains are needed for constructing the

Electrophoresis 110
active protein. The third zymogram is the outcome of a locus with two codominant alleles,
where four protein chains are needed for making the active protein.

Figure 9. Depiction of three different proteins with monomeric, dimeric and tetrameric structure, each
protein has two allozymes 1 and 2. Phenotype indicates the phenotypic interpretation of the zymogram
and genotype the genetic interpretation.
As shown in Fig. 9, the genotypic interpretation is determined from the phenotypic
interpretation. In theory, it is expected that the two bands seen for the genotype 1/2 are of
equal strength for a monomeric enzyme, but that is not always the case. If the two allozymes
for the dimeric enzyme combine randomly, the three banded phenotypes seen for the
heterozygous individual should come out as 1:2:1. The representing allozyme 1 consists of
two units of allozyme 1, the band in the middle consists of one unit of allozyme 1 and one
unit of allozyme 2, and the band representing allozyme 2 consists of two units of allozyme 2.
In a similar way, the five bands for the tetrameric enzyme should come out in the ratio
1:4:6:4:1, when the two units combine randomly.
Some times more zones are revealed in the zymogram. An example of this is shown in Fig.
2, where two zones are seen - one in the upper end of the gel and one in the lower end of the
gel. Furthermore, there is a zone in the middle. The active protein is dimeric and the
interpretation is that the enzyme is determined by two loci, one with three codominant
alleles, located in the upper zone, and one with two codominant alleles, respectively. Fig. 10
depicts a more simple case for a dimeric enzyme determined by two loci with one and two
codominant alleles.
Monomeric
Dimeric
Tetrameric
Allozyme 1
Phenotype
Allozyme 2
Allozyme 1
Allozyme 2
Genotype
2 12 1
2/2 1/2 1/1
Allozyme 2
Allozyme 1


Figure 10. Zymogram of a dimeric enzyme determined by two loci with one and two codominant
alleles, respectively. The hybrid zone consists of one unit from zone 1 and one from zone 2
Normally the fastest anodic migrating zone is determined by locus 1, the next by locus 2 and
so on. The most common allele is designated 100 and the others are named according to the
migration distance, e.g. for allele 75, 75% of the distance from application to the location of
allele 100, see Fig. 11. However, the nomenclature of the alleles is not very rigid. The only
feature is that a reference is needed, so the nomenclature is consistent all the way through
the study.

Figure 11. Designation of alleles for a monomeric enzyme
Zone 1
Zone 2
Hybrid
zone
Allozyme 1
Allozyme 1
Allozyme 2
Phenotype Zone 1
Zone 2
1 1 1
1 12 2
Genotype Locus 1
Locus 2
1/1 1/1 1/1
1/1 1/2 2/2
Allozyme 1
Allozyme 2
Allozyme 3
Allozyme 4
Phenotype
Genotype
100 125 50 75
Application
100/100 125/125 50/50 75/75

Electrophoresis 112
In general, an enzyme is written with capitals, the enzyme esterase as EST. If you speak
about the locus, the designation is EST and if you speak about an allele, the name is written
as EST-1-100 or EST-1
100
, this means esterase locus No. 1 and allele 100. However, there is
no strict rule about how to do this. The abbreviations of the enzymes may be found in many
books on enzyme electrophoresis.
5.2. Expression
Due to the fact that enzymes are produced within an individual, the physiology of the
individual as well as the environment may have an impact on expression of the enzyme. All
cells have the same chromosomal set with the exception of semen and eggs, so in theory all
enzymes could be expressed in all cells. However, due to cell differentiation not all enzymes
are important for all cells, so a regulation of the expression in the various tissues is found.
An example is shown in Fig. 12.

Figure 12. Zymogram of lactate dehydrogenase from four eelpouts. From left to right, 4 samples of
heart tissue, 4 brains, 4 eyes without vitreous body, 4 vitreous bodies and 4 muscles. For further
information, see Simonsen & Christiansen (1985)
The lactate dehydrogenase is a tetrameric enzyme determined by three loci in the eelpouts,
where only one locus is expressed in all tissues investigated. The interpretation of the
zymogram is difficult and is only possible when finding rare variants of the three loci
(Simonsen & Christiansen 1985).

Another feature may be age, and a good example of this is haemoglobin, as described by
Harris (1980). As the expression may vary due to tissue and age, it is very important to
collect the samples from individuals of similar age and use the same tissue throughout an
investigation.
Sometimes null alleles or silent alleles are observed. To be sure that it is a true silent allele
and not just a poor sample, it is important that at least two zones are seen for the enzyme. If
one zone is present and the other zone is absent in a particular individual, the conclusion
regarding the presence of a silent allele is reliable. Remember that the electrophoretic
procedure is a tough treatment of the proteins, and even if the allozyme is not working
when developed, it may function in the individual.
Expression of an enzyme may be controlled by other genes, see e.g. Poulsen et al. (1983).
5.3. Modification
Modification of the gene product can be done in several ways. Regarding the protein
structure and folding, various small molecules may have an impact on the charge of the
protein. These molecules may be a part of the protein, but may not be necessary for the
enzymatic function. This may result in a copy of the original product, and an example is
depicted in Fig. 13.

Figure 13. Depicted zymogram of adenosine deaminase from the vitreuos body of eelpouts. Allozyme 1
and 2 are seen in many tissues, but allozymes 1 and 2 are only seen in the vitreous body, see Simonsen
& Christiansen (1981)
The small molecules in the buffer may also have an impact on the zymogram, see Fig.14.
For the slice to the left and the one in the middle, each allozyme consists of two bands,
whereas the last buffer only resolves one band for each allozyme.
Among many others, these examples merely illustrate how important it is to find the right
tissue and electrophoretic procedure for the individuals studied.
Allozyme 1
Allozyme 2
Allozyme 2
Allozyme 1
Application

Electrophoresis 114

Figure 14. The enzyme phosphoglucomutase from six individuals of earthworms, analysed by using
three different buffers for electrophoresis
6. Conclusion
In present times, analysis of proteins by electrophoresis is considered an old fashioned
method despite the fact that it was a great improvement for studying genetic variation in
populations fifty to sixty years ago. However, nowadays, the method is overruled by the
DNA analytic methods. Despite this fact, the study of proteins together with the
development of analytical software initiated an enormous step forward for our knowledge
of population genetics and much of the software can be used for analysing the results from
DNA methods.
Keep in mind that protein electrophoresis only reveals about 30% of all the genetic variation.
Also keep in mind that the method is only sufficient for showing dissimilarities. When you
have two allozymes with same migration distance on a gel, it cannot be concluded that they
have identical amino acid composition, but at least they have the same electric charge.
Finally, remember that the result of your study is not any better than the primary data, i.e.
the zymogram.
Author details
Vibeke Simonsen
Aarhus University, Institute of Bioscience, Denmark

Acknowledgement
I would like to thank Aarhus University for giving me 43 years of working with allozymes.
Many of the examples used in the chapter have been done in co-operation with many
colleagues. It has been exiting to follow the development of the electrophoretic procedures
for protein studies. Furthermore, I wish to thank Charlotte Kler for the great help with the
language.
7. References
Axelsen, N.H., Krll, J. & Weeke, B. (1973) A manual of quantitative immunoelectrophoresis:
Methods and applications. Universitetsforlaget, Oslo, 169 p.
Ferguson, A. (1980) Biochemical systematics and evolution. Blackie & Son, Glasgow, 194 p.
Harris, H. (1980) The principles of human biochemical genetics. Elsevier/North-Holland,
Amsterdam, 554 p.
Harris, H. & Hopkinson, D.A. (1976) Handbook of enzyme electrophoresis in human
genetics. - North-Holland, Amsterdam. Pages are numbered for each section.
Hedrick, P.W. (1983) Genetics of populations. Science Books International, Boston, 629 p.
Manchenko, G.P. (1994) Handbook of detection of enzymes on electrophoretic gels. CRC
Press, Boca Raton, 341 p.
Murphy, R.W., Sites Jr., J.W., Buth, D.G. & Haufler, C.H. (1996). Proteins: Isozyme
electrophoresis. In D.M Hillis, C. Moritz & B.K. Mable (Eds.), Molecular systematics.
Sinauer Associates, Inc., Sunderland, Massachusetts, pp. 51-120
Pasteur, N., Pasteur, G., Bonhomme, F., Catalan, J. & Britton-Davidian, J. (1989). Practical
isozyme genetics. - Translated by M. Cobb, Ellis Horwood Limited, Chichester, 216 p.
Peakall, R. & Smouse, P.E. (2006) GENALEX 6: Genetic analysis in Excel. Population genetic
software for teaching and research. Molecular Ecology Notes 6, 288-295.
Poulsen, H.D., Simonsen, V. & Wellendorf, H. (1983) The inheritance of six isoenzymes of
Norway spruce (Picea abies (L.) Karst). Forest Tree Improvement 16, 12-33.
Richardson, B.J., Baverstock, P.R. & Adams, M. (1986) Allozyme electrophoresis: A handbook
for animal systematics and population studies. Academic Press, London, 410 p.
Simonsen, V. & Christiansen, F.B. (1981) Genetics of Zoarces populations. XI. Inheritance of
electrophoretic variants of the enzyme adenosine deaminase. Hereditas 95, 289-294.
Simonsen, V. & Christiansen, F.B. (1985) Genetics of Zoarces populations. XIV. Variation of
the lactate dehydrogenase isoenzymes. Hereditas 103, 177-185.
Simonsen, V. (1992) Genetic polymorphism of esterase in plasma of American mink (Mustela
vison L.). Animal Genetics 23, 553-555.
Soltis, D.E. & Soltis, P.E. (1990) Isozymes in plant biology, Springer, New York, 268 p.
Tanksley, S.D. & Orton, T.J. (1983). Isozymes in plant genetics and breeding. Part A -
Elsevier, Amsterdam, 516 p.
Westermeier, R., Gronau, S., Beckett, P., Berkelman, T., Blles, J., Schickle, H. & Thesseling,
G. (2001) Electrophoresis in practice. A guide to methods and applications of DNA and
protein separations, 3rd edition, Wiley-VCH, Weinheim, 349 p.

Electrophoresis 116
Whitmore, D.H. (1990). Electrophoretic and isoelectric focusing techniques in fisheries
management. - CRC Press, Boca Raton, 360 p.
Chapter 7
Electrophoresis as a Useful Tool in
Studying the Quality of Meat Products
Dario G. Pighin
http://dx.doi.org/10.5772/45761
1. Introduction
The study of meat quality usually involves a great number of methodologies at the
laboratory. Thus, new technology and automatic equipments are used in combination to
well-known methodologies in order to improve the study of the constituents of a given
sample. Several methodologies are usually carried out in combination, especially when
composition and functionality of food compounds are studied.
Meat and meat products are mainly composed of water, proteins, lipids, minerals and
carbohydrates (Bender, 1992). Most sensory and functional properties depend on the
quantity and quality of these compounds. In the last years, several studies aimed its efforts
on the improvement of sensory quality of meat products by means of the incorporation of
additives (Szerman et al., 2012).
At present, ready-to-cook and pre-cooked foods are installed in life. Regarding meat market,
sous vide processing appears to be an interesting option for offering different kinds of beef
products for consumption. It consists of the cooking/pasteurization process to a raw
material packaged in a heat-stable vacuum container. The products are stored at 0-3 C and
are able to be re-heated and consumed even after 5 weeks (Vaudagna et al., 2002). Besides
the enhancement of shelf life, several other advantages have been associated to sous vide
processing, such as increased flavour profile, increased tenderness and nutritional loss
reduction (Church & Parsons, 2000; Vaudagna et al., 2002).
The sous vide system has been extensively studied in meat and meat products (Church &
Parsons, 2000; Garca-Segovia et al., 2007). Nevertheless, there is still a lack of knowledge
regarding the application of this mild cooking system to whole-muscle beef. Moreover, this
pre-cooked system presents important technological inconvenients like juice retention inside
the packaging, which would affect profitability and sensorial and nutritional characteristics

Electrophoresis 118
of the final product (Church & Parsons, 2000; Vaudagna et al., 2002). On this regard,
additives such as sodium chloride and alkaline phosphates have being frequently used for
meat products manufacturing in order to increase water holding capacity (WHC) of meat
and, consequently, reducing cooking weight loss (Baublits et al., 2006; Pietrasik & Shand,
2004, 2005; Vaudagna et al., 2008). Results obtained showed that the incorporation of NaCl
(0.70% or 1.20% w/w) plus TPP (0.25% w/w), and posterior cooking at mild temperatures
(60-65 C) increase the WHC of Semitendinosus muscles and reduce cooking weight losses.
The increased WHC would be responsible for the tenderness increment found, probably as a
consequence of the swelling of myofibrils. Nevertheless, the exact physicochemical events
involved have not been completely elucidated. Thus, the study of the structure and
functionality of meat proteins emerges as an important issue to take into account in order to
elucidate the effect of additives incorporated into meat products and the mechanisms
involved in the improvement of sensorial and physical properties.
Regarding meat proteins, it is known that they have a widely range of size between
approximately 20 kDa to 3,000 kDa (Warris, 2000). Among them, high molecular weight
proteins display a key role in conferring functional properties to meat (Warris, 2000). Its
approach usually represents a challenge at the laboratory. Myosin, actin, titin and nebulin
are four of the major proteins of the skeletal sarcomere, and possibly the most important
myofibrillar proteins having interactions with each other in the muscular tissue. Myosin
(aprox. 540 kDa) and actin (aprox. 43 kDa) are the most noticeable contractile proteins of the
thick and thin filaments, respectively (Clark et al., 2002). Titin (aprox. 3,000 kDa) and
nebulin (aprox. 800 kDa) are two of the giant myofibrillar proteins, acting as protein rulers
by regulating the assembly of myosin and actin filaments in the sarcomere (Wang, 1996). At
present, several methods from different sources have been reported for their isolation.
However, most of them are complex, tedious and mainly focused on the isolation of one
protein at a time.
Currently, electrophoresis represents one of the most and reliable methods used in studying
meat proteins in the laboratory. It does not require expensive equipment and its outcomes
allow to be analyzed by means of several techniques like densitometry and differential
scanning calorimetry (DSC), among others. In general terms, electrophoresis refers to the
migration of charged particles in a particular medium under the influence of an electric
field. It constitutes a powerful and broadly used method for the analysis of complex
mixtures of analytes from different sources. Separated molecules can be visualized by means
of specific staining. One remarkable advantage is that the dried support medium can be
finally kept as a permanent record. It has been stated that the migration rate (cm/s) of a
given particle submitted to electrophoresis relies on several factors related to the shape,
charge and size of the particle and to the electrical field strength and the temperature of the
system as well. Thus, the mobility of the particle migration rate per unit of field strength
(V/cm)- results directly proportional to net charge of the particle and inversely proportional
to its molecular size at a given viscosity of the electrophoretic support (Karcher & Landers,
2006). Several support mediums had been described. Among them, polyacrylamide gels
(PAGE) offer several advantages when compared to others matrixes, especially when

Electrophoresis as a Useful Tool in Studying the Quality of Meat Products 119
proteins mixtures are been analyzed (Karcher & Landers, 2006). In this case, protein
separation takes place on the basis of charge-to-mass-ratio and molecular size. The addition
of sodium dodecyl sulphate to the system (SDS) denatures proteins conferring to them a
constant charge-to-mass ratio. Thus, under described conditions, proteins separation only
depends on its relative molecular weights (MW). Gradient SDS-PAGE is usually
recommended when proteins mixture spans wide MW range. In this case, the decreasing
pore size also contributes to sharpen the proteins bands.
2. Practical applications
A great deal of attention has been paid to the most important proteins of the myofibrillar
system because of their potential role in meat processing. Several authors have succeeded in
the isolation and/or enrichment of some of these proteins (Ho et al., 1994; Pan &
Damodaran, 1994; Wang, 1982; Wang & Greaser, 1985). Nevertheless, most of the described
methodologies use to be difficult to carry out because of their complexity, associated in part
with the exerted interactions among these proteins throughout the myofibrils (Houmeida et
al., 1995; Linke et al., 2002).
The purpose of the present chapter is to compile own published data which illustrate the
usefulness of electrophoresis analysis in meat research, either used alone or in combination
to other techniques. The first part describes a simple methodology to simultaneously co-
enrich myosin, actin, titin and nebulin from beef muscles. It is believed that the described
method will provide enriched myofibrillar proteins, which could be used for more specific
analysis, like microscopy studies, immunological and/or differential scanning calorimetric
analysis. The second component of this chapter is focused on the study of the effect of saline
additives on muscle proteins structure to improve meat quality. Since DSC has become one
of the most useful techniques for studying the thermal behavior of components of biological
systems, its approach in combination to SDS-PAGE is also described. The methodologies
used provide useful information in order to explain the improvement of sensorial properties
observed in meat treated with saline additives and submitted to sous vide system.
3. Methodology
3.1. Materials
Beef Semitendinosus muscles were dissected from British breed steer carcasses 48 h post
slaughter, following the Guidelines of National Sanitary Authority (SENASA, 1969).
Slaughter procedure was carried out in a commercial meatpacking and processing plant
with good manufacturing practices. High molecular weight (HMW) standard was
purchased from Amersham-Pharmacia (Buckinghamshire, England). Bovine serum albumin
(BSA, fraction V), enzyme inhibitors and mouse monoclonal anti-nebulin antibody were
obtained from SIGMA (St. Louis, MO, USA). Goat anti-mouse IgG antibody conjugated to
alkaline phosphatase was purchased from Bio-Rad (Cambridge, MA). All other reagents
were of the purest grade available (analytical grade) and obtained from SIGMA or Bio-Rad.

Electrophoresis 120
Semitendinosus muscles were trimmed free of fat and connective tissue before measuring pH
with a portable puncture pHmeter (Metrohm AG CH-9101, Herisau, Switzerland). Muscles
with pH 24 h values ranging between 5.5 and 5.7 were selected for the studies carried out.
3.2. Methods
3.2.1. Isolation of titin, nebulin, MHC and actin
3.2.1.1. Separation of myofibrillar proteins
The separation of myofibrillar proteins of Semitendinosus muscles was conducted according
to Boehm et al. (1998), and as detailed in Pighin & Gonzalez (2008). Protein concentration
was assayed according to Lowry et al. (1951), using BSA as standard. Discontinuos SDS-
PAGE (see details below) was performed to evaluate the protein profile. The rest of the
extracted fraction was diluted as described in Pighin & Gonzalez (2008) to reach a final
concentration of 5 g protein/L.
3.2.1.2. SDS-PAGE analysis and enrichment of titin, nebulin, MHC and actin
Partial isolation of proteins was achieved by means of ammonium sulfate salt precipitation,
using four different saturation ranges: 020, 2040, 4060 and 60100 g/L. After each
precipitation step, protein solution was centrifuged at 23,000 x g for 20 min at 4 C. The
resulting precipitate was resuspended in SDS buffer (3% SDS, 10% glycerol, 1 mM EDTA, 5
mM DTT, 10 mM Tris-HCl, pH 8.0), and the saline extract was dialyzed overnight against
buffer SDS (nitrocellulose membrane, MWCO: 1214 kDa). Discontinuous SDS-PAGE (see
details below) was used to monitor the protein composition of each precipitate.
SDS-PAGE analysis conducted according to Laemmli (1970) with some modifications was
performed in order to monitor the protein profile of extracts and to continue the isolation of
proteins. Electrophoresis was run in a lineal gradient of acrylamide concentration (312%) in
a Mini Protean III system (Bio-Rad), at constant voltage (130 V) during 90 min. For
comparison purposes, stacking and resolving gel solutions were prepared both with and
without glycerol (50 mL/L). A HMW calibration stained kit was used to estimate the
molecular weight (MW) of the different proteins. In order to monitor protein composition,
each line was loaded with 50 mg of proteins, and with 120 mg for isolation purposes. Gels
were stained with 1 g/L Coomassie Brilliant Blue R-250 solution. Images were captured with
a Bio-Rad GS-800 Imaging Calibrated Densitometer and processed by Quantity One 1-D
Analysis software (Bio-Rad, version 4.4.1) to determine relative front (Rf), MW and relative
amount of each protein. When a protein was not accurately identified in the electrophoretic
gel by staining, the Western blot technique was utilized as a tool to reveal the presence of
this protein. This analysis was applied to nebulin. For this purpose, a non-stained SDS-
PAGE gel was transferred overnight onto a nitrocellulose membrane (0.45-mm pore size) at
90 mA using Mini Trans-Blot Cell (Bio-Rad). Then, the presence of nebulin was detected
with the commercial mouse monoclonal anti-nebulin clone NB2 antibody (diluted 1:400),
followed by the secondary antibody anti-mouse IgG raised in goat and conjugated to
alkaline phosphatase (dilution 1:1,000). Once the protein of interest was located on the

nitrocellulose membrane, its position on an electrophoretic gel could be established by
comparison.
Once MHC, actin, nebulin and titin were identified, they were excised from a non-stained
gel, and individually eluted in an Electro-Eluter model 422 (Bio-Rad, MWCO: 1215 kDa) as
detailed in Pighin & Gonzalez (2008). Finally, each protein was collected in a volume of
approximately 500 mL, dialyzed overnight, lyophilized and stored at -80 C. The purity of
the isolated extracts was finally monitored by discontinuous SDS-PAGE as previously
described.
3.2.2. Study of protein contribution to the effect of saline addition to meat
3.2.2.1. Incorporation of saline additives
Brines containing NaCl and/or TPP were incorporated into the selected muscles (10% w/w)
with an automatic multi-needle injector (36 needles, Fricor, Buenos Aires, Argentina) to give
the final concentrations (g/100 g injected muscle) of 0.70 NaCl; 0.25 TPP; 0.70 NaCl + 0.25
TPP, and 1.20 NaCl + 0.25 TPP. Non injected (NI) muscles were used as control. Injected and
NI muscles were vacuum packaged (Cryovac CN510, Sealed Air Co., Buenos Aires,
Argentina) and submitted to continuous tumbling at 5.0 rpm, 8 h, 2-4 C (Lance Industries
tumbler, model LT-15, Allenton, USA) in order to improve brine sorption and protein
extraction. Two slices (2.0 cm wide) of each muscle were frozen at -80 C until thermal
analysis and the rest of the muscles were kept at 2-4 C until protein extraction.
3.2.2.2. Myofibrillar and sarcoplasmic proteins extraction and myofibrils isolation
Myofibrillar proteins extraction was conducted as mentioned in 3.2.1.1. After centrifugation,
the supernatant containing sarcoplasmic proteins was separated and stored at -80 C until
electrophoretic analysis. The precipitate was suspended in SDS buffer and filtered through a
home-made nylon strainer to remove connective tissue. An aliquot of the filtrate, which
included proteins of the myofibrillar system, was subjected to SDS-PAGE analysis. Protein
concentration of both sarcoplasmic and myofibrillar extracts was measured according to
Lowry et al. (1951).
Myofibrils isolation from minced meat of the different saline treatments was conducted
according to Culler et al. (1978), and as detailed in Pighin et al. (2008). The filtered
myofibrils collected were used for DSC analysis. Protein concentration of isolated myofibrils
and of whole muscles was determined by a macro-Kjeldahl method (Foss Tecator
equipment, Sweden), with a conversion factor of N = 6.25.
3.2.2.3. SDS-PAGE analysis and thermal analysis
Electrophoretic profile of protein extracts was monitored by discontinuous SDS-PAGE
(Laemmli, 1970), carried out under the same conditions previously described in 3.2.1.2.
A Perkin-Elmer Pyris-1 differential scanning calorimeter (DSC) was employed to study the
thermal properties of proteins of the whole muscle and of the isolated myofibrils. For

Electrophoresis 122
temperature and heat flow calibration, Indium was used as standard (melting point 156.6
C, enthalpy 28.46 J/g). Five to fifteen milligrams of each sample, accurately weighed, were
placed into an aluminum pan, which was hermetically sealed and equilibrated for 2 min at
the initial scanning temperature. During the study, a heating rate of 10 C /min was applied
over a 20-90 C temperature range. All samples were re-scanned (submission to a second
thermal analysis) to check for the irreversibility of the denaturation process. Endotherm
(mW vs. temperature), denaturation enthalpy (DH) and endothermic peak (Td) were
obtained using the software Pyris-7 (Perkin-Elmer). The total enthalpy change (DHT) was
estimated by the algebraic sum of the individual denaturation enthalpies.
3.2.2.4. Statistical analysis
The experiments described were conducted in duplicate. The study of saline incorporation
included 5 treatments (NI; 0.70% NaCl; 0.25% TPP; 0.70% NaCl + 0.25% TPP; 1.20% NaCl +
0.25% TPP). A set of five muscles were assigned at random to each treatment. SDS-PAGE
analysis and DSC measurements were performed in all muscles included in the treatments.
Analysis of variance (ANOVA) for DSC measurements was conducted using the statistical
package SAS (version 8, SAS Institute Inc., 2004, Cary, NC). Duncans Multiple Range Test
(with a significance of a = 0.05) was used to determine differences among treatments.
4. Results and discussion
4.1. Isolation of titin, nebulin, MHC and actin
4.1.1. Separation of myofibrillar proteins
Figure 1 shows the electrophoretic pattern of supernatant and precipitate fractions
obtained after the extraction of myofibrillar proteins. It can be seen (lane 2) that most of the
soluble proteins (2870 kDa) remained in the supernatant fraction. Instead, proteins of the
precipitate (lane 3) have a wider MW range (252,500 kDa). The precipitate included
almost exclusively myofibrillar proteins, yielding 64 g of extracted protein per kilogram of
muscular tissue. It can also be observed that the most intense band is MHC (MW: 220 kDa,
Rf: 0.47), which represents approximately 35 % of total myofibrillar proteins extracted.
Actin (MW: 43 kDa) is the second most intense (20 %) having one of the largest Rf (0.92).
Titin band represents approximately 4 % of the proteins extracted. It has the lowest Rf
(0.06) in the SDS-PAGE analysis with an estimated MW of 2,500 kDa. A similar MW for
titin was found by Kellermayer & Grama (2002) and Kurzban & Wang (1988). A
comparable electrophoretic pattern of myofibrillar proteins has been reported previously
by Chen et al. (2005).
Nebulin could not be precisely identified on the present SDS-PAGE due to the presence of a
set of several proteins in the range of 600800 kDa. Therefore, the use of monoclonal
antibodies appeared to be a useful tool to corroborate nebulin localization in the gel. Figure
2 shows the Western blot analysis of protein extract (precipitate fraction). Only one band
was detected by the commercial kit against nebulin, with an Rf of 0.20 and an estimated MW
of 710 kDa. It is important to denote that the MW of titin and nebulin were calculated in

approximation because of the absence of suitable HMW standards for SDS-PAGE.
Nevertheless, the MW found for nebulin is consistent with previous published data
reporting a value of 600 900 kDa for this protein (Locker & Wild, 1986; Wang, 1982).

Figure 1. Myofibrillar protein isolation and SDS-PAGE (312%) analysis of protein fractions
Lane 1 = HMW standard. Lane 2 = supernatant. Lane 3 = precipitate. (Pighin & Gonzalez, 2008)

Figure 2. Western blot analysis of myofibrillar protein extract Lane 1 = HMW standard.
Lane 2 = myofibrillar protein extract. (Pighin & Gonzalez, 2008)
It is important to remark that the use of gradients is usually recommended when SDS-PAGE
analysis is applied to high-MW proteins, in order to improve separation and resolution of
bands. The addition of glycerol has been reported to facilitate gradient formation and also to
improve resolution of the SDS-PAGE technique (Sobieszek, 1994). However, Chen et al.

Electrophoresis 124
(2005) did not find a great improvement of the gel system with the use of this reagent.
Present research supports the first statement, given the fact that the addition of glycerol both
to the resolving and the stacking gel solutions has successfully improved band resolution
(comparison data not shown).
4.1.2. Enrichment of titin, nebulin, MHC and actin
The isolation of myofibrillar proteins requires time-consuming procedures that usually
separate them individually. Thus, myosin isolation generally involves the removal of
sarcoplasmic proteins by means of washings with diluted phosphate buffer and subsequent
protein extraction with specific solutions containing elevated amounts of KCl (Dudziak &
Foegeding, 1988; Hermanson et al., 1986). Actin isolation requires a prior separation of G-
actin by breaking intermolecular linkages of F-actin. One of the most commonly used agents
is acetone, which can break the linkages without denaturing the protein (Syrovy, 1984).
After that, a second isolation stage involves several steps including washing, filtration,
centrifugation, polymerization/depolymerization cycles, dialysis and centrifugation (Pardee
& Spudich, 1982; Spudich & Watt, 1971). Gel filtration chromatography can also be
employed for further actin purification (Kuroda, 1982). More recently, Perez-Juan et al.
(2007) have succeeded in the isolation of actomyosin and actin in an interesting attempt to
amalgamate myofibrillar protein isolations in a single extraction process.
High-molecular weight proteins (i.e. titin and nebulin) extraction specifically involves the
addition of protease inhibitors to avoid degradation during processing. The isolation
methodology initiates with myofibrils extraction and posterior proteinSDS solubilization,
followed by the precipitation of titin/nebulinSDS complex by salt fractionation. For further
purification, the use of gel filtration is recommended because of the great size of these
proteins (Wang, 1982). A more exhaustive extraction of titin was proposed by Pospiech et al.
(2002), which demanded several centrifugation steps followed by suspension/
homogenization of the sediments in specific solutions, dialysis, and hydroxyapatite and ion-
exchange chromatography. Previous description shows the requirement of time and effort
to achieve the isolation of one or another myofibrillar protein. For that reason, the present
research was focused on looking for a more simple methodological strategy.
Salting out fractionation is believed to be a useful step for protein isolation because the
procedure is inexpensive and relatively easy to apply. Sodium chloride had been proposed
for the precipitation of the giant proteins, titin and nebulin (Wang, 1982; Wang & Wright,
1988). However, in the present research, the use of ammonium sulfate as precipitating agent
produced a proper isolation of these two proteins and also of myosin and actin. In order to
select the appropriate saturation range, different ammonium sulfate ranges were assayed: 0
20, 2040, 4060 and 60100 g/L. After each centrifugation step, the protein profile of the
precipitated fractions was monitored by SDS-PAGE. The different saturation ranges showed
the following protein pattern (Fig. 3): the band corresponding to MHC (MW: 220 kDa) was
the most noticeable one, showing similar concentrations in all saline ranges (see numbers
between brackets). Actin (MW: 43 kDa) was also detected in all fractions, being its intensity

higher in the saline ranges of 4060 g/L and 60100 g/L. Protein profiles of the ranges 020
g/L and 2040 g/L were similar, except for the amount of the titin band (2,500 kDa), which is
about four times higher in the second saline range. A saturation range of 4060 g/L showed
that the amount of titin and nebulin extracted is the highest. A higher saturation range (60
100 g/L) did not include the giant proteins in the precipitated fraction.

Figure 3. SDS-PAGE analysis of the different ammonium sulfate precipitates
Lane 1 = HMW standard. Lane 2 = range 020 g/L. Lane 3 = range 2040 g/L. Lane 4 = range 4060 g/L.
Lane 5 = range 60100 g/L. T, titin; N, nebulin; MHC, myosin heavy chain; A, actin. *Numbers between
brackets mean times increment. (Pighin & Gonzalez, 2008)
If this protein profile (Fig. 3) is densitometrically compared to the myofibrillar protein
extract (Fig. 1), it becomes evident that titin and nebulin have been greatly enriched in the
saline range of 4060 g/L, having 3.6 and 4.0 times increment, respectively.
Even though the analysis of the gel in Fig. 3 indicates that the saline ranges of 2040 g/L and
4060 g/L were appropriate for isolation and enrichment of titin, and 4060 g/L was so for
the enrichment of nebulin, the unified range of 2060 g/L appeared to be an interesting
option to increase the amount of these proteins in subsequent experiments. Instead, the
saline range of 60100 g/L was suggested to isolate actin and MHC. Once the saline
precipitation ranges were selected, SDS-PAGE was chosen to continue the purification
protocol. For this purpose, each ammonium sulfate precipitate was resuspended and
electrophoretically developed in an SDS-PAGE gel (312%). After the identification of the
proteins on the gel by means of its Rfs, they were first excised, and then submitted to
electroelution.
Figure 4 shows the SDS-PAGE of each electroeluted protein (lanes 14) and the Western blot
analysis of nebulin (lane 5). It can be seen that MHC (lane 2) was obtained in an almost pure
state. Actin and titin (lanes 3 and 4) were obtained enriched but not in a pure state. The
reason for this result is that actin and titin concentrations obtained after the salt precipitation

Electrophoresis 126
and the electrophoresis steps were low and made difficult the removal of a thinner band
from the electrophoretic gel to improve their isolation by electroelution. Nebulin band -
stained with Coomassie Brilliant Blue dye- was not accompanied by any other protein band
(data not shown). However, the band was vaguely stained and it required the identification
and confirmation by the previously mentioned immunoblotting technique (lane 5).

Figure 4. SDS-PAGE and Western blot analysis of the electroeluated proteins
Lane 1 = HMW standard. Lane 2 = myosin heavy chain extract (SDS-PAGE analysis). Lane 3 = actin (A)
extract (SDS-PAGE analysis). Lane 4 = titin (T) extract (SDS-PAGE analysis). Lane 5 = nebulin (N)
extract (Western blot analysis).
(Pighin & Gonzalez, 2008)
4.2. Study of protein contribution to the effect of saline addition to meat
4.2.1. Electrophoretic analysis of proteins extracts
The electrophoretic analysis of sarcoplasmic proteins extracts belonging to saline treated
muscles are shown in Fig. 5. As can be seen, proteins bands ranged between 30 and 75 kDa. It is
worth noting that the intensity of the band with an estimated MW of 70 kDa was increased
about 2.5 times in samples of muscles containing NaCl alone or combined with TPP (see
arrows, lanes 3-A, 3-B and 4-B) with respect to NI. Even more, an additional band with an
estimated MW of 65 kDa was revealed in these saline treated muscles (see dashed arrows, lanes
3-A, 3-B and 4-B). On the contrary, the presence of TPP alone did not modify the protein pattern
found in NI muscles (lanes 2-A and 2-B). These findings suggest that the presence of NaCl
collaborates in the solubilization of myofibrillar proteins, releasing them to the soluble fraction.
Myofibrillar proteins were separated by SDS-PAGE into a broad range of molecular weight
(Fig. 6). Bands corresponding to MHC (MW 220 kDa, see arrow) and actin (MW 46 kDa, see
dashed arrow) were the most noticeable ones. No differences among treatments were verified
by means of densitometric analysis. This result does not agree with the one previously shown
in Fig. 5, which had suggested an augmented solubilization of myofibrillar proteins as a

consequence of the application of NaCl into muscles. Hence, an attenuated intensity of any of
the bands in Fig. 6 was expected. It is probable that the increment of 70 kDa or the appearance
of 65 kDa band found in Fig. 5 is a result of the degradation of myofibrillar proteins.

Figure 5. SDS-PAGE analysis of sarcoplasmic proteins obtained from muscles treated with NaCl and/or TPP
Gel A: Lane 1, HMW-SDS; Lane 2, NI; Lane 3, 0.70 % NaCl; Lane 4, 0.25 % TPP.
Gel B: Lane 1, HMW-SDS; Lane 2, NI; Lane 3, 0.70 % NaCl + 0.25 % TPP; Lane 4, 1.20 % NaCl + 0.25 % TPP.
(Pighin et al., 2008)

Figure 6. SDS-PAGE analysis of myofibrillar proteins extracted from muscles treated with NaCl and/or TPP
Gel A: Lane 1, HMW-SDS; Lane 2, NI; Lane 3, 0.70 % NaCl; Lane 4, 0.25 % TPP.
Gel B: Lane 1, HMW-SDS; Lane 2, NI; Lane 3, 0.70 % NaCl + 0.25 % TPP; Lane 4, 1.20 % NaCl + 0.25 % TPP.

Electrophoresis 128
4.2.2. Thermal analysis of whole muscle
Thermal analysis of NI whole muscles (Fig. 7) showed three characteristic endothermic peaks.
Published data allowed to relate them to myosin and its subunits denaturation (I; 57.0 C),
sarcoplasmic proteins and collagen denaturation (II; 67.4 C) and actin denaturation (III; 80.3
C) (Graiver et al., 2006; Kijowski & Mast, 1988; Stabursvik & Martens, 1980). Enthalpies
involved in each thermal transition were 1.32, 0.65 y 1.74 J/g protein, respectively. Re-scanned
samples did not show any thermal response, corroborating the irreversibility of the transitions.
The calculated DHT of NI muscles was 3.71 J/g protein. It can be seen in Table 1 that NaCl
treatment (0.70 %) significantly reduced myosin DH and actin Td. Only slight decreases were
found in myosin Td and actin DH. These results make evident that actin was destabilized by
NaCl, becoming the more susceptible protein to thermal denaturation (decreased Td). Instead,
the effect of NaCl upon myosin could be seen on the DH, which reduction could be related to
the decrease of hydrogen bonds and/or to the increment of protein aggregation (exothermic
event) exerted by the salt. Total enthalpy (DHT) for NaCl-treated muscles (calculated by
adding DH of individual values related to transitions I, II, and III) decreased from 3.71 J/g
protein (NI muscle) to 3.04 J/g protein, demonstrating system instability.

Figure 7. Thermal analysis of NI whole muscles

Table 1. Endothermic peaks (Td) and denaturation enthalpies (DH) of whole muscles treated with
NaCl and/or TPP
a-c Means within a column having different letters are significantly different (p < 0.05).

The described findings reveal an important effect of the salt in decreasing the thermal
stability of proteins. In order to support this fact, it was proposed that addition of neutral
salt causes anions to compete with water molecules for specific sites of proteins, altering its
hydration properties, and consequently requiring lower denaturation energies (Von Hippel
& Wong, 1965). Treatment with TTP (0.25 %) significantly increased sarcoplasmic
proteins/collagen Td. Also, Td of myosin and DH of actin were slightly increased. Whole
muscle treated with 0.25 % TPP increased the DHT from 3.71 (NI muscles) to 4.28 J/g
protein. These findings show an important stabilizing effect exerted by TPP, probably by
altering hydrophobic interactions rather than affecting pH or ionic strength (Trout &
Schmidt, 1986). With regard to the effect of phosphates on meat, Kijowski & Mast (1988)
studied the effect of different phosphates (sodium pyrophosphate or sodium
tripolyphosphate) on chicken breast muscles and its isolated myofibrils. They stated that
low levels of sodium pyrophosphate or sodium tripolyphosphate (0.25 % or 0.50 %) caused
the maximum stabilization (DHT) of chicken muscle and isolated myofibrils by means of
myosin stabilization (increased DH). These results agree with present ones, even they were
obtained in beef muscles.
When both additives were used together (0.70 % NaCl + 0.25 % TPP), Td corresponding to
sarcoplasmic proteins/collagen was significantly increased in comparison to NI muscles,
suggesting stabilization of the related proteins. This stabilization went along with a
significant decreased of the DH, probably associated to an increment of the exothermic
protein aggregation. On the contrary, actin Td showed a significant reduction in comparison
to NI. Destabilization of actin in the presence of NaCl and phosphates was also described by
Kijowski & Mast (1988) in chicken breast muscles treated with 2 % NaCl and 0.25/0.50 %
pyrophosphate. When the effect of this combination of salts is compared to the one
previously described, 0.70 % NaCl effect, it can be speculated that two effects appear to
coexist: the same stabilizing effect of TPP on sarcoplasmic proteins/collagen and the
destabilizing effect of NaCl on actin. Even though, the last effect is smaller in magnitude
than the one produced by NaCl alone. It is important to denote that DHT of the muscle
treated with the combination of salts (0.70 % NaCl + 0.25 % TPP) was slightly higher than
the NI one (3.83 vs. 3.71 J/g protein, respectively). Comparing the DHT obtained
individually for NaCl and TPP treatments, it can be seen that the effect of the addition of
both salts is contrary to the effect of NaCl alone and in the same direction as TPP. Hence, the
final increase could be assigned to the increase (even non significant) of myosin DH induced
by TPP. Even more, when NaCl was raised to 1.20%, a maximal drop in actin stability was
achieved (higher reduction of Td and DH). This event was accompanied by a maximal drop
in DHT (from 3.71 to 2.75 J/g protein). It becomes evident that the increased amount of NaCl
in the brine produced a destabilizing effect similar to the one found for NaCl alone, which
also turned to be predominant to expenses of the effect of TPP.
Regarding this issue, Findlay & Barbut (1992) had stated that the addition of NaCl
(0.5-2.0 %) reduced myosin Td and conversely TPP (0.2-0.6 %) increased its stability.
Additionally, DHT varied depending on salts concentration. At low NaCl levels (less than
1.00 %) the effect of TTP on the increment of DHT was predominant. Instead, as NaCl

Electrophoresis 130
increased the phosphate effect was minimized. In accordance with such observations,
present results indicate that NaCl exerts a destabilizing effect in the presence of TPP (0.25 %)
that becomes stronger as NaCl level raises.
4.2.3. Thermal analysis of isolated myofibrils
Thermal analysis of isolated myofibrils from NI muscle showed only two endothermic
peaks (Fig. 8), corresponding to denaturation enthalpies of myosin and its subunits
(I: 58.1 C) and actin (II: 73.5 C). Data obtained from Table 2 showed a significant decrease
of myosin and actin DH in myofibrils isolated from 0.70 % NaCl-treated muscles and
washed in the same brine solution. No changes in Td of these proteins were found. The
destabilizing effect of NaCl was in agreement with data obtained from the whole muscle.
TPP (0.25 %) treatment produced a significant increase of both, myosin and actin Td. In
contrast, DH of myosin was strongly reduced (p < 0.05) by the treatment. These findings
indicate that the salt is capable of stabilizing actin and myosin structures. However, the
smaller energy required to denature myosin (lower DH) suggests a small amount of protein
available in the myofibril fraction. Supporting this comment, it had been proposed that
phosphates can induce myosin solubilization by promoting both, thick filament
depolymerisation and actomyosin dissociation (Granicher & Portzehl, 1964; Xiong, 2005).
Even though, an increased protein aggregation (exothermic event) taking place immediately
after myosin denaturation, might not be discarded. In the case of whole muscle treated with
TPP, myosin and actin Td did not suffer any change and their DH were only slightly
increased. Therefore, it can be affirmed that the effect of TPP on the whole muscle and
myofibrils is almost alike.

Figure 8. Thermal analysis of myofibrils isolated from NI muscles
When NaCl and TPP were used together (0.70 % NaCl + 0.25 % TPP) both, myosin and actin
Td of isolated myofibrils significantly decreased (p < 0.05). Enthalpy of the proteins was also

decreased, being the myosin DH reduced in almost 2.60 J/g protein with respect to NI
muscles (NI: 3.87 vs. 1.27 J/g protein; p < 0.05). These findings confirm the destabilizing
effect of NaCl previously described for actin in the whole muscle. Even more, this effect
seems to be stronger in the myofibril fraction since myosin transition was also affected.
Further increments of NaCl (1.20 %) -in the presence of TPP- produced similar effects on
both proteins, with the exception of an intensified effect on actin DH. It is important to
remark that the effect of both additives together was definitively higher than the effect of
any of the additives individually. Offer & Knight (1998) proposed that polyphosphates -at
the concentrations used in the industry- may assist NaCl in causing depolymerisation of
thick filaments and dissociation of actomyosin, facilitating in consequence the myosin
extraction. The action of NaCl as a mild structure-breaker aided by the presence of TPP
could be the reason of the significant reduction of protein stability found.

Table 2. Endothermic peaks (Td) and denaturation enthalpies (DH) of myofibrils isolated from muscles
treated with NaCl and/or TPP
a-d Means within a column having different letters are significantly different (p < 0.05).
As it was mentioned, the physicochemical mechanisms involved in water binding and retention
in the protein matrix of cooked meat were not completely elucidated. These results showed that
the combined presence of NaCl (0.70 % or 1.20 %) and TPP (0.25 %) changes the heat
susceptibility of muscle proteins (especially actin) so that they denature and coagulate at a lower
cooking temperature. These modifications suggest the occurrence of protein conformational
changes due to the salts, probably by altering hydrophobic and electrostatic interactions that
stabilize the protein structure (Franks & Eagland, 1975). Evidently, these changes collaborate in
the binding and posterior retention of water inside the tissue by any of the mechanisms proposed
for the action of NaCl and polyphosphates (Offer & Knight, 1998). Increased solubilization of
proteins found in whole muscles treated with NaCl (alone or in combination with TPP) could be
related to the removal of transverse myofibrillar proteins which may act as structural constrains
to myosin extraction. Also, some other low molecular weight myofibrillar proteins could be
removed. The softening of the structure together with conformational modifications of
myofibrillar proteins would allow entrapping water inside the tissue. This event would be the
reason of the increased WHC of Semitendinosus muscle found by Vaudagna et al. (2008).
5. Conclusion
The isolation protocol previously described in this chapter has been demonstrated to be a
successful methodology for individually isolating four of the main myofibrillar proteins.

Electrophoresis 132
Despite the proteins were not isolated in a complete pure state, this approach seems to be
useful for using them for further analysis. The estimation of proteins Rf by means of an
immunological method looks like an interesting tool to complement the identification of
proteins in unclear electrophoretic patterns.
The thermal behavior of the myofibrillar proteins is quite involved in the thermal behavior
of whole muscle. NaCl incorporation into meat leads to an important protein destabilizing
effect upon myofibrillar proteins, even at relative low doses when compared to industrial
usage. The use of NaCl in combination with TPP produces a destabilizing global effect,
suggesting that TPP may assist NaCl in acting as structure-breaker. The increase of WHC of
Semitendinosus muscles injected with saline additives and submitted to sous vide cooking
would be mainly associated to the conformational modifications of myofibrillar proteins and
to the weakening of myofibrillar structures due to protein removal.
Taken together, the use of gel electrophoresis demonstrated to be a useful tool not only in
isolating major muscle proteins but also in contributing to study the effect of saline
additives in meat, in order to improve sensorial properties. Its combination with advanced
technologies like DSC represents an interesting issue to look forward in the meat science
approach.
Author details
Dario G. Pighin
Institute of Food Technology, National Institute of Agricultural Technology INTA, Argentina
Acknowledgement
Special thanks are given to the Instituto Nacional de Tecnologa Agropecuaria (INTA),
Consejo Nacional de Investigaciones Cientficas y Tecnolgicas (CONICET) and
Universidad de Morn (UM) for their financial support in the studies carried out and
compiled in the present chapter.
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nebulin filaments anchored at the Z line. The Journal of Cell Biology, Vol.107, pp. 2199
2212. ISSN: 0021-9525.
Wang, S.M. & Greaser, M.L. (1985). Immunocytochemical studies using a monoclonal
antibody to bovine cardiac titin on intact and extracted myofibrils. Journal of Muscle
Research and Cell Motility, Vol.6, pp. 293312. ISSN: 0142-4319.
Warris, P.D. (2000) The Chemical Composition and Structure of Meat, In Meat Science. An
Introductory Text. pp. 37-67. CABI Publishing, ISBN: 0-85199-424-5, Oxfordshire, UK.
Xiong, Y.L. (2005). Role of myofibrillar proteins in water-binding in brine-enhance meats.
Food Research International, Vol.38, pp. 281287. ISSN: 0963-9969.
Chapter 8
Application of the Different Electrophoresis
Techniques to the Detection and Identification
of Lactic Acid Bacteria in Wines
Luca Gonzlez-Arenzana, Rosa Lpez,
Pilar Santamara and Isabel Lpez-Alfaro
http://dx.doi.org/10.5772/45759
1. Introduction
The microorganisms play an essential role in winemaking since a mixed culture of
numerous microorganisms including fungal, yeast, and bacteria species are involved in this
process and are the responsible for the final quality of the wine (Bisson et al., 1993).
Therefore, in order to control the fermentation processes knowing and understanding the
complex microbiota involved in them is necessary.
Yeast are able to convert sugar from grapes into ethanol and many other changes that lead
to wine. Lactic acid bacteria (LAB) that are often present on the surface of the grapes and
can represent significant populations in musts (Lonvaud-Funel, 1999) play dual roles in
wine fermentations: as wine spoilage agents and as the main effectors of malolactic
fermentation (MLF). Numerous studies have been conducted on the LAB that occur on
grapes, grape musts and wines and it is generally agreed that a succession of species
happens during the different stages of winemaking and conservation of wines (Ribreau-
Gayon et al., 2006). Most bacterial species present in wine fermentations have been
identified by traditional microbiological techniques involving cultivation. However, as it
was observed with microbial ecology studies of other environments, cultivation-dependent
methods often exhibit biases resulting in an incomplete representation of the true present
bacterial diversity (Amann et al., 1995; Hugenholtz et al., 1998). Applications of culture-
independent molecular techniques to monitor the microbial successions of various food and
beverage fermentations have revealed microbial constituents and microbial interactions not
witnessed by previous plating analyses (Giraffa & Neviani, 2001).
Denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel
electrophoresis to separate bacterial 16S ribosomal DNA (rDNA) amplicons are common

Electrophoresis 138
culture independent methods employed to characterize microbial communities from specific
environmental niches (Lopez et al., 2003; Muyzer & Smalla, 1998). These approaches are
attractive since they enable to detect individual species as well as to get overall profiling of
community structure changes with time.
Otherwise, ecology, interactions and development of the different bacterial strains during
alcoholic fermentation (AF) and MLF are still a field of active research. Efficient and precise
methods of strain identification and discrimination have been developed during the last
years, either to prepare well-defined starters of biotechnological interest in winemaking, or
to quickly assess the presence of certain strains in a wine, or to gain insight of such a
complex ecosystem as wine.
Pulsed field gel electrophoresis (PFGE) has proved to be an useful tool for the identification of
a wide LAB strains variety and especially for species belonging to the genus Lactobacillus
(Charteris et al., 1997). Several restriction enzymes have been used for obtaining profiles of
Oenococcus oeni strains: NotI (Kelly et al., 1993; Prevost et al., 1995; Sato et al., 2001), ApaI and
SfiI (Daniel et al., 1993; Kelly et al., 1993; Lopez et al., 2007). Other used methods for LAB strain
identification include ribotyping or restriction fragment length polymorphism (RFLP) of genes
encoding rRNAs (Charteris et al., 1997; Zavaleta et al., 1997), random amplified polymorphic
DNA (RAPD-PCR) analysis with arbitrary primers (Cocconcelli et al., 1997; Lopez et al., 2008;
Spano et al., 2002), rep-polymerase chain reaction (rep-PCR) analysis (Parry et al., 2002) and
multilocus sequence typing (MLST) system (de las Rivas et al., 2004; de las Rivas et al., 2006).
RAPD-PCR and PFGE of macrorestriction fragments are the most frequently used (Guerrini et
al., 2003; Tenreiro et al., 1994; Viti et al., 1996; Zapparoli et al., 2000; Zavaleta et al., 1997), and
more recently Ruiz et al. (Ruiz et al., 2008) have obtained a better discrimination in the study of
bacterial diversity by the combination of results from those two techniques.
For all these reasons, the aims of this work were: (a) applying the DGGE, PFGE and RAPD-
PCR techniques to the analysis of the LAB species diversity and the intraspecific diversity of
Oenococcus oeni in a winery of La Rioja region during three consecutive vintages, (b) getting
a better knowledge of bacterial ecology throughout both AF and MLF in wine elaborated
with Tempranillo, the classic red grape variety of Spain and native of Appellation of Origin
Rioja, (c) evaluating the occurrence of genotypes from commercial Oenococcus oeni strains
between the autochthonous Oenococcus oeni strains isolated from non inoculated fermenting
wines, and (d) contributing to the maintenance of the LAB biodiversity in Rioja red wines.
2. Methodology
2.1. Wine production and wine samples
Traditional red wine fermentations from c.v. Tempranillo local grapes of 2006, 2007 and
2008 vintages at one winery of the Spanish northern region of Rioja were studied.
Winemaking practices were the typical of this wine-producing area: AFs were conducted in
the presence of grape skins, seeds and stalks, after the addition of sulphur dioxide and until
the residual reducing sugar content was under 2 g/L. At this final point of AF, wines were
Application of the Different Electrophoresis Techniques
to the Detection and Identification of Lactic Acid Bacteria in Wines 139
drawn off into tanks and were allowed to undergo spontaneous MLF with the endogenous
microbiota. The sampled winery had never used commercial starters for MLF. One
fermentation tank was sampled in each vintage. Wine samples were collected aseptically for
chemical and microbiological analysis at different times: must (stage 1), tumultuous AF
(density around 1,025; stage 2), at final AF (< 2 g/L glucose + fructose; stage 3), initial MLF
(consumption of 10% of the initial malic acid; stage 4), tumultuous MLF (consumption of
60% of the initial malic acid; stage 5) and at final MLF (L-malic acid concentration < 0.5 g/L;
stage 6).
2.2. Commercial Oenococcus oeni starter samples
Sixteen commercial starter cultures employed to induce MLF derived from six different
companies were analyzed. These commercial cultures were selected between the most
frequently used in Spain.
2.3. Chemical analysis of the musts and wines
Alcohol degree, pH, total acidity, volatile acidity, reducing sugars, free and total sulphur
dioxide and L-malic and L-lactic acid content were measured according to the European
Community Official Methods (European Community, 1990).
2.4. Culture dependent methods
2.4.1. Bacterial enumeration and isolation
Must or wine samples were diluted in sterile saline (0.9% NaCl) solution and plated on
modified MRS agar (Scharlau Chemie S.A., Barcelona, Spain) plates supplemented with
tomato juice (10% v/v), fructose (6 g/L), cysteine-HCl (0.5 g/L), D,L-malic acid (5 g/L) and
pymaricine (50 mg/L) (Acofarma, S. Coop., Terrassa, Spain). Samples were incubated at 30
C under strict anaerobic conditions (Gas Pak System, Oxoid Ltd., Basingstoke, England) for
at least ten days, and viable counts were reported as the number of CFU/mL. Fifteen
colonies from each wine sample were selected for reisolation and identification. Isolates
were stored in 20% sterile skim milk (Difco) at 20 C.
Every commercial lyophilized starter culture was hydrated in saline solution (0.9% NaCl)
and then 100 L aliquot from the appropriate dilution was plated at the surface of modified
MRS agar without pymaricine. Because of their low viability in laboratory conditions
(Maicas et al., 1999a; Maicas et al., 1999b) glycine (40 mM) and ethanol (10% v/v) were
added to this medium. The plates were incubated for at least 10 days at 30 C under
anaerobic atmosphere (Gas Pak System, Oxoid Ltd.) and five colonies were isolated from
each one.
2.4.2. Species identification
Species identification was carried out by previously recommended methods, which
included bacteria morphology, Gram staining, and catalase (Holt et al., 1994). Oenococcus

Electrophoresis 140
oeni, Lactobacillus plantarum and Lactobacillus brevis species were confirmed by the species-
specific PCR method (Beneduce et al., 2004; Zapparoli et al., 1998). In case of the
identification of other species, PCR amplification of partial 16S rRNA genes was performed
with WLAB1 and WLAB2 as previously described (Lopez et al., 2007). PCR products were
sequenced by Macrogen Inc. (Seoul, South Korea) and sequences were used for comparison
to the data in GenBank using the Basic Local Alignment Search Tool (BLAST) (Altschul et
al., 1990).
2.4.3. Oenococcus oeni typification by PFGE
PFGE was carried out according to the method described by Birren et al. (1993) with some
modifications (Lopez et al., 2007) for agarose block preparation. Because of the difficulty of
typing commercial strains first of all these cells underwent to fifteen minutes of ultrasounds,
moreover a higher quantity of lysozime (100 L/block) was added and incubated for 2 h.
Macrorestriction analysis was performed with two endonucleases: SfiI, following the
method reported by Lpez et al. (2007), and ApaI by the method reported by Larisika (2008)
with the following modifications for optimal separation of fragments: 1.2% (w/v) agarose
gels were submitted to 24 h with a pulse ramping between 0.5 and 20 s at 14 C and 6 V/cm
in a CHEF DRII apparatus (Bio-Rad).
2.4.4. Oenococcus oeni typification by RAPD-PCR
RAPD-PCR was carried out following the procedure described by Ruiz et al. (2010b) with
some modifications: MgCl 100 mM, dNTP 50 mM and primer M13 100 mM. RAPD-PCR
reaction was developed in a total volume of 50 L and it was carried out with a Perkin
Elmer, GeneAmp PCR System 2400 thermocycler. 20 L of amplified products were
resolved by electrophoresis in a 1.4% agarose gel in 0.5x TBE (45 mM Tris base, 89 mM, boric
acid, 2.5 mM EDTA pH 8) for 3 h at 70 V.
2.4.5. Numerical analysis of PFGE and RAPD-PCR images
The conversion, normalization and further processing of images were carried out by
InfoQuest
TM
FP software version 5.10 (Bio-Rad, USA). Comparison of the obtained PFGE
patterns was performed with Pearsons product-moment correlation coefficient and the
Unweighted Pair Group Method using Arithmetic averages (UPGMA). Comparison of the
pulse types from the PFGE and RAPD was made by composite data set comparison with
average molecular analysis by Unweighted Pair Group Method using Arithmetic averages
(UPGMA) (Ruiz et al., 2008).
2.5. Culture independent methods
2.5.1. Direct DNA extraction from wines samples
A volume of 10 mL of each must or wine sample was centrifuged (30 min, 10000xg, 4 C).
The supernatant was discarded and 1.2 mL of saline solution (NaCl 0.9%) and 2.4 mL of
zirconium hydroxide (7 g/L) were added to the pellet to facilitate pelleting of the bacteria in
wine (Lucore et al., 2000). After horizontal shaking during 10 min at room temperature, the
suspension was again centrifuged (10 min, 500xg, 7 C) and finally DNA was purified from
the cell pellet by using a PowerSoil
DNA isolation kit (MO BIO Laboratories, Inc.,

Carlsbad, CA USA) as per the manufacturers instructions.
2.5.2. PCR conditions
PCR was performed using an Applied Biosystem, GeneAmp
PCR System 2700

thermocycler at a final volume of 50 L. To amplify the region V4 to V5 of 16S rDNA gene,
primers WLAB1 and WLAB2
GC
were used as Lpez et al. described (2003). Moreover,
primers rpoB1, rpoB1o, and rpoB2
GC
were employed to amplify the region of the rpoB gene as
it was described by Renouf et al. (2006) with the next modifications: 0.5 M of each primer, 1
mM dNTP mix and 0.5 L of PfuUltra II Fusion HS DNA Polymerase (Stratagene).
2.5.3. PCR-DGGE analysis
The separation of the respective PCR products was performed with the D-CODE
TM
universal
mutation detection system (Bio-Rad, Hercules, Calif.). PCR products obtained from WLAB1-
WLAB2
GC
primers were run on 8% (wt/V) polyacrilamide gels in a running buffer
containing 2 M Tris base, 1 M Glacial acetic acid and 50 mM EDTA pH 8 (TAE), and a
denaturing gradient from 35 to 55% of urea and formamide. The electrophoresis was
performed at 20 V for 10 min, and 80 V for 18 h at a constant temperature of 60 C. PCR
products generated with the rpoB1, rpoB1o, and rpoB2
GC
primers were separated with 8%
polyacrylamide gels containing a 32 to 50% urea-formamide gradient. Electrophoresis was
performed for 10 min at 20 V, and 16 h at 60 V at a constant temperature of 60 C. The
DGGE gels were stained in ethidium bromide after the electrophoresis and then were
visualized with UV transiluminattion (GelDoc, Bio-Rad). Blocks of polyacrylamide gels
which contained selected DGGE bands were excised and later incubated overnight in 20 L
of sterile and pure water at 4 C to make DNA bands diffuse to the liquid. One microliter of
this solution was used to reamplify the PCR product.
2.5.4. DNA sequencing and phylogenetic analysis
PCR products were sequenced by Macrogen Inc. (Seoul, South Korea). The quality and
characteristics of the obtained sequences were analyzed with the software InfoQuest
TM
FP
5.10, only those ones considered as appropriate were used for comparison to the GenBank
database with the Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990). After
this preliminary study, our sequences and their homologous ones (obtained from the
Nucleotide Database: http://www.ncbi.nlm.nih.gov/nuccore) were assembled and submitted
to phylogenetic and evolutionary analysis with MEGA version 4.0.2 (Tamura et al., 2007).
The Neighbor-Joining analysis (Saitou & Nei, 1987) allowed to get information about the
relations between the gotten sequences and the reliability of the identifications provided by
the Nucleotide Database. The bootstrap test was based on 1000 replicates (Felsenstein, 1985).

Electrophoresis 142
The evolutionary distances were computed using the Maximum Composite Likelihood
method (Tamura et al., 2007) that allowed to calculate the equivalent units to the base
substitutions per site.
3. Results and discussion
3.1. Oenological parameters of wine samples and fermentation development
Results for analytical composition of wines during three vintages are displayed in Table 1.
Data were within the usual range of Tempranillo wines from this Spanish region (Gonzlez-
Arenzana et al., 2012b). After completion AF, alcohol content ranged between 13.0% and
14.0%, pH was between 3.32 and 3.64 and free SO2 level was between 4.24 and 18.1 mg/L.
During MLF a decrease in total acidity and a subsequent increase in pH were observed. In
addition, an increase in volatile acidity was noted as it was expected. The wine from 2006
vintage showed less restrictive parameters for microbial growth, so it presented higher pH
and the lowest values of alcohol content and SO2.

Year 2006 2007 2008
Stage 3 6 3 6 3 6
Alcohol content (% v/v) 13.0 - 13.8 - 14.0 -
pH 3.64 3.83 3.41 3.57 3.32 3.50
Total acidity (g/L tartaric acid) 7.98 5.91 7.63 6.71 9.00 7.20
Volatile acidity (g/L acetic acid) 0.25 0.46 0.37 0.49 0.26 0.37
Total SO2 (mg/L) 28.4 - 38.1 - 31.6 -
Free SO2 (mg/L) 4.24 - 18.1 - 13.2 -
L-malic acid (g/L) 2.60 0.04 1.48 0.16 2.61 0.21
L-lactic acid (g/L) - 1.81 - 1.10 - 1.72
-: not analyzed
Table 1. Analytical composition of wines at final AF (stage 3) and final MLF (stage 6) at each vintage.
AF completion lasted for six, sixteen and eleven days in 2006, 2007 and 2008 vintages,
respectively. Viable LAB counts during AF were in the range of 10
2
- 10
3
CFU/mL, increasing
to 10
7
10
8
CFU/mL during MLF, similar to spontaneous MLF results reported by other
authors (European Community, 1990; Lopez et al., 2008). The development of the MLF was
related to the viable population of LAB and there was a relation between bacterial
population and decrease in L-malic acid (data not shown). Important differences in MLF
duration were observed between vintages and MLF completion lasted for 21, 239 and 136
days in 2006, 2007 and 2008 vintages, respectively. Different temperatures at each vintage
(wine temperature below 12 C after AF in 2007) and the lack of temperature control in the
winery were the determinant factors in these differences, but factors such as pH,
composition of the wine and the interaction with other microorganisms implicated in the
fermentation could also influence, as it has been reported by other authors (du Plessis et al.,
2004; Lonvaud-Funel, 1999; Reguant et al., 2005a; Reguant et al., 2005b).
3.2. Species identification
3.2.1. Culture dependent microbiological analysis
Figure 1 shows the number of isolates of the viable LAB species identified at each stage and
year of vinification. A total of 251 LAB isolates were recovered and identified as belonging
to eight different species. The greatest diversity of LAB species was detected during the AF.
Oenococcus oeni was present in all studied stages of the fermentation process except in 2007.
It was isolated in must and tumultuous AF in 2006 and 2008 vintages, and it was the only
species isolated at MLF in the three years, being therefore the predominant species, followed
by Lactobacillus plantarum, Leuconostoc mesenteroides and Lactobacillus mali. The other non-
Oenococcus oeni species appeared at stages 1-2 in variable rates in the three vintages. A
similar distribution of species has been also reported by other authors (Fugelsang &
Edwards, 2007; Ruiz et al., 2010) and they also concluded that Oenococcus oeni was the main
responsible species for MLF. The diversity of species found at each year was different, being
the number of species isolated in 2007 almost double that in 2006 and 2008, and missing
Oenococcus oeni until the end of the 2007 AF, a fact that could also have influence in the MLF
duration, as it was indicated above.

Figure 1. Number of isolates of the viable LAB species identified at each stage of vinification (1, must; 2,
tumultuous AF; 3, final AF; 4, initial MLF; 5, tumultuous MLF; 6, final MLF) and in each vintage.
3.2.2. Culture independent microbiological analysis and comparison with culture
dependent method
PCR-DGGE analysis of the sampled wine fermentations in the three studied vintages using
primers WLAB1/2 (16S rDNA-based primer sets) and primers rpoB1/1o/2 ( subunit of the
0
2
4
6
8
10
12
14
16
1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6
Stage 2006 vintage Stage 2007 vintage Stage 2008 vintage
N

o
f

i
s
o
l
a
t
e
s

o
f

e
a
c
h

L
A
B

s
p
e
c
i
e
s
O.oeni L. plantarum L. mali L. brevis L. coryniformis L. uvarum Ln. mesenteroides P. parvulus

Electrophoresis 144
RNA polymerase-based primer sets) (Figure 2a and 2b, respectively) revealed different
species and a profile of bacterial community structure changes during AF and MLF.

Figure 2. DGGE gels of the sampled wines at each vintage and stage of vinification (1, must; 2,
tumultuous AF; 3, final AF; 4, initial MLF; 5, tumultuous MLF; 6, final MLF). Letters indicate bands
excised for each gene, 16S rRNA (a) and rpoB (b).
The sequences obtained from the DNA excised DGGE-bands of each sample and their
homologous ones from Nucleotide Database (Altschul et al., 1990) constituted a tree for each
studied gene (Figure 3). Figure 3a shows a tree based on 16S rDNA gene composed by four
ramifications or branches belonging to the genus Oenococcus, Lactobacillus, Weissella and
Leuconostoc; and Figure 3b shows a tree based on rpoB gene composed by three branches
belonging to Oenococcus, Leuconostoc and Lactobacillus.
2006 2007 2008
1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6
a
b
a
a
a a a
a
a a a
2006 2007 2008
1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6
a
b
c
d
a
a a
b
b b
a
a
a a
b
a
b
a
b b
a
b
c
a
a a
a
a
a
b
a
c
(b)
(a)

(a)

Electrophoresis 146

Figure 3. Phylogenetic trees generated from the method of 16S rDNA (a) and rpoB (b) sequences of
recovered bands were inferred using the Maximun Parsimony method (Felsenstein, 1985). Numbers over
the branches are bootstrap values (1000 repetitions). Escherichia coli and Listeria inocua were used as
outgroups. Scale bar represents (calculated) distance. Reference strains are closely related to the sequenced
bands and accession number of each gene is indicated. Band number isolated from fermenting wines are
indicated by the sampled stage of vinification (1, must; 2, tumultuous AF; 3, final AF; 4, initial MLF; 5,
tumultuous MLF; 6, final MLF) the letter of the position in the gel and the isolation year.
The species identification at each stage of vinification in the three studied years with culture
independent techniques (16S rDNA/PCR-DGGE and rpoB/PCR-DGGE) and culture
dependent method (plating on modified MRS) are recovered in Table 2.
A total of fourteen different LAB species were identified in the three studied vintages by
traditional and culture independent methods. PCR-DGGE analysis allowed to identify nine
species in comparison to the eight ones detected by culture in plate of the sampled wine.
Thus, Fructobacillus ficulneus, Fructobacillus tropaeoli, Lactobacillus buchneri, Leuconostoc
pseudomesenteroides and Weissella were not detected in the employed culture medium, while
Lactobacillus coryniformis, Lactobacillus brevis, Lactobacillus mali, Lactobacillus uvarum and
Pediococcus parvulus were not detected by PCR-DGGE. Therefore, results obtained by both
methods were complementary and demonstrated the importance of using a combined
analytical approach to explore microbial communities as other authors have concluded in
different ecological niches (Iacumin et al., 2009). In relation with DGGE analysis, PCR-
amplified bands from 16S rDNA gene gave better results than the rpoB bands amplification,
with eight and three identified species, respectively. Nevertheless, the results obtained with
the two genes were again complementary so Lactobacillus buchneri was only detected by
rpoB/PCR-DGGE.
Results about diversity of LAB species found at each year and stage of vinification were
very similar to those described above for culture dependent method. The greatest diversity
was detected again during AF, opposite to MLF were only two species were present,
Oenococcus oeni and Leuconostoc mesenteroides. The presence of Leuconostoc mesenteroides in
(b)
2007 at stage 4 confirmed again the possible competition of other LAB with Oenococcus oeni
and their influence in MLF duration.

Detected LAB species
2006 2007 2008
1
*
2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6
Fructobacillus ficulneus - - - - - - - - w - - - - - - - - -
Fructobacillus tropaeoli - w w - - - - - - - - - - - - - - -
Lactobacillus coryniformis - - - - - - c - - - - - - - - - - -
Lactobacillus brevis c - - - - - - - - - - - - - - - - -
Lactobacillus buchneri - - - - - - - - - - - - - b - - - -
Lactobacillus mali c - - - - - c - - - - - c c - - - -
Lactobacillus plantarum c c c - - - - c - - - - - c w - - -
Lactobacillus uvarum - - - - - - c - - - - - - - - - - -
Lactobacillus sp. - - - - - - - - - - - - - w - - - -
Leuconostoc mesenteroides w - - - - - bc c - w - - - - - - - -
Leuconostoc pseudomesenteroides w - w - - - - - - - - - - - - - - -
Oenococcus oeni wc c c wc w w wc wc wc wc
Pediococcus parvulus - - - - - - c - - - - - - - - - - -
Weissella sp. w - - - - - - - - - - - - - - - - -
UB.1 - - - - - - - - - - - - w - - - - -
UB.2 - - - - - - - - - - - - - w - - - -
UB.3 - - - - - - - - - - - - - - w - - -

Total species n 7 3 4 1 1 1 6 3 2 2 1 1 3 6 3 1 1 1
Plate species n 4 2 2 1 1 1 5 2 1 1 1 1 2 3 1 1 1 1
Total DGGE species n 4 1 2 1 1 1 2 1 2 2 1 1 2 4 3 1 1 1
16S rDNA/DGGE species n 4 1 2 1 1 1 1 1 2 2 1 1 2 3 3 1 1 1
rpoB/DGGE species n - - - - 1 1 1 - - 1 1 1 - 1 - 1 1 1
*
stage of vinification (1, must; 2, tumultuous AF; 3, final AF; 4, initial MLF; 5, tumultuous MLF; 6, final MLF)
-; not detected
UB; uncultured bacterium
w; detected with 16S rDNA/DGGE
b; detected with rpoB/DGGE
c; detected in plate after culture
; LAB species detected with all the employed methods
Table 2. LAB species detected with culture independent techniques (16S rDNA/PCR-DGGE and
rpoB/PCR-DGGE) and culture dependent methods at each stage of vinification (1, must; 2, tumultuous
AF; 3, final AF; 4, initial MLF; 5, tumultuous MLF; 6, final MLF) during three vintages.
3.3. Strain typing of Oenococcus oeni
3.3.1. PFGE analysis of the strains of this study
Identification of the Oenococcus oeni strains of this study was successfully achieved by PFGE
of DNA digested with SfiI and each strain presented a characteristic PFGE pattern.
Digestions with ApaI enzyme were not more discriminating than SfiI restriction (data not

Electrophoresis 148
shown). Cluster analysis and visual inspection of the PFGE patterns from the 187 Oenococcus
oeni isolates recovered from wine fermentations in the three years gave a total number of 37
distinct genotypes (Figure 4). Twenty-four of them were detected in 2006, five in 2007 and
fifteen in 2008 vintages (Figure 5). The lower clonal diversity found in 2007 vintage would
be related with the low temperatures during MLF (below 12 C) what could make few
genotypes be able to get adapted to the difficult conditions.

Figure 4. UPGMA dendrogram based on the SfiI PFGE patterns of the 37 Oenococcus oeni genotypes.
Figure from Gonzlez-Arenzana et al. (2012a).
1
0
0
9
0
8
0
7
0
6
0
5
0
4
0
3
0
2
0
1
0
2
26
31
11
25
14
27
24
17
20
29
30
7
19
18
36
15
34
35
13
10
33
16
21
12
9
37
1
32
22
23
5
3
4
6
8
28
% Similarity
Comparing coincident genotypes for the three vintages, it was observed that between the
genotypes isolated in 2006 vintage two were found in 2007 (genotypes 18 and 20) and four
in 2008 (genotypes 3, 13, 17 and 18). Moreover, two genotypes isolated in 2007 (genotypes
18 and 25) were also detected in 2008 vintage. Only one genotype (18) was identified in
the three studied years. The frequency of participation of each genotype varied from year
to year, thus dominant genotypes one year were minority or not present at other one
which suggested the adaptation of Oenococcus oeni strains to the winery conditions every
year. Similar results were reported by other authors in studies about bacteria and yeast
populations (Gutierrez et al., 1999; Reguant et al., 2005a; Ruiz et al., 2010; Santamara,
2009).
Interestingly, no genotype was isolated in all fermentation stages so fourteen genotypes
appeared only at AF (stages 1-3), six were present at all MLF stages (4, 5 and 6) and three of
them were also detected at the end of AF. Most fermentation stages showed mixed
Oenococcus oeni strains populations, which confirmed that several Oenococcus oeni strains
occurred in a single spontaneous MLF (Lopez et al., 2007; Renouf et al., 2009; Ruiz et al.,
2010). The number of different genotypes identified at each stage ranged from 0 to 5, and
from 3 to 9, at stages 1-3 and 4-6, respectively. Genotypes 9 and 18 in 2006 vintage, 20 and 26
in 2007, and 13 in 2008 were the predominant ones during MLF. Three out of these
genotypes (13, 18 and 20) could be considered as interesting Oenococcus oeni strains for the
selection of new malolactic starter cultures as individual or mixed strains, because in
addition to be dominant in most of the MLF stages at each vintage, they were isolated at
more than one year in quality wines.

Figure 5. Frequency of appearance (%) of Oenococcus oeni genotypes at each stage (1, must; 2,
tumultuous AF; 3, final AF; 4, initial MLF; 5, tumultuous MLF; 6, final MLF) and vintage. Genotypes
without color only appeared once, genotypes with grey color appeared only in one year at more than
one stage and genotypes with color appeared in more than one vintage.
1
1
2
2
4
4
3
3
3
5
6
7
11
11 11
8
9
10
12
34
34
36
36
13
13
13
13
17
17
17
26
26
26
26
18
18
18
18
18
20
20
20
20
20
14
15
16
19
21
22
23
24
25
25
25
27
28
29
30
31
32
33
35
37
0%
20%
40%
60%
80%
100%
1 2 3 4 5 6 3 4 5 6 1 2 3 4 5 6
Stage 2006 vintage Stage 2007 vintage Stage 2008 vintage
A
p
p
e
a
r
a
n
c
e

(
%
)

Electrophoresis 150
3.3.2. Comparison of the PFGE and RAPD-PCR profiles from the wine fermentation
strains and commercial strains
The thirty-seven genotypes of the indigenous Oenococcus oeni strains from wine
fermentations and fourteen PFGE and RAPD-PCR patterns obtained of strains from several
commercial cultures were submitted to comparison by bioinformatics and visual analysis
(data not shown). Figure 6 shows that genotypes I, II, III and IV from commercial starter
cultures resulted indistinguishable from four indigenous genotypes (23, 13, 21 and 18,
respectively), despite commercial malolactic cultures had never been employed in the
sampled cellar.

Figure 6. Consensus dendrogram obtained by combination SfiI-PFGE, ApaI-PFGE and M13 RAPD-PCR
patterns corresponding to the commercial strains (I to IV) and their respective indistinguishable
genotypes from indigenous Oenococcus oeni strains from this winery.
These all four autochthonous genotypes were detected in 2006 vintage, year which showed
the greatest strain diversity, two of them occurred in 2008 and one in 2007, with an
important frequency of appearance in some cases (Table 3).

Genotype Isolation stage
Frequency of appearance at each vintage (%)
2006 2007 2008
13 4-5-6 2 - 45
18 3-4-5-6 15 14 3
21 6 2 - -
23 6 2 - -
Table 3. Oenococcus oeni genotypes indistinguishable to commercial patterns; isolation stage (1, must; 2,
tumultuous AF; 3, final AF; 4, initial MLF; 5, tumultuous MLF; 6, final MLF) and frequency (%) of their
appearance (frequency of appearance (%) = n of isolates that presented a specific PFGE pattern
100/total n of isolates per vintage) at each vintage.
Therefore, despite two of these strains had been previously considered as interesting for the
selection of new malolactic starter cultures, the possible identical strain identification with
already marketed strains suggested reject these two indigenous Oenococcus oeni isolates from
a future selection process regardless of their oenological properties as malolactic starter
cultures.
4. Conclusion
This study has been a contribution to a better description of the LAB ecology along the
process of Tempranillo wines winemaking.
The study about the microbial diversity of viable LAB populations showed that the species
diversity was higher at the AF stage where eight different species were identified.
Oenococcus oeni was detected during AF in variable proportions and it became the majority
species during spontaneous MLF.
This work allowed to increase the endogenous strain collection of LAB isolated from
fermenting wines of the Appellation of Origin Rioja what meant a contribution to the
preservation of biodiversity and wine peculiarity of this region and a starting point for
future research.
The analysis of the total LAB populations by culture independent techniques (PCR-DGGE)
showed that the species diversity detected along the winemaking process was higher than
the one found by the study of viable LAB, identifying up to nine different LAB species. The
LAB species variability was also higher at the previous stages to the MLF. Once
spontaneous MLF started this variability was greatly reduced, with Oenococcus oeni and
Leuconostoc mesenteroides as the only detected species.
The results obtained with culture dependent and independent techniques were
complementary so in studies conducted in microbial ecology they both should be used to
achieve a broader view of the studied ecosystem.
PFGE has shown to be a suitable method for strain differentiation, for monitoring individual
strains and determining which strains actually survive and carry out MLF. The results of
Oenococcus oeni typification indicated the high diversity of indigenous Oenococcus oeni
strains responsible for MLF of the wines of this study and the complexity of the ecology
involved in a fermentating wine. The frequency of participation of each genotype varied
from year to year, thus dominant genotypes one year were minority or not present at other
one, which suggested the adaptation of Oenococcus oeni strains to the winery conditions
every year.
Several genotypes could be considered as interesting Oenococcus oeni strains for the selection
of new malolactic starter cultures as individual or mixed strains because, in addition to be
isolated at more than one year in quality wines, they were dominant in most of the MLF
stages at each vintage. The comparison of the patterns from commercial cultures and the
genotypes from indigenous Oenococcus oeni strains showed four indistinguishable
genotypes. The presence of these four genotypes for one to three years, and in some cases
with a high frequency of appearance, demonstrated the significance of this study in order to
exclude these genotypes from a future selection process.

Electrophoresis 152
Author details
Luca Gonzlez-Arenzana, Rosa Lpez, Pilar Santamara and Isabel Lpez-Alfaro
*

ICVV, Instituto de Ciencias de la Vid y del Vino (Gobierno de La Rioja,
Universidad de La Rioja and CSIC) C/ Madre de Dios 51, Logroo (La Rioja), Spain
Acknowledgement
This work was supported by funding and predoctoral grant (B.O.R. 6
th
March, 2009) of the
Government of La Rioja, the I.N.I.A. project RTA2007-00104-00-00 and FEDER of the
European Community and was made possible by the collaborating winery.
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Electrophoresis 156
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Chapter 9
Isoenzyme Analyses Tools
Used Long Time in Forest Science
http://dx.doi.org/10.5772/45756
1. Introduction
1.1. What kind of tool are isoenzymes
A specific group of proteins enzymes, characterized by similar chemical properties are
isoenzymes (Hunter & Markert, 1957, Tanskley & Orton, 1983, Goncharenko, 1988,
Mejnartowicz, 1990, Sukowska, 1995). They are characterized by similar chemical, structural
and catalytic properties almost identical within their functional group. They are
characterized the same function in cell reactions, however they are products of different
genes (described as allozymes).
Their origin is explained by gene duplication of polyploidisation mutation, which can create
kinds of pseudogene or nucleic acid hybrydisation. Different forms of an enzyme that are
coded by variant alleles at the same locus are called allozymes, while isozymes are products
coded by genes located at different loci. Even then, isozymes and allozymes are variants of
the same genes and they possess the same catalytic functions they can be easily identified in
biochemical way. First of all the substitutions of different amino acids are responsible for
their variant electric charge and this can be the way how to identify them by electrophoresis
process. This charge characteristics of isozymes and allozymes is the basis to use them as
molecular markers.
An important feature of this group of proteins is that the presence of both alleles of a gene is
disclosed in a manner independent of each other so cold co-dominant character, so it is
possible to request a degree of heterozygosity within the study population characteristics
(Rider & Taylor, 1980). In practice, this means that there is a simple way to distinguish
heterozygotes from homozygotes. The isoenzyme molecules are proteins. It is important to
define them as the first products of gene translation, so they possess information about their
coding region of DNA. Their heredity of responsible genes is explained by Mendelian

Electrophoresis 158
character. A large number of genes encoding enzymes shows a significant degree of
polymorphism. The process of natural selection is still important factor, because genes are
specialised to different functions.
Long time in forest sciences, the isoenzyme markers were the best tools to analyse genetic
variation of populations despite of the different limits and restrictions of this method.
Nowadays we have more informative tools based on DNA markers such as sequencing,
microsatellites, PCR-RFLP and single genes analysis like SNPs. Most of this DNA based
markers is still developed to make them more suitable in analysis of plant organisms. The
level of complication of plant genome is higher comparing to animal. Average of size of
Pinus genome is about 30 billion base pair (Grotkopp et al., 2004). We have up to now many
possibilities to analyse the polymorphism of DNA fragments of plant material but not the
particular genes. So, the isoenzyme markers represent still one of the best markers close to
DNA level. It is possible to assess the variation of individuals at different level: within
species, within population, and among populations within species. It is worth to add they
are quick and cheap marker systems and good alternative to assay and identify level of
genetic variation as pilot study of populations (Bakshi & Knnert, 2011) as well as
conservation biology activities e.g. gene bank enables choosing of proper sample for long
time conservation (Bednorz et al., 2006) or as well as in quantifying mating system analysis
(Mrazikowa & Paule, 1990).
The proteins of enzyme are consisted of polypeptides and are molecules characterised the
specific conformation. The quaternary structure of particular enzyme forms may be different
in relation to combination of peptides involved to build their molecules. We can observed
(Figure 1) monomeric enzymes with two the same allozymes, dimeric forms with three
allozymes and tetrameric proteins with fives allozymes. We can sometimes observe on gel
after electrophoresis bands of some intermediate mobility reaction.
The isoenzyme analysis method enables the assessment of variability of isoenzymes in
different types of tissues: young leaves, buds, pollen and seeds. There is possibilities to
analyse diploid as well as haploid tissue. Especially it is important, when we study
coniferous species genetic variation, then it is great tool to analyse mating system of trees,
because of possibilities of using haploid tissue of mother trees and diploid tissue of
embryos. The analysis requires very small amounts of plant material and is a very sensitive
method. Simultaneously, it is possible to identify many samples/or individuals. The great
advantage of this method is low cost of chemicals used to performed the studies.
1.2. Advantages of isoenzymes and week points of the utilisation
The using of isoenzyme electrophoresis method is useful tool especially in assessment of
gene frequency of specific genes, determining of genetic similarities and genetic distances
between the two objects e.g.:
- Good tools to support conservation and management of forest trees genetic resources
To characterize forest tree stands genetic structure

Isoenzyme Analyses Tools Used Long Time in Forest Science 159
To asses initial gene pole
To present selection processes of forest stands and to maintain rich of natural
diversity of stands
- Genetic characteristics of different forest tree species reproductive material:
Mother stands and progeny stands gene flow analysis
Seed orchards and progeny plantations mating system
The representation of populations selected for preservation in gene banks or in situ
or ex situ measures
- Solving of problems from seed stands management point of view:
clonal/pedigree identification/selection process
pollen contamination especially important in management of artificial tree stands
like forest seed plantations
patterns of gene flow and mating system in natural and artificial stands

Enzyme/Gene Type Homozygote Heterozygote Homozygote
Monomeric

Dimeric

Tetrameric

Figure 1. The graphic illustration of bands of proteins on the gel after electrophoresis process when we
study various types of isoenzyme proteins.
1.3. Methodical problems
The isoenzyme studies are suitable only when their heritability is explained by Mendelian
character often the same enzyme systems are not suitable when we study different species.
The enzyme loci are not randomly distributed over the genome, so they are not
representative for total genome variation (Hubby & Lewontin, 1966). We have problems to
compare analysed data with reference studies when the number of analysed enzyme system
is different. At the first studies some enzyme systems were described by different numbers
enzyme gene loci systems e.g. peroxidases or number of loci controlling analysed enzyme.
For example GOT (transaminase glutamine oxylo-acetate) was assumed to be coded by 2 or

Electrophoresis 160
3 or 5 gene loci for Scots pine (Mller-Starck et al., 1992). Some alleles are not identify as the
bands on the gel (null alleles). It is not possible to use particular enzyme markers when
linkage disequilibrium is occurred for analysed loci, so it obvious to verify the genetic
control system and inheritance of chosen enzyme markers.
Still seems to opened the question are allozyme neutral or adaptive?
Some researchers showed the results studies only for polymorphic enzyme systems without
any information about monomorphic ones, so the genetic variation was described as higher
than it was real.
The obtained information about isoenzyme variation is not a representative sample of the
total variability of the analyzed species, which illustrates the genetic variability within the
ranges of their occurrence, due to too small amount of tested samples. Evaluation of
variation by this method applies only to a very small part of the genome.
2. Methods
2.1. The idea of isoenzyme electrophoresis
The charge characteristics of isoenzymes enables to use them as genetic markers, which can
be distingished. First of all the substitutions of different amino acids are responsible for their
variant electric charge and this can be the way how to identify them by electrophoresis
process. The identification of proteins molecule characterised a specific electric charge can
be dissolved in a buffering solution, after homogenisation of plant or animal tissue. The
non-denatured proteins are separated on the carrier gel and they migrate under the
influence of an applied electric field. The molecules of enzyme proteins can change
conformation shape and it will be followed by modification of net charge. This changes of
molecules charge can be detect using electrophoresis method.
The rate of migration of molecules in the gel and segregation of bands of enzymes in the gel
is the result of interaction of: the applied electric field, pH of gel and electrode buffers
(Concle et al., 1982). After completion of electrophoresis on gel plate, the proteins are
stained by using the appropriate staining reactions.
2.2. Migration process of proteins in different media and their visualization
possibilities
The carrier of proteins in electrophoresis may be different types of substances:
polyacrylamide gels and potato starch gels. The second one were historically used, but the
great advantage of them still is the possibility of cutting them to obtain slices, which can be
analise to identify different protein markers using the same gel and tissue material, because
all the proteins are active in the gel.
Proteins separated in the gel during electrophoresis can be detected by staining or UV
analysis e.g. fluorescent esterase enzyme. Methods of detection of enzymes are relatively

well developed and reported in many publications (Concle et al., 1982, Goncharenko, 1988,
Wang & Szmidt, 1989, Liengsiri et al., 1990). The visualisation of enzymes is possible by
precipitation of soluble indicators like tetrazolium salts using cofactors of NAD or NADP to
transfer them into reduced insoluble forms (Figure 2). It is important to put attention that
the staining reactions require the active forms of enzymes and they simulate work of
proteins in functional tissues of organisms. Most of reactions are performed in the
temperature 37 C. During relatively short time one day of laboratory work we have result
and evaluation of the analysis for about 50 samples e.g. individuals using 10 or more
enzyme systems represented by one or more loci each of them. It is important as well that
the staining process does not requires complicated laboratory equipment. The resolving of
proteins during electrophoresis can be performed in horizontal or vertical chambers, but the
crucial is to protect the activity of enzymes by providing of cooling system. The power of
electric field applied for migration of proteins is often up to 280V, so the heating of gel is
observed and have to be reduced.

Figure 2. Menadione reductase electrophoretic pattern in European beech. We can observed The
monomeric enzyme form with two the same allozymes (homozygote form), dimeric form with three
allozymes (heterozygote form) and tetrameric proteins with fives allozymes (heterozygote form). Photo
M. Sulkowska
2.3. Genetic differentiation evaluation process
2.3.1. Analysed parameters to describe population genetic variation and differentiation
The basic parameters evaluated to measure population genetic variation and differentiation
are usually as it follows: number of alleles per locus, heterozygosity (H), percentage of
polymorphic loci, genetic distance (GST). The terms of heterozygosity, diversity and
differentiation are explained in different way by various researchers. Most of people used to
work with genetic parameters elaborated by Nei (1973, 1978), when we consider
heterozygosity observed or expected, what is estimated as well as a measure of genetic
diversity heterozygosity observed (Ho), heterozygosity expected (He or D gene
diversity). The value of this parameter is calculated as values from zero (no heterozygosity)
up to nearly 1.0 (when we observe a large number of nearly equally frequent alleles).
Instead of average number of alleles per locus more precise used to be measure of effective
number of alleles per locus (ne) Crow & Kimura (1970).

Electrophoresis 162

2
1
1
n
i
He pi
(1)

2
1
1
e
n
i
n
pi
(2)
pi frequency of n allele occurrence in population
Heterozygosity is often one of the most important "parameters" when we describe the
genetic data. Using this measure we explain the general trends in the structure of
analysed populations even is their history and future genetic structure is concerned.
Low values of heterozygosity is influenced by small population size and processes of
genetic drift e.g. bottlenecks effect. A lot of heterozygotes in population is equal high
genetic variability and the opposite. When we compare the level of the observed and
expected heterozygosity in balanced populations concerning random and open mating
system it means under Hardy-Weinberg equilibrium and heterozygosity is higher than
expected we can expect flow of alien pollen outside of population. If the observed
heterozygosity is lower than expected we can assume that some inbreeding processes may
occur in the population.
The interpopulational variation is described very often as GST (Nei, 1973) and it is used as
equivalent of FST statistics (1969, 1978) and it enables to asses for each population the
distance from other populations.

T S
ST
T
H H
G
H
(3)
HT - heterozygosity interpopulational
HS - heterozygosity intrapopulational

IT T I T
F H H H (4)
FST, cold as fixation index is the measure of proportion of the total genetic variance within
subpopulations in relative to the total genetic variance. The values of this parameter can
range from 0 to 1. High FST implies a considerable degree of differentiation among
populations.
FIS (inbreeding coefficient) is the proportion of the variance in the subpopulation contained
in an individual. High FIS implies a considerable degree of inbreeding. Values can range
from -1 (outbred) to +1 (inbred).

ST T S T
F H H H (5)

IS S I S
F H H H (6)

HT - heterozygosity total for population
HS - heterozygosity within subpopulation
HI - heterozygosity of individual
2.3.2. Software
One of the oldest programs enabling computing of isoenzyme analysis data is BIOSYS-1.
This program was elaborated to help biochemical population geneticists to describe the
analysis of electrophoretically detectable allelic variation. It can be utilised to study allele
frequencies and genetic variability measures, to test for deviation of genotype
frequencies from Hardy-Weinberg, expectations, to calculate F-statistics, to perform
heterogeneity of chi-square analysis, to calculate a variety of similarity and distance
coefficients, and to construct dendrograms using among others cluster analysis
procedures. The program, documentation, and test data are available from the authors
(Swofford & Selander, 1981).
Another one interesting software enabling as well analysing of DNA data markers is
POPGENE (Yeh & Boyle, 1997). The current version of POPGENE is designed specifically
for the analysis of co-dominant and dominant markers using haploid and diploid data. It
performs most types of data analysis encountered in population genetics and related fields.
It can be used to compute summary statistics, including: allele frequency: estimates gene
frequencies at each locus from raw data, effective number of alleles per locus, percentage of
all loci that are polymorphic, observed homozygosity, expected homozygosity, Shannon
Index, gene diversity Neis (1973), F-Statistics, Gene Flow: estimates gene flow from the
estimate of GST or FST and many others parameters.
POPGENE is a good tool in analysing and simulations studies of population genetics,
including: Hardy-Weinberg Equilibrium, multiple allele and loci inheritance, natural
selection, genetic drift, migration, mutation and inbreeding.
Applied procedure of isoenzyme analysis is consisted of the following steps:
Sample preparation (collecting of samples, homogenization and extraction of proteins
from the tissue)
Preparation of gels and running buffers requirements regarding analysed enzyme
systems
Development of isoenzyme electrophoresis
Detection and staining of proteins
Computing of the obtained data base with utilisation of proper software
3. Results
3.1. Genetic variation characteristics
The investigations of beech variation and differentiation in Europe showed (Gmry et al.,
2003, Sukowska et al., 2012). The aim of this study was the assessment of genetic diversity

Electrophoresis 164
and differentiation patterns of European beech (Fagus sylvatica L.) populations within its
natural range in Poland and to compare them to those in other neighbouring European
countries including Slovakia, the Czech Republic, Ukraine, and even Romania, which was
reported previously (Paule et al. 1995). These stands cover 5.2% of the forest area in Poland,
and form the predominant forest tree communities throughout the Carpathians and Sudety
Mountains, and the moraine landscape of the Pomeranian Lake District. Varying
environmental conditions have resulted in a great number of ecotypes and populations
which are characterised by various ecological requirements. Poland represents the
northeastern limit of the beechs natural distribution. Genetic diversity and differentiation
was assessed using allozyme gene markers employing 9 enzyme systems: glutamate-
oxaloacetate transaminase (GOT - EC 2.6.1.1), leucine aminopeptidase (LAP- EC 3.4.11.1),
isocitrate dehydrogenase (IDH - EC 1.1.1.42), malate dehydrogenase (MDH - EC 1.1.1.37),
menadione reductase (MNR - EC 1.6.99.2), phosphoglucomutase (PGM - EC 2.7.5.1),
phosphoglucose isomerase (PGI - EC 5.3.1.9), peroxydase (PX - EC 1.11.17) and shikimate
dehydrogenase (SKDH - EC 1.1.1.25). The data revealed: high genetic diversity of beech,
similar like in other neighboring European populations, slight decrease of average number
of alleles per locus and level of differentiation towards the North of the natural range limit,
which generally confirm the migration paths after glaciations but it is not the basis to
distinguish geographic regions.
The population differentiation of beech provenances of selected seed stands and their
progenies for chosen genetic parameters and on the basis of soil characteristics of the
habitats were studied (Sulkowska et al., 2008, Sulkowska, 2010). Beech populations
occurring toward the northeast of the natural range were characterised by a decreasing the
average number of alleles per locus and percentage of polymorphic loci. The highest genetic
differentiation was found in the East Carpathians.
3.2. Ecotype variation characteristics
3.2.1. Geographic trends with an example of coniferous species - Scots Pine (Pinus
sylvestris L.)
Allozyme differentiation in chosen European populations of Scots Pine (Pinus sylvestris L.)
were studied in 17 populations from North and East-Central Europe (Prus-Gowacki et al.,
1993). The samples were collected from provenance trial in Lubie (Poland). The
provenances from Scandinavia, northern Poland, Netherlands and Belgium were more
heterozygotic, more polymorphic and characterised higher number of alleles per locus. The
source of seeds used to establish the provenance trial was unknown as far their
autochthonous or introduced origin is concerned, but the results indicate a degree of
coincident agreement with geographical distribution of stands. This effect seems to be
blubbered by human activity (uncontrolled seed transfer).
The studies of 11 enzyme systems concerned on the genetic variation of Pinus sylvestris from
Spain in relation to other European populations revealed genetic dissimilarity of
populations from this region. The differences were observed as far as slightly higher

number of alleles per locus, but lower heterozygosity level in populations from Spain (Prus-
Gowacki & Stephan, 1994).
In Poland, the isoenzyme studies of 5 systems variability of 8 populations revealed existence
of two groups of populations North and South groups (Krzakowa, 1979). There was a high
variability within all analysed loci. The importance of existence of this groups was
undertaken as well by field studies and low regulations (Dz.U. 04, nr 67, poz. 621, 2004,
Matras, 2005).
3.2.2. Site plasticity with an example of deciduous species European beech (Fagus
sylvatica L.
Present genetic structure of beech population in Europe was formed many different factors
not only environmental and genetic but also anthropogenic. Different environmental
condition resulted in great number of ecotypes and populations, that characterized various
ecological requirements (Dzwonko, 1990, Giertych, 1990). Very important factors affected
the gene pool were glacial epoch and the location of beech refugia, for postglacial migration
paths of species (Szafer, 1935, Huntley & Birks, 1983, Ralska-Jasiewiczowa, 1983, Hazler et
al., 1997). One of the first studies on genetic variability of isoenzymes of European beech -
Fagus sylvatica L. were conducted in France. The study of genes encoding peroxidase system.
There was a relationship between the frequencies of particular alleles encoding these
enzymes, and analyzed population, geographical and environmental factors (Cugen et al.,
1985). Genetic variation of beech - Fagus sylvatica L., was also analyzed on a wider scale
within the six enzyme loci, 130 population for southern and western Europe. Observed
correlation between the frequency of alleles of genes encoding peroxidase and climate
(Comps, et al., 1990).
Some genotypes are eliminated during natural selection process, when they are not efficient
in the environment, it is shown using isoenzyme markers (Mller-Starck, 1985). In most case
populations characterised higher level of genetic variation seems to be more tolerant to
harmful environmental factors (Starke et al., 1996).
In a continuous process of verifying the adaptation of individuals, which occurs in nature, it
is contributed to both human and natural selection. This was proven among other by
differences in the genetic structure of two provenances of beech German and Romanian,
grown in a greenhouse and natural conditions (Kim, 1985). It was found that beech
seedlings with Lap-A2 allele always were characterised a higher survival. Homozygous for
the allele Lap-A2 survived better in a greenhouse, while heterozygotes were characterized by
higher vitality in the natural environment.
3.3. Using of isoenzyme markers in coniferous seed orchard research
Isoenzyme markers are very important tool concerning genetic parental or progeny identity
(Wheeler & Jech, 1992). The estimation of gene flow in mating system of seed orchards is
crucial for proper use of seeds to asses and if possible avoid pollen contamination outside of

Electrophoresis 166
the stands and seed orchard genetic efficiency. It plays important role for gene conservation of
the stands and the improvement in forest tree selection (Concle, 1972, Rudin & Lingren, 1977).
The investigations of 122 trees in seed stand in Sweden were conducted for adult trees,
embryos of seeds and progeny of the stand (Yazdani et al., 1985). The analyses revealed
significant variation among different groups of studied objects at 5 enzyme loci. Genetic
frequencies of alleles were close to Hardy-Weinberg equilibrium, but the deviations were
found for embryos.
In Poland, also featured on the study of inheritance of a certain enzyme - GOT
(transaminase glutamino-oxylo-acetate) for selected two homozygous plus trees, under the
terms of this gene, and the pollination of these trees by surrounding neighbors. The study
provided evidence of contamination at least 40% of the seeds with pollen outside the stands,
which indicates a high out-crossing rate for pine (Krzakowa, 1980).
In the seed orchard in Slovakia, the mating system of trees was analysed on the basis of five
enzymes studies inheritance (Mrazikowa & Paule, 1990). The study was conducted
simultaneously for macrogametophytes of embryos and seeds. The degree of foreign seed
pollination with pollen from outside the plantation was shown.
4. Discussion
The isoenzyme markers are important tool in when we assay gene variability of forest trees.
The complicity of genome of this organism makes it impossible in most cases to obtain
information about particular genes. Nowadays, molecular markers are powerful tools,
which enables to study genetic variation of the organism, but we are usually able to work
with specific fragments DNA, not to assay the genes. The methods biochemical and
molecular should be taken into account in case of genetic variability analyses of forest trees
as complementary tools. It can be revealed by studies of migration paths of European beech
using both types of markers (Magri et. al., 2006), where existence of one common refugium
of the species was proved for most part of Central and West Europe.
Single genes are responsible to control proteins of particular features genetic traits
(Bergmann, 1991). They are not responsible to control the complex of morphological traits,
physiological and adaptability of individuals as reaction of needs of environment, that are
crucial for surviving of rooted in one site plants over their whole life.
Ecotype variation of forest tree species can be classified as relation to their geographic range
(Mller-Starck et al., 1992):
Species characterised large geographic range like Scots pine, Norway spruce and
European beech and little genetic differentiation among populations within regions
derived from the same refugia, but greater comparing various refugia
Species with large geographic ranges, but with many subspecies like Pine species
Pinus nigra, Pinus halepensis with small interpopulational variation within subspecies,
but differentiation among subspecies

Species characterised small geographically ranges like fir but with great
interpopulational differentiation and medium level of intrapopulational genetic
variation endemic species
Species with extremely small geographic range like Siberian dwarf pine characterised
relatively high interpopulational differentiation relic species.
Using of isoenzyme studies as one of DNA responsible marker to solve the problems of
reproductive and economic value of seed orchard stands is very crucial (Wheeler & Jech,
1992, Krzakowa, 1980).
A especially interesting seems to be using of isoenzyme analysis in estimation of gene flow
in natural and artificial populations of forest trees, when the genetic values of artificial
management stands is taking into account (Savolainen & Yazdani, 1991, Skrpa, 1994). The
reported by authors genetic diversities as well the level of outcrossing rates estimated on the
basis of allozymes differentiation were comparable in natural and artificial stands.
However, that was underlined the importance of possible changes in the important
quantitative traits not revealed by neutral enzyme markers. It was undertaken (Skrpa,
1994) the meaning of many aspects important for proper quality production of forest
reproductive material e.g.: seed collection procedure, seedlings sylviculture management,
progeny testing.
Low costs of the analyses are the reason why isoenzyme markers are good tools in pilot
studies of gene pool, as well as conservation biology activities e.g. gene bank enables
choosing of proper sample for long time conservation. Using of isoenzyme analysis was the
step to assess the genetic variation for Sorbus torminalis L. Krantz. natural populations in
Poland (Bednorz et al. 2006), what was the basis to establish in the progeny stands in next
step, as ex-situ measures for the species.
The present selection processes of forest stands should to maintain richness of natural
diversity and do not allowed to use the trees with high economic as in of case natural
populations of Pinus wallichiana A.B. Jacks (Blue Pine) in India (Bakshi & Knnert 2011).
5. Conclusions
The isoenzyme molecules are proteins, which are defined as first product of the first
products coding region of DNA activity. Their heredity is known, when it is explained by
Mendelian character of segregation it is possible to utilise as genetic markers.
Application of isoenzyme electrophoresis method is useful tool in forest trees genetic
diversity assessment, in spite of their long history of their utilization, elaboration of DNA
analysis markers as well as known limits of their possibilities to apply.
Especially the possibilities to use them on wild scale, because of low costs of analysis makes
them important tools in:
Genetic characteristics of different forest reproductive material of natural and artificial
stands e.g.

Electrophoresis 168
- Mother stands and progeny stands gene flow
- Seed orchards gene flow
- Gene banks representation of populations assessment
Solving of particular problems in case of:
- clonal/pedigree identification/selection process
- pollen contamination
- mating system
- patterns of gene flow
Good tools to support conservation and management of forest genetic resources e.g. to
support following activities:
- identification of migration path of species from postglacial refugia
- selection and protection of ecotypes
- to asses initial gene pole for needs of effective gene conservation measures
- to present selection processes of forest stands to maintain rich natural diversity.
Author details
Forest Research Institute, Sekocin Stary, Raszyn, Poland
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Wheeler, N. C. and Jech, K. S. (1992). The use of electrophoretic markers in seed orchard
research. New Forests 6: 31 l-328.
Wright, S. (1969). Evolution and the Genetics of Populations, Vol. II. The Theory of Gene
Frequencies. University of Chicago Press, Chicago.
Wright, S. (1978). Evolution and the Genetics of Populations, Vol. IV. Variability Within and
Among Natural Populations. University of Chicago Press, Chicago.
Yazdani, R., Muona, O., Rudin, D., Szmidt, A.E. (1985). Genetic structure of Pinus
sylvestris L. seed-tree stand and naturally regenerated understory. Forest Science, 31,
430436.

Electrophoresis 172
Yeh, F.C., Boyle, T.B.J. (1997). Population genetic analysis of co-dominant and dominant
markers and quantitative traits Belgian Journal of Botany 129: 157.
Chapter 10
Seminal Plasma Proteins as
Potential Markers of Relative Fertility
in Zebu Bulls (Bos taurus indicus)
Marcelo G.M. Chacur
http://dx.doi.org/10.5772/45758
1. Introduction
Progress has been made in developing reliable indicators of ejaculate quality that allow
exclusion of low quality ejaculates for use in artificial insemination (AI). Physical semen
characteristics and sperm morphology measurements are not always indicative of fertility and
reproductive performance in animals, and accurate and predictive genetic and protein
markers are still needed (Foxcroft et al., 2008). Two-dimensional polyacrylamide gel
electrophoresis (2-D PAGE) represents a valuable tool for the separation and characterization
of proteins from complex biological samples (Killian, 1992; Killian et al., 1993).
In the case of dairy sires, data sets are avaliable to assess fertility of individual males based
on insemination of large numbers of cows using semen that has been frozen by
standardizing procedures. This information has been used to demonstrate that fertility
associated proteins exist in bull seminal plasma (Killian et al., 1993). Seminal plasma is
composed of secretions from the male accessory sex glands and epididymis, which contains
many organic and inorganic components that have effects on sperm quality (Foxcroft et al.,
2008). The proteins secreted into seminal plasma may play an important role during sperm
capacitation and fertilization (Rodriguez-Martinez et al., 1998), and may also serve to protect
sperm from damage or to maintain their longevity.
Specific proteins in seminal plasma have been identified as potential markers of male
fertility or infertility in the human (Martinez-Heredia et al., 2008; Yamakawa et al., 2007).
Comprehensive proteomic analyses have been conducted in the bull (Moura et al., 2006a;
Moura et al., 2006b; Chacur et al., 2010a; 2011a). The literature on the effects of seasons on
the semen characteristics and upon seminal plasma proteins in Nellore (Bos taurus indicus)
cattle under natural conditions in Brazil has already been recently studied in Brazil (Chacur
et al., 2006b; 2007; 2010b; 2011a).

Electrophoresis 174
There is evidence revealing that seminal plasma prevents premature capacitation of sperm
(Eng & Oliphant, 1978) and protects sperm from peroxidative damage (Jones et al., 1979;
Schnech et al., 1996). Its well known that low temperatures alter the function of
spermatozoa (Watson, 1995). Cold shock results in the destabilization of sperm membranes
and impairment of sperm function, and it is also well known that animal spermatozoa are
sensitive to cold-shock stress as the bull (Schnech et al., 1996).
Seminal plasma has also been shown to have deleterious effect on bovine sperm during
semen storage at ambient temperatures and a damaging effect during semen cooling and
freezing. Recent studies have shown that proteins from bovine seminal plasma (BSP) may
modulate sperm properties. Two proteins (26kDa, pI 6.2; 55kDa, pI 4.5) predominate in
higher-fertility bulls and two proteins (16kDa, pI 4.1; 16 kDa, pI 6.7) predominate in
lower-fertility bulls. The major protein fraction of bovine seminal plasma is represented
by three acidic proteins, designated as BSP-A1/-A2, BSP-A3 and BSP-30kDa (collectively
called BSP proteins). These proteins are secretory products of the seminal vesicles and
ampullae, and their biochemical characteristics have been well-described (Moura et al.,
2006a; 2006b).
Although many autors believe that the seminal plasma proteins may function to stabilize the
sperm against premature capacitation and spontaneous acrosome reaction. Significantly,
these proteins may also protect the sperm from cooling-induced damage, such as
cryocapacitation. The establishment of 2-D PAGE reference map could represent a usefull
tool for the study of the still poorly understood nature and functions of the seminal plasma
proteins, for the identification of previously unknown proteins, and for the comparison of
seminal plasma protein composition between males of differing fertility. Many autors
believe that the bull seminal plasma contains fertility associated protein markers.
Comparison of individual 2-D PAGE maps with the reference map could provide a useful
key to relate protein pattern changes to some physiopathological events influencing the
reproductive sphere (Mortarino et al., 1998).
The present chapter describes the use of electrophoresis in animal reproduction:
Electrophoresis and fertility in animals; Approaches to use of electrophoresis; Effect of
seasonal changes in seminal plasma proteins of zebu bulls (Bos taurus indicus).
2. Electrophoresis and fertility in animals
The development of genetic markers to identify bulls of high breeding value represents one
of the ways of genetic gain achievement in dairy farming. Several studies have been
performed in an attempt to uncover the relationship between semen quality and fertility
(Linford et al., 1976; Saacke et al., 1994). Thus, sperm morphology and motility, the number
of sperm of sperm per insemination, percentage of acrosome-reacted sperm, and in vitro
fertilization have been extensively evaluated as an indication of sperm ability to fertilize an
egg. Evidence suggests that seminal plasma, which is a complex mixture of secretions
originating from the testis, epididymis, and accessory glands, contains factors that modulate
the fertilizing ability of sperm (Amann & Griel, 1974; Henault et al., 1995).

Seminal Plasma Proteins as Potential Markers of Relative Fertility in Zebu Bulls (Bos taurus indicus) 175
Recent studies have shown that proteins from bovine seminal plasma (BSP) may modulate
sperm properties (Killian, 1992; Bellin et al., 1994). Two proteins (26kDa, pI 6.2; 55kDa, pI
4.5) predominate in higher-fertility bulls and two proteins (16kDa, pI 4.1; 16 kDa, pI 6.7)
predominate in lower-fertility bulls (Killian et al., 1993).
2.1. Electrophoresis and seminal plasma
Secretions from the accessory sex glans are mixed with sperm and ejaculation and
contribute to the majority of semen volume and components. Some accessory sex glands
proteins are known to bind to the spermatozoa membrane and affect its function and
properties (Yanagimachi, 1994).
Mammalian seminal plasma is constituted by secretions of the male accessory glans in
which the spermatozoa are suspended in semen. Seminal plasma is an extremely complex
fluid containing a wide variety of both organic and inorganic chemical constituents, among
which only proteins present high molecular masses. The protein composition varies from
species to species but in all the cases investigated so far these components have important
effects on sperm function (Killian et al., 1993). Two-dimensional polyacrylamide gel
electrophoresis (2-D PAGE) represents a valuable tool for the separation and
characterization of proteins from complex biological samples. Seminal plasma, a
physiological secretion from multiple glands of the male reproductive tract, is the natural
medium for maturation of the spermatozoa through hormonal, enzymatic, and surface-
modifiyng events (Killian et al., 1993).
The establishment of a 2-D PAGE reference map could represent a useful tool for the
study of the still poorly understood nature and function of the seminal plasma proteins,
for the identification of previously unknown proteins, and for the comparison of seminal
plasma protein composition between males of differing fertility. In particular, it seems
likely that bull seminal plasma contains fertility-associated protein markers (Killian et al.,
1993).
3. The use of the SDS-PAGE and 2-D PAGE
3.1. Proteins of the male accessory glands and fertility
In the case of dairy sires, data sets are avaliable to assess fertility of individual males based
on insemination of large numbers of cows using semen that has been frozen by
standardizing procedures. This information has been used to demonstrate that fertility
associated proteins exist in bull seminal plasma (Killian et al., 1993).
The ability to analyze the components secreted exclusively from the accessory sex glands
has provided unique information about proyeins they secret and that are correlated with
fertility indexes. Relating expression levels of specific proteins to fertility phenotype should
serve as a sound foundation to evaluate their role in sperm function and fertilization (Moura
et al., 2006b).

Electrophoresis 176
3.1.1. Heparin binding proteins (HBPs)
Heparin, a commercially available, sulfated glycosaminoglycan, induces capacitation/
acrosome reactions in sperm from bulls (Lenz et al., 1983). Sperm from high-fertility bulls
have a greater frequency of acrosome reactions in response to heparin-like material (Lenz et
al., 1988). Heparin-binding proteins (HBPs) are produced by the male accessory glands,
secreted into seminal fluid (Nass et al., 1990). Fertility for Group 1 (HBP-B5) in sperm
membranes but undetectable HBP-B5 in seminal fluid) was 82% of 1,692 cows. Group 2
(HBP-B5 detectable in seminal fluid as well as in sperm membranes) had 67% of 919 cows
pregnant. Group 3 (detectable HBP-B5 in the seminal fluid and undetectable HBP-B5 in the
sperm membranes) and Group 4 (undetectable HBP-B5 in the seminal fluid and sperm
membrane) had 63% of 747 and 1,208 cows pregnant, respectively (Bellin et al., 1994). These
trials indicated that grouping bulls according to the presence or absence of the greatest
affinity herarin-binding protein (HBP-B5) on sperm membranes and in seminal fluid was an
effective means of identifying fertility potential. Thus, understanding the protein
composition of sperm membranes can be directly applicable to field situations and result in
a more efficient production of calves (Bellin et al., 1994).
Other studies have shown that accessory glands produced and secreted HBP into seminal
fluid , and HBP bound to sperm at ejaculation (Miller et al., 1990; Nass et al., 1990). Miller et al.
(1990) reported that HBP constituted 28% of the protein component of seminal fluid or
approximately 19.6 mg HBP/mL of ejaculate, and HBP-B5 constituted 6% of the total HBP in
fluid from vasectomized bulls. The average concentration of total HBP represents 19.2 mg and
0.14 mg per mL of ejaculate in bovine seminal plasma and sperm membrane, respectively
(Bellin et al., 1994). Although all HBPs may bind to sperm surfaces (Bellin et al., 1996). The
concentration of the 30-kDa HBP, namely fertility associated antigen (FAA), on bovine sperm
has been paired with a greater fertility potential (Bellin et al., 1998). FAA represents 0.8% of
total HBP and has been identified as a deoxyribonuclease I-like protein (McCauley et al., 1999).
Studies have shown that these BSP proteins facilitate capacitation by promoting cholesterol
efflux from sperm membranes (Thrien et al., 1998). Because sperm membrane cholesterol
has an important role in modulating membrane bilayer fluidity and stability (Bloch, 1985;
Yeagle, 1985), its efflux may perturb membrane structure and thereby lead to capacitation
(Davis, 1981; Ehrenwald et al., 1988). In the context of sperm cryopreservation may lead to a
decrease in sperm resistance to cold shock (Darin-Bennett & White, 1977; White, 1993). Thus,
changes induced by the BSP proteins in the sperm membrane may have influence on sperm
fertilizing ability and the success of the cryopreservation process (Nauc & Manjunath, 2000).
The characterization of seminal plasma proteins including osteopontin (OPN) 55 kDa and
the study of their binding proteins (HBPs) will be the first step in understanding their role in
the fertilization and identification of HBPs would provide information that could improve
the knowledge of this aspect of reproductive physiology in Nellore bulls (Fernandes et al.,
2008). In Nellore bulls (HBPs) bands with molecular weights ranging from 15 to 63 kDa
were observed: 15 kDa, 17 kDa, 22 kDa, 25 kDa, 39 kDa, 53 kDa, 58 kDa and 63 kDa
(Fernandes et al., 2008).

3.1.2. Spermadesin
In dairy cattle the average intensity of spermadesin (14 kDa) was higher in bulls of low
fertility. The most basic isoform of accessory sex gland fluid is equivalent to the one
originally found as an antifertility factor in the seminal plasma (Moura et al., 2006b). Z13 is a
seminal plasma protein made up of two disulfide-linked 13-kDa subunits. The data indicate
that the protein is a dimer of 26 kDa in native conditions and a monomer of 13 kDa in the
presence of reductants. Therefore it antifertility peptide reported by can be suggested that
Z13 presents at least one intermolecular S-S bridge (Tedeschi et al., 2000).
The intensity of apermadhesin Z13 in AGF showed an inverse relationship with fertility
(Moura et al., 2006b). The low-molecular-weight antifertility peptide reported by Killian et
al. (1993) in the seminal plasma of Holstein bulls was also identified as spermadhesin Z13.
The isoform originally described in the seminal plasma by those authors appears to be more
basic (pI 6.7) than the ones found in the AGF as antifertility factors pI 6.5 and 6.3 (Moura et
al., 2006b).
The 2-D PAGE reference map of bull seminal plasma proteins provides information about
the presence, in this particular fluid, of polypeptides of specifical biological significance
(Mortarino et al., 1998). The PDC-109 represents more than 30% of the total protein
contained in bull seminal plasma (Manjunath & Sairam, 1987). Comparison of individual 2-
D PAGE maps with the reference map could provide a useful key to relate protein pattern
changes to some physiopatological events influencing the reproductive sphere (Mortarino et
al., 1998).
Spermadhesin Z13 is a peptide yhat displays 50% and 43% homology with the acidic
seminal fluis protein and seminal plasma motility inhibitor (SPMI), respectively (Tedeschi et
al., 2000). The former has positive effects on bovine sperm in vitro when as average
concentratons, but it can inhibit both sperm motility and mitochondrial activity when at
high levels (Schoneck et al., 1996).
3.1.3. Family of proteins (BSP proteins)
Bovine seminal plasma contains a family of proteins designated BSP-A1/-A2, BSP-A3, and
BSP-30-kDa (collectively called BSP proteins). These proteins are secretory products of
seminal vesicles that are acquired by sperm at ejaculation, modifying the sperm membrane
by inducing cholesterol efflux. Because cholesterol efflux is time and concentration
dependent, continuous exposure to seminal plasma that contains BSP proteins may be
detrimental to the sperm membrane, which may adversely affect the ability of sperm to be
preserved (Manjunath et al., 2002). These proteins coat the surface of the spermatozoa after
ejaculation and are believed to play an important role in membrane modifications occurring
during capacitation. Isoforms of each BSP protein were found when purified iodinated
proteins analysed by 2D-PAGE. BSP-A1was found at a M(r) of 16.5 kDa and in the range of
pI of 4.7-5.0; BSP-A2 at 16 kDa and at a pI of 4.9-5.2; BSP-A3 at 14 kDa and a pI of 4.8-5.2,
and BSP-30-kDa at 28 kDa and at a pI of 3.9-4.6 (Desnoyers et al., 1994). BSP protein are

Electrophoresis 178
acidic and have several isoforms. Furthermore, they heterogeneity of BSP-30-kDa is mainly
due to its sialic acid content (Desnoyers et al., 1994).
The concentration of BSP-A1/-A2 was much higher compared with other seminal plasma
proteins, and this protein alone represented a average of 38% of the total protein fraction,
whereas BSP-A3 and BSP-30 kDa represented 3% to 4% of the total protein fraction (Nauc et
al., 2000). The determination of BSP protein content on sperm surface may be an index of
individual bull fertilizing ability or post-thaw status of sperm membranes (Nauc et al.,
2000).
3.1.4. Osteopontin (OPN)
Osteopontin (OPN) is an acidic glycoprotein of about 41.5 kDa that has been isolated from
rat, human and bovine bone. It is rich in aspartic acid, glutamic acid and serine and contains
about 30 monosaccharides, including 10 sialic acids (Butler, 1989). The 55 kDa protein,
shown to be more prevalent in higher-fertility males, was determined to be osteopontin
(OPN) (Cancel et al., 1997). Results of immunofluorescence analyses of the male
reproductive tract paralleled results for tissue extracts and fluids, indicating that bovine
OPN is secreted by the ampulla and seminal vesicle. Tissue sections of the testis, epididymis
(caput, corpus, cauda), vas deferens, prostate and bulbourethral gland were negative when
reacted with antibodies against bovine seminal plasma OPN (Cancel et al., 1999).
Brown et al. (1992) studied the expression of OPN in normal adult human tissues. They
showed that OPN was present on the luminal surfaces of epithelial cells of the
gastrointestinal tract, gall bladder, pancreas, urinary and reproductive tracts, lung, bronchi,
mammary and salivary glands, and sweat ducts. In general Brown et al. (1992) found that
OPN accumulated on surfaces of epithelia bordering the luminal compartment.
Bulls with the highest fertility scores had 2.3 times more of a 55 kDa osteopontin than bulls
with above-average fertility and at least 4 times more than bulls with below average (Moura
et al., 2006b). A secreted form of phospholipase A2 (PLA2) 58 kDa present in the accessory
gland fluid was more prevalent in bulls of high fertility (Moura et al., 2006b).
3.2. Proteins of the cauda epididymal fluid and fertility
Factors isolated from epididymal fluid or epithelial cells in culture have been linked to
sperm motility and protection of membranes against damage caused by cryopreservation
(Reyes-Moreno et al., 2002), anticapacitation effects (Roberts et al., 2003), or sperm number
(Gatti et al., 2004), but evidence linking epididymal proteins to male fertility indexes is
limited (Moura et al., 2006a).
Because most proteins from the rete testis are not present in the milieu of the epididymis
lumen (Olson & Hinton, 1985; Dacheux et al., 1989), there is a general assumption that
proteins of the epididymal fluid are mainly the product of the epididymis itself. Numerous
proteins have been detected in the epididymal milieu of mammalian species (Cornwall et

al., 2002; Dacheux & Dacheux, 2002) but the exact roles of most of them in sperm maturation
are yet to be determined (Gatti et al., 2004).
Immature spermatozoa newly formed in the seminiferous tubules have a period of transit
through the epididymis where they become motile and undergo a series of events that
include changes in the composition of membrane lipids and proteins (Sullivan et al., 2005).
The epididymal epithelium secretes proteins that potentially affect not only sperm
maturation (Dacheux and Dacheux, 2002) but also other aspects of sperm physiology while
these cells are stored in the cauda compartment (Hinton et al., 1995).
Fluid produced by the epididymis is diluted about 8- to 10- fold when mixed with accessory
sex glands secretions at ejaculation (Gerena et al., 1998). This makes it difficult to accurately
identify epididymal proteins in the seminal plasma milieu, particularly those secreted in
low abundance or if they are also secreted by other organs, such as the accessory sex glands
(Moura et al., 2006a).
An average of 118 spots was detected in the 2-D maps of the cauda epididymal fluid (CEF)
in Holstein bulls. The intensity of alfa-L-fucosidase and cathepsin D was 2.3- 2.4-fold greater
in high-fertility bulls than in low-fertility bulls (Moura et al., 2006a). The intensity of 3
isoforms (24-27 kDa, pI 6.3-5.8) of prostaglandin D-synthase (PGDS) were from 3.2 to 2.2
fold greater in low-fertility sires. The findings suggest that molecular markers of male
fertility are associated with both epididymal sperm physiology and postejaculation eventus
regulated by accessory sex gland components (Moura et al., 2006a). PGDS could influence
male fertiling by mediating the action of hydrophobic molecules on sperm during
epididymal transit or cauda epididymal storage (Moura et al., 2006a).
P25b, a protein with predictive properties for bull fertility, is transferred from prostasome-
like particles present in the cauda epididymal fluid (PLPCd) to the sperm surface. The
pattern of distribution of the PLPCd transferred varied from one sperm cell to the other,
with a bias toward the acrosomal cap (Frenette et al., 2002).
4. Seasonal variation of zebu bull seminal plasma proteins
4.1. Heat-shock proteins and seminal quality in (Bos taurus indicus)
There were a number of suggestions in the earli er literature exposed to heat can produce
sperm which do not produce normal offspring in unexposed females (Setchell, 1998). Bulls
were subjected to scrotal insulation for 48 hours and semen collected and cryopreserved 2 or
3 weeks later, Following in vitro fertilization with swin-up sperm from these samples, there
were decreased rates of spermpenetration, pronuclear formation (Walters et al., 2006)
embryo cleavage, development and blastocyst formation (Walters et al., 2005) with semen
collected from two of the bullsthree weeks after the insulation, but not which semen from
two others bulls, or with semen collected after two weeks.
In bulls the heat is lost from the testis and scrotum to the environment through the scrotal
skin, which is well endowed with sweat glands (Setchell, 2006). The temperature on the

Electrophoresis 180
surface of the scrotum is lower at its base than near the neck, but the temperature inside the
testis is almost uniform, even slightly warmer at the base (Kastelic et al., 1996).
There is considerable variation between individual animals in their response to heat
exposure. Of the six bulls subjected to scrotal insulation by Vogler et al. (1993), two showed
a large increase in abnormal spermatozoa (to more than 60%) whereas others had as few as
23% abnormal cells. Likewise, 4 bulls used for semen collection for in in vitro fertilization
showed widely variable effects of 48h scrotal insulation on pronuclear formation, embryo
development and apoptosis, with two bulls classed as severe responders, one a moderate
responder and one showing no response to scrotal insulation (Walters et al., 2006).
Progress has been made in developing reliable indicators of ejaculate quality that allow
exclusion of low quality ejaculates for use in artificial insemination (AI). Physical semen
characteristics and sperm morphology measurements are not always indicative of fertility
and reproductive performance in animals, and accurate and predictive genetic and protein
markers are still needed (Foxcroft et al., 2008).
There is evidence revealing that seminal plasma prevents premature capacitation of sperm
(Eng & Oliphant, 1978) and protects sperm from peroxidative damage (Jones et al., 1979;
Schnech et al., 1996). It`s well known that low temperatures alter the function of
spermatozoa (Watson, 1995). Cold shock results in the destabilization of sperm membranes
and impairment of sperm function, and it is also well known that animal spermatozoa are
sensitive to cold-shock stress as the bull, rabbit and man (Schnech et al., 1996).
Comprehensive proteomic analyses have been conducted in the bull Bos taurus taurus
(Moura et al., 2006b). The literature on the effects of seasons on the semen characteristics
and upon seminal plasma proteins in Nellore and Tabapua (Bos taurus indicus) and
Limousin, Brown-Swiss and Brangus cattle under natural conditions in Brazil has already
been recently studied (Chacur et al., 2003; 2004; 2006a; 2006b; 2007; 2008; 2009; 2010a; 2010b;
2011a; 2011b; 2012).
4.2. Potential markers of relative fertility in Bos taurus indicus
Bos indicus bulls are less sensitive to the effects of high temperatures than Bos taurus or
crossbred bulls, but as they are actually more sensitive to the effects of scrotal insulation
(Brito et al., 2003). This would appear to be due to the greater ability of indicus animals to
keep their testes cool (Brito et al., 2002). Bos indicus bulls have greater testicular artery length
to testicular volume ratios, and smaller testicular artery wall thickness and arterial to
venous distances, which may be responsible for greater cooling of the arterial blood in the
spermatic cord (Brito et al., 2004).
In Brazil sixty-eight Nellore (Bos indicus) bulls were used, with twenty of the padron variety
and forty-eight of the mocho variety with mean of 4 years old. There was no difference
(P>0.05) for the spermatic morphology between padron and mocho variety, respectively

with 5.068.20% and 5.326.40% of major defects; 9.916.7% and 8.36 6.06% for minor
defects; and 14.7613.20% and 13.8212.61% for the total defects. The electrophoresis of the
seminal plasma showed protein bands with weights between 5- and 105-kDa. In 100% of the
bulls with good semen the 13kDa protein were present, the same happens with the 18- and
20-kDa bands. The varieties padron and mocho revealed similar reproductive adaptation in
front of the handling conditions and weather and looking very efficient (Chacur et al.,
2006b).
Disruptions in sperm production include decreased sperm motility and increased of
abnormal sperm. Seminal plasma appears to exert important effects on sperm function. The
objective was to evaluate the dry and rainy season influence on the seminal characteristics
and semen plasma proteins. Eleven bulls (Bos taurus indicus) with ages ranging from 34 to 38
months were submitted each one to 12 semen collect with eletroejaculation 6 on dry season
and 6 on rainy season with 14 days interval, totalizing 144 samples. Qualitative and
quantitative semen characteristics were evaluated. Samples of semen were centrifuged
(1.500 g / 15 minutes) and conditioned and stored (20C) until further processing. The
proteins were extracted and quantified to electrophoresis performed. Variance analysis and
Tukey test 5% was used. The semen vigor (P<0.01), minor defects and total defects (P<0.05)
showed statistical difference between seasons, while the volume, motility and minor defects
did not (P>0.05). The number of bands occurred between 6- and 125-kDa, see Table 1. The
molecular band of 26 kDa was present in 100% of bulls in rainy season. The molecular bands
of 6-, 9- and 125-kDa showed a high frequency in dry and rainy season. In conclusion, these
results showed a band distribution variation throughout the season and the year seasons
changed the semen quality with increase sperm vigor and reduction of abnormal sperm on
dry season (Chacur et al., 2011b).

bulls Proteins (kDa) Dry season (n=144) Rainy season (n=144)
a, b, c, d, e, f, h, i, j, k, 6 9/11 (81.81%) 10/11(90.90%)
a, b, c, d, e, f, g, h, j, k 9 10/11(90.90%) 8/11(72.72%)
d, e, f, h, i, j, k 12 7/11(63.63%) 5/11(45.45%)
a, b, c, d, e 13 5/11(45.45%) 1/11(9.09%)
a, b, c, e, f, g, j 17 3/11(27.27%) 7/11(63.63%)
a, d, e, f, j 20 3/11(27.27%) 3/11(27.27%)
a, b, c, d, e, f, g, h, i, j, k 26 6/11(54.54%) 11/11(100%)
a, b, c, e, f, h, i, j, k 35 4/11(36.36%) 7/11 (63.63%)
a, b, e, f, g, h, j, k 44 2/11(18.18%) 8/11(72.72%)
a, c, d, e, f, h, i, j 55 7/11(63.63%) 3/11(27.27%)
b, d, h, i, k 66 2/11(18.18%) 4/11(36.36%)
a, c, d, e, f, i, j, k 75 6/11(54.54%) 6/11(54.54%)
a, b, c, i 80 1/11(9.09%) 4/11(36.36%)

Electrophoresis 182
bulls Proteins (kDa) Dry season (n=144) Rainy season (n=144)
a, c, f, i, j 105 4/11(36.36%) 3/11(27.27%)
a, b, c, e, f, g, h, i, j, k 125 9/11 (81.81%) 10/11(90.90%)
Chacur et al. (2011).
Table 1. Frequency of proteins bands in dry season (may-july) and rainy season (october-december).
Seminal plasma is a complex of secretions of the male accessory reproductive organs and
appears to exert important effects on sperm function (Shivaji et al., 1990). The protein
quality of the seminal plasma may affect positively the bulls' fertility (Killian et al., 1993).
Peptides of 55- and 66-kDa were present in bulls with excellent spermatic conditions for
example motility and vigor. On the other hand, 16- and 36-kDa peptides were observed with
unfavorable spermatic conditions (Chacur et al., 2009). The objective was to determine the
influence of season on seminal plasma proteins in Brown Swiss bulls. Semen from 33 Brown
Swiss bulls 24 months of age were collected by electroejaculation during winter (from June
to August) and summer (from December to February) in the southern hemisphere in 2008.
Semen samples were collected with 14-day intervals totalizing 196 ejaculates. Samples of
semen were centrifuged (1500g/15 min) and the seminal plasma was conditioned in
cryotubes and stored at 20C until further processing. Proteins were extracted from 200 L
of each sample in 2 mL of extraction buffer composed of 0.625 M Tris-HCl, at pH 6.8, in 2%
SDS, 5% -mercaptoethanol, and 20% of glycerol. Percentages of different plasma proteins
by season were statistically compared by the chi-square test with significance level (P <
0.05). Proteins were quantified according to Bradford (1976) and electrophoresis was
performed according to Laemmili (1970). Gels were fixed with isopropanol:acetic acid:water
(4:1:5 v/v) for 30 minutes and stained in the same solution with 2% of Coomassie Blue R250.
In 26 bulls, the absence of high molecular weight (HMW; 55 kDa, 66 kDa, and 80 kDa)
proteins was found in the summer. There was a significant increase (P < 0.05) in total
spermatic defects, acrosome defects, and distal cytoplasmatic droplets in these bulls. The 40-
kDa protein that reflected low fertility was observed in 10 bulls in the summer with semen
quality decreases. The 11 bulls showed presence of HMW (55 kDa) in the winter. In 11 bulls,
HMW (55 kDa, 66 kDa, or 80 kDa) proteins were present with a satisfactory semen condition
according to Killiam et al. (1993). In conclusion, the seasons of the year may influence the
presence of proteins in seminal plasma. There was a direct relationship of the season with
seminal plasma proteins. The presence of the proteins of 20 kDa, 55 kDa, 66 kDa, and 80 kDa
suggested an increase of the semen quality during the winter (Chacur et al., 2010a; 2011a).
In Brazil semen from eleven Tabapua bulls, 30 months old, were collected by
electroejaculation during winter (from June to August) and summer (from December to
February) of 2007. From each bull a total of 132 semen samples were collected in an interval
of 14 days. Samples of seminal plasma were centrifuged (1500g/15min) and conditioned in
criotubes and stored at 20C until further processing. Proteins were extracted from 200 L
of each sample in 2 mL of extraction buffer composed by 0.625 M Tris-HCl, pH 6.8, 2% SDS,
5% -mercaptoethanol and 20% of glycerol. Proteins were quantified according to Bradford
(1976) and electrophoresis was performed according to Laemmli (1970). Gels were fixed

with isopropanol: acetic acid: water (4:1:5 v/v) for 30 minutes, and stained in the same
solution with 2% of Comassie Blue R250. Percentage of different seasons including plasma
proteins were statistically compared by the Chi-square test with significance level at P<0.05.
In two bulls, the absence of high molecular weight (HMW 55kDa, 66kDa and 80kDa)
proteins was verified in the summer. There was a significant increase (P<0.05) in total
spermatic defects in these two bulls. The protein of 40kDa which suppose to be of low
fertility was observed in eight bulls in the summer with semen quality decrease. The eight
bulls showed presence of HMW (55kDa) in the winter. In nine bulls HMW (55kDa, 66kDa or
80kDa) proteins were present with a satisfactory semen condition in accordance with
Chacur et al. (2006a). The two bulls showed presence of HMW proteins (66kDa and 80kDa)
in the summer. The results suggest that different seasons of the year may influence the
presence of a variety of proteins in seminal plasma. There was a direct relationship of the
season upon seminal plasma proteins. The presence of the proteins of 20kDa, 55kDa, 66kDa
and 80kDa suggests an increase of the semen quality during the winter (Chacur et al., 2008).
Peptides of 55- and 66-kDa were present in bulls with excellent spermatic conditions for
example motility and vigor (Chacur et al., 2009a). On the other hand, 13- and 33-kDa
peptides were observed in association with unfavourable spermatic conditions (Chacur et
al., 2009b). The objective of this study was to determine the profile SDS-PAGE of seminal
plasma and evaluate the semen characteristics in Brangus and Brown-Swiss bulls. Semen
from 14 Brangus, 36 months old, was collected by electroejaculation during summer of 2009-
2010. A total of 84 semen samples were collected in an interval of 14 days. Semen volume,
motility, vigor, major defects and minor defects were evaluated according to Brazilian
College of Animal Reproduction (Manual..., 1998). Animals were divided in two groups:
poor semen (motility <50% and major defects >10%) and good semen, and subsequently
compared regarding the composition of seminal plasma proteins. Samples of seminal
plasma were centrifuged (1500g/15min) and conditioned in criotubes and stored at 20C
until further processing. Proteins were extracted from 200 L of each sample in 2 mL of
extraction buffer composed by 0.625 M Tris-HCl, pH 6.8, 2% SDS, 5% -mercaptoethanol
and 20% of glycerol. Proteins were quantified according to Bradford (1976) and
electrophoresis was performed according to Laemmli (1970). Gels were fixed with
isopropanol: acetic acid: water (4:1:5 v/v) for 30 minutes, and stained in the same solution
containing 2% of Comassie Blue R250. Each semen collection was used in duplicate. The
concentration of proteins was measured using a spectrophotometer PF-901(Chemistry
Analyser Labsystems). Gels were submitted to a photodocumentation system (Bio Doc-IT
and Visidoc-IT Gel Documentation systems, UVP) and analysed by Doc-IT-LS 6.0 software.
GLM from SAS, version 6, was used in order to evaluate possible variations of seminal
variables and protein molecular mass. Statistical significance was accepted from P<0.05%.
The means of semen variables were: volume (51 mL), motility (755%), vigor (4), major
defects (72%) and minor defects (124%). The results of analyses of gels revealed a variety
of proteins in each animal and among bulls. There were 28 different major polypeptides,
ranging from 15 to 24 bands in each individual bull. In six Brangus bulls the presence of low
molecular weight (LMW 13kDa and 33kDa) proteins was associated with low motility (35-
40%) in accordance with Chacur et al. (2009a). There was a significant increase (P<0.05) in

Electrophoresis 184
major spermatic defects in these six bulls (20.33.7%) associated with presence of proteins
that had molecular weights of (23, 35 and 72KDa). In eight Brangus bulls, 55KDa, 66KDa or
80KDa proteins were present and associated with a satisfactory semen condition (motility
776% and major defects 52%) in accordance with Chacur et al. (2009b). In cattle, the 55-,
66- and 80-kDa proteins are associated positively with camp-dependent progressive motility
(Shivaji et al., 1990). Consistently, in the present experiment, there was a positive
relationship of presence of seminal plasma proteins 55kDa, 66kDa and 80kDa and semen
quality (motility and major defects). The presence of these proteins suggests an increase in
semen quality (Chacur et al., 2010b).
4.3. Interation between year seasons on the semen and hormones in Bos indicus
and Bos taurus in Brazil
In Brazil the influence of four year seasons was study on semen characteristics and levels of
testosterone and cortisol in Nelore and Simmental bulls. Five Nelore and five Simmental
bulls with 48-72 months old, extensively managed were evaluated for sexual soundness
using physical and morphological characteristics of semen and serum levels of testosterone
and cortisol. There was decreased motility and vigor semen (P<0.05) during winter in
Simmental bulls (Table 2). There was correlation (P<0.01) between testosterone x motility
(0.69) and testosterone x vigor (0.57) in Simmental breed (Table 4) and cortisol x motility
(0.68) and cortisol x vigor (0.65) in Nelore breed (Table 3). The effect of year seasons changed
the semen quality with increase sperm motility and vigor on springer-summer in Simmental
bulls. The cortisol level decreased on autumn in Nelore bulls (Chacur et al., 2012).

characteristics breed spring summer autumn winter
Volume (mL) S 8.800.65 Aab 9.850.65 Aa 8.760.58 Aab 7.350.65 Ab
N 7.100.65 Aa 7.550.65 Ba 6.260.58 Ba 6.050.65 Aa
Motility (%) S 70.005.83Aa 70.005.83Aa 60.808.21Aab 48.005.83Ab
N 63.505.83Aa 60.005.83Aa 54.405.21Aa 53.505.83Aa
Vigor (1-5) S 3.350.28 Aa 3.550.28 Aa 3.000.25 Aab 2.200.28 Ab
N 3.050.28 Aa 2.950.28 Aa 2.320.25 Aa 2.500.28 Aa
Major defects (%) S 11.331.31 Aab 8.001.07 Ab 10.750.98 Aab 12.181.20 Aa
N 6.301.07 Ba 6.311.10 Aa 9.920.96 Aa 9.421.10 Aa
Minor defects (%) S 7.880.83 Aa 7.250.79 Aa 8.250.72 Aa 10.250.88 Aa
N 7.600.79 Aa 5.630.81 Aa 7.160.70 Aa 6.840.81 Ba
Total defects (%) S 19.221.63 Aab 15.251.54 Ab 19.001.41 Aab 22.431.73 Aa
N 13.901.53 Ba 11.971.59 Aa 17.081.38 Aa 16.361.59 Ba

characteristics breed spring summer autumn winter
Concentration
(x10
9
/mL)
S 1.350.13 Aa 1.380.13 Aa 1.320.12 Aa 1.000.13 Aa
N 0.880.13 Ba 1.140.13 Aa 0.950.12 Ba 0.910.13 Aa
Significance level 5% (P< 0,05); A, B distinct letters in column (P<0,05); a, b distinct letters in line (P<0,05).
Table 2. Semen characteristics in spring, summer, autumn and winter for Simmental (S) and Nelore (N)
bulls.

breed spring summer autumn winter correlations
cortisol S 0.6 0.6 0.5 1.1
Volume (mL) S 8.800 9.444 10.800 7.500 -0.87
color S 1.700 2.111 1.400 1.200 -0.60
aspect S 1.800 1.889 1.400 1.900 0.55
Mass moviment
(1-5)
S 1.800 4.000 2.800 1.800 -0.52
Motility (%) S 67.000 80.000 64.000 46.000 -0.83
Vigor (%) S 3.000 4.111 3.200 2.100 -0.79
Concentration
(x10
9
/mL)
S 1.093 1.697 1.360 0.960 -0.66
Major defects (%) S 13.889 6.222 10.800 10.875 0.10
Minor defects (%) S 7.889 6.222 7.400 8.250 0.60
Total defects (%) S 21.778 12.444 18.200 19.125 0.22
cortisol N 1.68 3.10 1,36 3.06 correlations
Volume (mL) N 8.278 8.286 5.700 5.800 0.14
color N 1.444 3.000 1.600 2.200 0.87
aspect N 1.889 2.429 1.500 2.000 0.85
Mass moviment
(1-5)
N 2.222 3.714 1.300 2.200 0.75
Motility (%) N 63.333 80.000 33.000 56.000 0.75
Vigor (1-5) N 3.111 4.000 1.500 2.600 0.65
concentration
(x10
9
/mL)
N 0.913 1.329 0.701 1.080 0.91
Major defects (%) N 5.889 5.857 10.700 9.200 -0.31
Minor defects (%) N 7.000 4.571 8.000 7.500 -0.60
Total defects (%) N 12.889 10.429 18.700 16.900 -0.43
color: 1 white, 2 White-Milk and 3 White-yellow; aspect: 1 aquous, 2 viscous and 3 cremous.
Table 3. Correlations between cortisol (g/dL) and semen characteristics on year season in Simmental
(S) and Nelore (N) bulls.

Electrophoresis 186
breed spring summer autumn winter correlations
testosterone S 879.5 901.1 584.0 648.2
Volume (mL) S 8.800 9.444 10.800 7.500 -0.16
color S 1.700 2.111 1.400 1.200 0.86
aspect S 1.800 1.889 1.400 1.900 0.62
Mass moviment (1-5) S 1.800 4.000 2.800 1.800 0.31
Motility (%) S 67.000 80.000 64.000 46.000 0.69
Vigor (1-5) S 3.000 4.111 3.200 2.100 0.57
Concentration
(x10
9
/mL)
S 1.093 1.697 1.360 0.960 0.37
Major defects (%) S 13.889 6.222 10.800 10.875 -0.19
Minor defects (%) S 7.889 6.222 7.400 8.250 -0.47
Total defects (%) S 21.778 12.444 18.200 19.125 -0.26
testosterone N 430.41 234.71 420.31 329.15 correlations
Volume (mL) N 8.278 8.286 5.700 5.800 -0,28
color N 1.444 3.000 1.600 2.200 -1.00
aspect N 1.889 2.429 1.500 2.000 -0.89
Mass moviment (1-5) N 2.222 3.714 1.300 2.200 -0.87
Motility (%) N 63.333 80.000 33.000 56.000 -0.71
Vigor (1-5) N 3.111 4.000 1.500 2.600 -0.70
Concentration
(x10
9
/mL)
N 0.913 1.329 0.701 1.080 -0.93
Major defects (%) N 5.889 5.857 10.700 9.200 0.36
Minor defects (%) N 7.000 4.571 8.000 7.500 0.82
Total defects (%) N 12.889 10.429 18.700 16.900 0.56
color: 1 white, 2 White-Milk and 3 White-yellow; aspect: 1 aquous, 2 viscous and 3 cremous.
Table 4. Correlations between testosterone (ng/dL) and semen characteristics on year season in
Simmental (S) and Nelore (N) bulls.
5. Summary and conclusions
Seminal plasma is composed of secretions from the male accessory sex glands and
epididymis, which contains many organic and inorganic components that have effects on
sperm quality (Foxcroft et al., 2008). The proteins secreted into seminal plasma may play an
important role during sperm capacitation and fertilization (Rodriguez-Martinez et al., 1998),
and may also serve to protect sperm from damage or to maintain their longevity.

Comprehensive proteomic analyses have been conducted in the bull (Moura et al., 2006a;
Moura et al., 2006b; Chacur et al., 2010a; Chacur et al., 2010b; Chacur et al., 2011a; Chacur et
al., 2011b). The literature on the effects of seasons on the semen characteristics and upon
seminal plasma proteins in Nellore (Bos taurus indicus) cattle under natural conditions in
Brazil has already been recently studied in Brazil (Chacur et al., 2003; 2004; 2006a; 2007;
2009a; 2010a; 2010b; 2011a; 2011b; 2012).
Its well known that low temperatures alter the function of spermatozoa. Cold shock results
in the destabilization of sperm membranes and impairment of sperm function, and it is also
well known that animal spermatozoa are sensitive to cold-shock stress as the bull (Watson,
1995).
Seminal plasma has also been shown to have deleterious effect on bovine sperm during
semen storage at ambient temperatures and a damaging effect during semen cooling and
freezing. Recent studies have shown that proteins from bovine seminal plasma (BSP) may
modulate sperm properties. Many autors believe that the bull seminal plasma contains
fertility associated protein markers. Comparison of individual 2-D PAGE maps with the
reference map could provide a useful key to relate protein pattern changes to some
physiopathological events influencing the reproductive sphere.
Factors isolated from epididymal fluid or epithelial cells in culture have been linked to
sperm motility and protection of membranes against damage caused by cryopreservation
(Reyes-Moreno et al., 2002), anticapacitation effects (Roberts et al., 2003), or sperm number
(Gatti et al., 2004), but evidence linking epididymal proteins to male fertility indexes is
limited (Moura et al., 2006a).
6. Future prospects
The determination of protein content on sperm surface and seminal plasma may be an index
of individual bull fertilizing ability or post-thaw status of sperm membranes (Nauc &
Manjunath, 2000). A reference map of seminal plasma proteins could be useful in relating
protein pattern changes to physiopathological events influencing the reproductive sphere
(Mortarino et al., 1998). PGDS could influence male fertility by mediating the action of
hydrophobic molecules on sperm during epididymal transit or cauda epididymal storage
(Moura et al., 2006a; 2006b). Although known functional attributes of these proteins provide
some understanding of how they may influence male reproductive performance (Moura et
al., 2006b; Chacur et al., 2010a; 2011a).
Author details
Marcelo G.M. Chacur
Animal Reproduction Department, University of Oeste Paulista (UNOESTE), Pres. Prudente-SP,
Brazil

Electrophoresis 188
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Chapter 11
Identification of Polymorphism in the Keratin
Genes (KAP3.2, KAP6.1, KAP7, KAP8)
and Microsatellite BfMS in Merino
Sheep Using Polymerase Chain Reaction-Single
Strand Conformational Polymorphism
(PCR-SSCP) Analysis
http://dx.doi.org/10.5772/45732
1. Introduction
Wool production is a major agricultural industry world-wide, the most important wool-
growing countries include Australia, China, New Zealand, South Africa and countries within
South America. In Australia for example, the world's largest producer of wool accounting for
~ 30% of the world production, wool industry is among the top industries in export revenue.
While Australia has long been associated with the production of high-quality wool, the
importance of this industry and the value of wool exports have been steadily declining.
1.1. Challenges facing the wool industry
The wool industry is faced with many challenges that require innovative solutions. The major
competitors to the wool industry, cotton and synthetics, have developed new fibres that meet
consumer needs such as being lightweight, soft and easy to care. These competitors have also
made better productivity gains than wool, which has resulted in lower prices for all textile
products. Today, there is much instability in wool prices, with a major problem facing the
industry in faulty wool production. It has been observed that considerable variation exists both
within and between fleeces across sheep breeds, as well as within inbred lines of sheep. Since
the efficiency of wool processing is dependent on the consistency of wool fibre, it is of prime
importance to wool producers that this variation is controlled. The wool characteristics that are

Electrophoresis 194
of economic importance include fibre diameter (or fineness), grease and clean fleece weight,
fleece strength and length, colour, yield, crimp and bulk. For Merino and halfbred wools, fibre
diameter is the major factor that contributes to price variation as it significantly influences both
fibre processing properties and ultimate product quality. The colour of wool is also important
because superior colour (bright and white) can be dyed to the maximum range of shades and
consequently is worth more than poorer coloured wool. Furthermore, the quantity of wool is
important in overall wool production and in the efficiency of the production system.
1.2. Classical selective breeding Not a simple solution
For many years, farmers have been using classical selective breeding, where by selection of
breeding animals was traditionally based on the phenotype (that is appearance) of the
individual animal, a rather slow method of selection. Each animal is assigned a breeding value
(BV), which describes the future genetic potential of an animal. The BV is calculated by
adjusting phenotype to exclude factors such as birth rank, lambing status and sex in order to
give an estimate of the genetic merit. The desired goal of this strategy is the accumulation of
good forms of genes for that particular trait in the population, over time. This has resulted in
many breeds that are commercially important today. The domestic sheep Ovis aries today
comprises over 500 different domestic breeds. However, wool characteristics, like many
production traits (such as milk yield, growth rate, meat tenderness), do not exhibit simple
Mendelian inheritance patterns (recessive or dominant). Instead, they are controlled by not only
many genes, but also the interaction of these genes, each having small additive effects on the
phenotype observed. Environmental and management factors also play a role. Thus, wool traits
are quantitative and show continuous variation in phenotype, a fact that makes it difficult to
deduce the genotype of an animal from its phenotype, and to relate genetic variation to
differences in the phenotype. In other words, genetic improvement breeding programme select
for phenotypic superior animals, without the knowledge of the actual genes that are being
selected which I will term as blind selection in this paper. Furthermore, other strategies to
control environmental factors such as nutrition, time of shearing or mineral supplementation
tend to be costly. In addition, wool production traits tend to only be fully expressed when an
animal is mature, at least three years old, and therefore genetic progress using phenotypic
selection and pedigree information is relatively slow.
1.3. Identification of gene markers: A possible solution
The answer to sidestepping this blind selection, inaccuracy in describing the genetic
potential of an animal and slow progress may lie in identifying specific genetic markers that
are associated with wool production traits. Some sheep consistently produce quality or
faulty wool, suggesting that genetic factors are an important key in determining wool
characteristics. In addition, estimates for the heritability (h
2
) of most wool traits are
generally high (h
2
= 0.3 - 0.6), indicating that wool traits are under genetic control and that
they can be selected for. A gene is a segment of DNA that provides the genetic information
necessary to produce a protein. For almost all of the genes, there are two copies (alleles), one

Photocatalytic Hydrogenation on Semiconductor Particles 195
inherited from the mother and the other from the father. In any population of animals, there
can be many different alleles. This is termed polymorphism or genetic variation.
Polymorphism results from DNA mutation. It is this polymorphism that is taken advantage
of, in order to identify genetic markers. A genetic marker for a particular characteristic can
be defined as a piece of DNA that directly affects a phenotype and shows polymorphism. It
can also be a piece of DNA that is closely linked to another piece of DNA that affects a
phenotype. Genetic markers can either be genes or non-functional DNA segments such as
microsatellites or minisatellites.
A number of different types of genetic markers are commonly used, including restriction
fragment length polymorphisms (RFLPs), microsatellite and minisatellite DNA, and polymerase
chain reaction-single strand conformational polymorphism (PCR-SSCP) variants. Restriction
fragment length polymorphism results from the alteration of the restriction site(s) recognised by
a specific restriction endonuclease or by the insertion or deletion of sequence between two
restriction sites. The variation in fragment lengths is detected using gel electrophoresis.
Although RFLPs were the first genetic markers developed, they are losing popularity as a
screening method to identify genetic markers because they have the disadvantages of not
identifying all of the polymorphism with a length of DNA, are time-consuming and restriction
enzymes and consumables tend to be expensive. Simpler marker systems have subsequently
been developed, many of these systems are now based on satellite DNA sequences.
Throughout the genome of higher eukaryotic organisms, there are a variety of different short
DNA sequence repeats known as satellite DNA. These sequences do not code for protein and
are highly variable from individual to individual in both the number and type of repeats
(Groth et al., 1987). Microsatellites are composed of DNA repeats in tandem at each locus. The
tandem repeats are usually simple, and consist of either a single nucleotide or dinucleotide
such as (CA)n, with each dinucleotide repeated about ten times. Minisatellites have longer
repeated sequences than microsatellites, such as (ACTG)n. Since microsatellites and
minisatellites show a substantial amount of polymorphism, they can serve as useful markers
for the identification of genetic variation of value to animal breeding. Although the variation
in the number of repeats can sometimes be detected using RFLP, PCR is generally used to
amplify the polymorphic region and the amplimer analysed for length variation (a technique
referred to amplified fragment length polymorphism AFLP).
1.4. Polymerase chain reaction-single strand conformational polymorphism
(PCR-SSCP) as a preferred type of genetic marker
PCR is also used in conjunction with SSCP. The PCR-SSCP technique offers a rapid, sensitive
and relatively inexpensive way to screen for sequence variation with minimal sequencing.
First described by Orita et al. (1989), this technique has become one of the preferred methods
for screening samples to detect polymorphism because it is both simple and sensitive. In this
techniques, regions of the gene of interest are amplified using PCR and the products
denatured and then cooled rapidly to promote the formation of secondary structures due to
internal base-pairing, which are in turn sequence dependent (Orita et al., 1989). The folded

Electrophoresis 196
single-stranded DNA molecules are separated by polyacrylamide gel electrophoresis under
non-denaturing conditions. The folded secondary structures are affected by physical
conditions such as temperature, percentage of polyacrylamide, ionic strength of the
electrophoretic buffer, glycerol concentration (Spinardi et al., 1991), ratio of acrylamide to bis-
acrylamide, run length and run voltage. This can be exploited when optimising an SSCP
protocol so that maximum variation can be detected in a given section of DNA. Molecules
that differ by even a single nucleotide may form different conformers under a given set of
conditions and, upon electrophoresis in a non-denaturing polyacrylamide gel, migrate
differently. Many methods for viewing the folded DNA conformers have been described.
These include the radioactive labelling of primers followed by autoradiography (Orita et al.,
1989), silver staining (Sanguinetti et al., 1994), ethidium bromide staining (Yap and McGee,
1993) and more recently the use of fluorescently labelled primers and fluorescent dyes.
1.5. Methods used to identify genetic markers
There are several ways to identify genetic markers, but the two approaches most commonly used
are the genome scanning or linkage analysis and the candidate gene approach. In the genome
scan approach, the whole genome is searched to identify Quantitative Trait Loci (QTL) that affect
any given trait. These are not necessarily the genes that are responsible for trait variation, but
give an indication of where such genes may lie. Linkage analysis is an involved process. A map
of the chromosomes, laying out the location, phase and order of genes and markers, and the
distance between them, is required before linkage analysis can be performed. Firstly, a selection
of about 200 markers distributed throughout the genome are genotyped, in the sire of the
animals. Only the informative markers are genotyped in the progeny and each marker tested for
suggestive linkage. Regions showing suggestive linkage are then studied by saturating the region
with markers to identify those that are tightly linked. Phenotypic variation is then linked to the
segregation of DNA markers within a population. Once the gene locus is identified by the tightly
linked markers, the DNA can be sequenced. Linkage analysis can be an expensive and lengthy
process requiring access to full chromosome libraries and arrays of markers.
In the candidate gene approach, known genes or gene markers that are thought to be
responsible for the phenotypic variance of a trait are targeted for investigation. In this case,
knowledge of the understanding of the genes that are likely to affect wool quality. The
method requires a good knowledge of the physiological and biochemical processes of the
gene product and can be a more direct method than the gene mapping approach, provided
the right initial assumptions are made. One of the limitations of this approach is its hit and
miss nature. A targeted gene may not be polymorphic in a population or genetic variation
within the targeted gene may not affect the trait (Goddard, 2002). For the candidate gene
approach to be useful, a quick and relatively inexpensive way to screen the target gene for
polymorphism is essential.
The wool fibre is a complex structure composed primarily of proteins from the keratin
family, which are the keratin intermediate-filament proteins (KRTs) and the keratin
intermediate-filament associated proteins (KAPs). The KRTs form the skeletal structure of

the wool fibre (microfibrils) and are embedded in a matrix of KAPs (Powell and Rogers,
1986), the different proteins being connected through disulphide cross-linkages (Powell,
1996). Therefore, genes that code for the KAPs and KRTs proteins are potential candidate
genes in the identification of genetic markers associated with wool quality traits.
1.6. Half sib analysis
Half-sib analysis is a tool that allows genetic effects to be ascertained from field trials while
controlling for environmental and management effects. Firstly, the gene being targeted must
be polymorphic, with at least two alleles. A good sire is selected and mated to many ewes
(at least 200 in number), that are selected at random from a range of environments, in order
to maximize phenotypic variation in wool traits. The sire must be informative at each locus
that is being investigated (i.e., the genotype of the sire must be heterozygous). If not, then
the progeny does not get genotyped for those loci that the selected sire is homozygous. For
those loci that the sire is heterozygous, the progeny born are genotyped soon after birth, and
allowed to grow until their wool measurements can be taken at (12, 24 and 36 months of
age). Suppose a sire has the genotype AB at the K33 locus, then all the progeny that have
inherited the A allele from the sire are put in one group, and those that have inherited the B
allele from the sire are put in another group. The means of the wool measurements from
both groups are then compared. If the group of progeny that inherited the B allele from their
sire are found to for example have a significantly stronger staple strength than those
progeny that inherited the A allele from their sire, then this would give an indication that
the K33 B allele might be associated with stronger staple strength.
1.7. Previously published association of genetic markers with wool traits
Numerous studies have described variation within both the KAP and KRT genes, including
the work of Rogers et al. (1994a); Parsons et al. (1994a; 1996); McLaren et al. (1997); Beh et al.
(2001); Itenge-Mweza et al. (2007). There are some reports associating variation in the KRT
and KAP genes with variation in wool traits. Parsons et al. (1994b) and Beh et al. (2001)
reported associations between variation in KAPs and mean fibre diameter in Merino sheep,
while Rogers et al. (1994b) reported association between staple strength in Romney sheep
and the region spanning the KAP1.1/KAP1.3/K33 loci on ovine chromosome 11. Itenge et al.
(2009; 2010) reported association between variation in the KAP1.1 gene with variation in
yield. In one of the half-sib families studied, variation in the K33 gene was associated with
variation in staple strength. Markers, other than the KRT and KAP genes associated with
wool traits have also been reported and these, together with reported keratin gene markers
are summarised in Table 1.1.
1.8. Gel electrophoresis
Gel electrophoresis is the process in which an electrical current is applied to a gel to separate
large molecules such as nucleic acids, from a mixture of similar molecules, based on
differences or how they react to the electrical current. The technique relies on the fact that

Electrophoresis 198

1
MFD = mean fibre diameter; CVD = coefficient of variation of fibre diameter.
2
A composite Romanov (prolific breed) and Berrichon du Cher (meat breed)
3
MRM = Merino X Romney X Merino backcross
Table 1. Potential genetic markers for wool quality traits reported by various researchers.
nucleic acids are negatively charged because of the phosphate groups on the phosphodiester
backbone of the nucleic acid strands (Nicholl, 1994). Nucleic acid molecules will migrate
from the negative (black) terminal to the positive (red) terminal if put in solution and an
electric field is applied, due to the net negative charge in solution. The gel matrix adds a
sieving effect so that particles can be characterized by both charge and size.
Agarose is a macromolecular substance that is derived from seaweed. It can be purified
to a whitish granular powder which, when mixed with water and heated, can be left to set
like a jelly. This is called a gel and it acts like a sieve for the DNA molecules. To separate
DNA molecules that are different lengths, agarose is used to produce a molecular sieve.
The speed that the DNA travels through the gel is inversely proportional to the size of the
DNA. In other words, small DNA particles migrate faster than large DNA molecules, as
they are less physically restrained by the gel matrix. The length of a piece of DNA can be
determined by comparing it to a molecular weight ladder. Agarose gel electrophoresis can
be affected by:
1. The percentage of agarose, which affects the sieving of the DNA molecules.
2. The voltage applied during the electrophoresis, which cause the DNA molecules to
move.
Typically, 1000 50,000 bp can be separated by 0.3% agarose, and 300 6000 bp can be
separated by 1.4% agarose, while base pairs less than 500 are better separated using
polyacrylamide gel, with gel percentage between 10-20. The polyacrylamide gel
electrophoresis works under non-denaturing conditions.

After the electrophoresis is complete, the molecules in the gel can be stained to make them
visible. Ethidium bromide, silver, or coomassie blue dye may be used for this process. Other
methods may also be used to visualize the separation of the mixture's components on the
gel. If the analyte molecules fluoresce under ultraviolet light, a photograph can be taken of
the gel under ultraviolet lighting conditions, often using a Gel Doc. A molecular weight
marker (MM) is often included on the gel to give an indication of the fragment size.

Figure 1. An example of a gel photo. MM is the molecular marker. Lane 1 has 341 bp DNA, while lane 4
has 280bp DNA. Lane 2 is blank.
1.9. Aim and objective of this paper
This paper discusses the identification of genetic variation in the KAP3.2, KAP6.1, KAP7,
KAP8, KRT2.10 and BfMS loci in Merino sheep using polymerase chain reaction-single
strand conformational polymorphism (PCR-SSCP) analysis. Polymorphism within these loci
is likely to be in part responsible for the observed variation in wool characteristics and could
result in the identification of gene markers to be used in gene marker-assisted selection
programmes within the wool industry.
2. Materials and methods
2.1. Sheep used in the study
This study used two half-sib families referred to as Sire Line 1 (SL1) and Sire Line 2 (SL2).
The SL1 half-sib was produced by mating a fine wool producer Merino ram to 150 Merino
ewes, selected at random from a range of New Zealand environments, in order to maximise
phenotypic variation in wool traits. In year one, the SL1 consisted of 131 pure New Zealand
Merino lambs, with 128 of these surviving to the second shearing at 24 months. Following
the second shearing the wether lambs and some of the ewe lambs were culled and only the
remaining ewe lambs (n = 37) were shorn at 36 months of age. The SL2 half sib consisted of

Electrophoresis 200
35 lambs (Merino x Romney ram x Merino ewes). Half-sib groups were kept as single flocks
to minimise environmental variation between individual progeny and provide control. All
lambs were tagged at birth to their dam and their gender and birth rank were recorded.
2.2. Wool shearing and sampling
Mid-side wool samples were collected at 12, 24 and 36 months of age for SL1 and at 12
months of age for SL2. Except for greasy fleece weight (GFW) which was determined at
shearing, wool measurements were performed by the New Zealand Wool Testing Authority
Ltd (NZWTA), Napier, New Zealand according to International Wool Textile Organisation
(IWTO) standards. Measurements included comfort factor or the percentage of fibres of
diameter greater than 30 m (F<30), mean fibre diameter (MFD, IWTO-12-03), fibre diameter
standard deviation (FDSD, IWTO-12-03), coefficient of variation of fibre diameter (CVD,
IWTO-12-03) and curvature, were all measured using a Sirolan Laserscan Fibre Diameter
analyser while the mean staple length (MSL, IWTO-30) and mean staple strength (MSS,
IWTO-30) of each sample was determined using Automatic tester for Length and Strength
(ATLAS). The colour (MY-Z) and brightness (MB) of the wool was measured using a
reflectance spectrophotometer, where the tristimulus values Y-Z indicate the yellowness of
the wool and the tristiulus value Y represents the brightness of the wool. The yield of wool,
the weight of clean wool after impurities such as vegetable matter have been removed,
expressed as a percentage of greasy wool weight was mathematically derived for the wool
base (IWTO-19) measurements. Once yield measurements were obtained from the NZWTA,
clean fleece weight (CFW) was calculated as the product of GFW and yield.
2.3. Blood sampling on FTA
TM
cards and DNA isolation
Blood samples (containing DNA) were collected from the progeny and sires onto FTA
TM

cards (Whatman, Middlesex, UK). These were stored at room temperature (See Figure 2.1).
A small punch (1.2 mm in diameter) was taken from the blood on the FTATM cards using a
Harris Micro Punch (Whatman International Ltd, UK) and put into a 200 L tube. The DNA
on the punches was isolated following a modified manufacturers protocol. 200 L of FTA
TM

reagent was added to each tube containing a 1.2 mm punch of FTA
TM
paper, containing the
sample DNA. The tubes were incubated at room temperature for 60 minutes. Each tube was
vortexed three times for about five seconds at the start of the incubation, half-way through
the incubation, and after the incubation period. The FTA
TM
reagent was aspirated, and the
cards were washed with 200 L of TE buffer (1 M Tris and 0.5 M Na2EDTA) for two
minutes. The TE buffer was aspirated and the tubes were left open, but covered with a tube
holder and stored at 4
o
C and used for the subsequent PCR reaction.
2.4. Amplification of the loci using PCR
The PCR conditions for the loci that are described in the literature were initially used.
However, re-optimisation was necessary for amplification in an i-Cycler PCR machine (Bio-
Rad Laboratories Inc., Hercules, CA, USA). The PCR protocols were optimised by using a

temperature gradient (to determine annealing temperature) coupled with a magnesium
titration.
All the primer sequences used in the study were obtained from the literature (Table 2.1), and
were synthesized by Invitrogen New Zealand Limited, Penrose, Auckland, New Zealand.
PCR amplifications were performed in a reaction mixture containing ~ 50 ng of genomic
DNA on a washed 1.2 mm punch of FTA
TM
paper, 1 PCR reaction buffer with 1U Taq
polymerase (Qiagen, GmBH, Hilden, Germany). Table 2.2 lists the total reaction volume
used along with the specific dNTP, primer, magnesium, and Q concentrations for each
locus.
Amplification consisted of 1 minute denaturation at 95
o
C, followed by 30 cycles of
denaturation at 95
o
C for 1 minute, annealing at temperatures specified in Table 2.3 for 1
minute and extension at 72
o
C for 1 minute, with a final extension of 72
o
C for 7 minute. All
the primer sequences used in the study were obtained from the literature, and were
synthesised by Invitrogen New Zealand Limited, Penrose, Auckland, New Zealand.

Figure 2. FTA
TM
cards of blood samples collected from the progeny of sire line 1

Electrophoresis 202

Table 2. Primer sequences and source references for each locus investigated.

Locus Total Primer dNTP Mg
2+
Q
volume (L) concentration (nM) concentration (M) concentration (mM)
Conce-
tration
()

KAP3.2 25 350 175 1.0 1
KAP6.1 25 400 200 1.0 1
KAP7 25 350 175 1.0 1
KAP8 25 350 175 1.0 1
KRT2.10 20 400 200 1.0 -
BfMS 25 350 175 1.0 1
Table 3. Optimised PCR conditions for each locus investigated.

Locus Annealing temperature (
o
C) Amplimer size (bp)
KAP3.2 58 424
KAP6.1 62 528
KAP7 63 413
KAP8 62 124*
KRT2.10 65 191
BfMS 58 200*
* = Variable length as amplification of a microsatellite region
# = Amplified with primers by Rogers et al. (1994b)
Table 4. Optimised annealing temperatures and predicted amplimer sizes for each locus investigated.

2.5. Agarose gel electrophoresis
Amplimers were analysed in 1.0% w/v SeaKem
LE agarose (FMC Bioproducts, Rockland,

Maine, USA) gels prepared with 1 TBE buffer (89 mMTris, 89 mM orthoboric acid, 2 mM
Na2EDTA; pH 8) containing 0.1 mg/L ethidium bromide. Five L of PCR product was added
to 2.5 L of loading dye (0.2% bromophenol blue, 0.2% xylene cyanol, 40% (w/v) sucrose)
and the gels were electrophoresed at a constant 10 Vcm
-1
for 30 minutes. A molecular
weight marker (Invitrogen Life Technologies) was included on the gel to give an indication
of the fragment size. DNA bands were viewed on a UV transilluminator (254 nm) and a
photograph taken for records.
2.6. Optimisation of SSCP gels
PCR-SSCP conditions were available in the literature for KAP3.2 (McLaren et al., 1997),
however these were deemed to be insufficiently stringent. For this reason, the PCR-SSCP
protocols used in this study were established empirically using template DNA from two
small half-sib families (to observe inheritance of allele-specific banding pattern) and DNA
samples of other unrelated Merino sheep (for increased genotypic variation). Many different
gel conditions (gel percentage, voltage, time of running, temperature, addition of glycerol)
were assessed to determine the optimum combination of conditions to resolve allele specific
banding patterns in a reproducible manner. Amplimers from sires of the SL1 and SL2 and
their selected progeny were also included on the optimising gels in order to ascertain allele
banding patterns by following inheritance, and to determine whether the sires were
heterozygous, and therefore informative, for the locus genotyped. Alleles were named in the
order they were identified using letters of the alphabet.
2.7. Detection of sequence variation using PCR-SSCP
Each locus used specific SSCP gel conditions, and these are summarised in Table 2.4.
Polyacrylamide (37.5:1 acrylamide / bis-acrylamide, Bio-Rad Laboratories, Hercules, Ca,
USA) vertical gels (Protean II 16 x 16 cm, 1.0 mm thick spacers, 28 well comb, Hoefer, Inc.,
San Francisco, Ca, USA) were prepared containing 0.5 TBE (44.5 mMTris, 44.5 mM
orthoboric acid, 1 mM Na2EDTA [pH 8.0]) and polymerised using 10% ammonium
persulphate and TEMED. Gels were pre-electrophoresed at running temperatures and
voltage for one hour. Amplimers were mixed with 50 L loading dye (95% formamide, 10
mM Na2EDTA, 0.025% bromophenol blue, 0.025% xylene cyanol), denatured by heating at
95
o
C for five minutes and immediately placed on wet ice before loading 15 L aliquots. The
gels were then electrophoresed at the optimum gel conditions with 0.5 TBE running buffer,
followed by silver-staining according to the method of Sanguinetti et al. (1994).
2.8. Cloning of allele standards
For KAP3.2, KAP7 and KRT2.10 loci, genomic DNA was obtained from the sire and this
DNA was amplified using the PCR conditions described above and the amplimers were

Electrophoresis 204
Locus Gel % (37.5:1)
1

Run length
(hours)
Temperature
2
(
o
C) Voltage (V)
KAP3.2 8 17 30 250
KAP7 10 4 20 200
KAP8 10 4 20 200
BfMS 12 7 15 300
1
Acrylamide/Bis-acrylamide ratio
2
The vertical gel electrophoresis tanks were connected to a circulating water chiller to maintain a constant gel
temperature. This is the temperature listed in the above table.
Table 5. Optimised SSCP conditions for the loci investigated.
subsequently cloned using the Promega pGEM

- T Easy Vector System I (Promega
Corporation, Madison, WI, USA). Since each plasmid can only accept one molecule of DNA
and therefore only one allele. Ligation reactions were performed in a total reaction volume of
10 L containing three units T4 DNA ligase, 50 ng of plasmid DNA and 1 ligation buffer, and
incubated overnight at 4
o
C. Constructs were transformed into competent E. coli cells
(Invitrogen
TM
, One Shot
TM
, INVF) using the manufacturers protocol. Sixty L and 150 L of
the transformation mix were spread on labelled LB (0.5 % casein hydrolysate, 0.25 % yeast
extract, 85.6 mM NaCl; pH 7.0) agar plates containing 100 g/mL ampicillin that had been
spread with 40 L of 40 mg/mL X-Gal (BDH Laboratory Suppliers, Poole, England). The plates
were incubated overnight at 37
o
C. Six colonies for each representative allele were selected and
cultured overnight in terrific broth (Invitrogen Corporation, Paisley, Scotland, UK),
supplemented with 50 g/mL ampicillin, for plasmid isolation. Colonies were screened for the
correct alleles using a rapid boiling-PCR method, where by Fifty L aliquot of an overnight
culture (bacterial cells with gene of interest cultured in terrific broth) was centrifuged at 13,000
rpm for 2 minutes, the supernatant was discarded and 30 L TE (1 M Tris, 0.5 M Na2EDTA)
buffer added, boiled for 10 minutes, centrifuged at 13000 rpm for 2 minutes and then 1L of
the supernatant was used as the template for the appropriate PCR. Amplimers were run on 2%
agarose gels next to the original genomic PCR amplimers for comparison. Plasmid DNA was
then isolated from clones, which had banding patterns corresponding to the original banding
pattern seen from amplimers of genomic DNA, using the FastPlasmid
TM
Mini Kits (Eppendorf,
Hamburg, Germany) following the manufacturers instructions. These amplified plasmid
DNAs were subsequently sequenced and used as standards for scoring unknown genotypes.
2.9. DNA sequencing
Plasmid standards were sequenced in the forward and reverse directions using the M13
forward and reverse primers at the Waikato University DNA Sequencing Facility, University of
Waikato, New Zealand or Lincoln University Sequencing Facility, Lincoln, New Zealand. The
sequences were compiled using DNAMANTM version 4.0 (Lynnon Biosoft, Quebec, Canada)
and the electropherograms. To minimise the likelihood of PCR and sequencing errors, sequence
data was derived from four separate colonies, at least two of which were from independent
PCR amplifications. When sequencing data was consistent, the sequences were submitted to
NCBI GenBank (http://www.ncbi.nlm.hih.gov). These were Ovis aries keratin intermediate-

filament Type II (KRT2.10) gene: Accession number AY437406; Ovis aries high-sulphur keratin
IF-associated protein 3.2 (KAP3.2) gene: Accession number AY483216 and Ovis aries high-
glycine/tyrosine Type II keratin protein 6.1 (KAP6.1) gene: Accession number AY483217.
2.10. Statistical analyses
In order for any of the loci to be informative, they have to be heterozygous in the chosen sires
allowing the segregation of the sire alleles to be followed in the progeny and segregation
analyses performed. Segregation of the sire alleles within SL2 was observed and a chi-square
goodness of fit test performed to ascertain whether the sire alleles inherited by the progeny
occurred in a 1:1 ratio within the population. Any progeny which had the same genotype as
both its sire and dam was excluded from the association analysis since it was not possible to
determine which of the alleles had been inherited from the sire. The association of alleles of
KAP8 with all measured wool traits (MFD, FDSD, CVD, curvature, yield, yellowness,
brightness, comfort factor, staple length, staple strength, GFW and CFW) was then analysed
for each year of phenotypic data using an analysis of variance (ANOVA) tests using SPSS
version 13 (SPSS Science Inc., Chicago, IL, USA). The ANOVA model included sire allele and
gender as factors and a full factorial model was used. The analysis used assumed that the
ewes alleles effects were distributed randomly in progeny. The date of birth was not included
in the ANOVA because the progeny were half-sibs born in a five weeks period, and it was
assumed that variation in birth date was balanced across the half-sib in the segregation
analyses, and that none of the genes analysed had a significant effect on gestation length.
3. Results
Six loci (KAP3.2, KAP6.1, KAP7, KAP8, KRT2.10 and BfMS) were included in the study. All
of them were amplified successfully using PCR and polymorphism was detected in three
loci (KAP3.2, KAP8 and BfMS). Of the loci which were polymorphic, only KAP8 was
heterozygous for SL2 (Tables 3.1), and thus potentially informative as a genetic marker. The
remaining loci appeared to be homozygous in the sires, and thus uninformative. Table 3.2
shows the genotype of SL2 progeny at KAP8 locus.

Locus
No. of alleles
detected
SL1
genotype
SL2
genotype
Informative
1
(Yes / No)

KAP3.2 3 AA AA No
KAP6.1 1 AA AA No
KAP7 1 AA AA No
KAP8 4 AA AB Yes
2

KRT2.10 1 AA AA No
BfMS 3 AA CC No
1
Heterozygous = informative; homozygous = uninformative
2
Informative for SL2 only.
Table 6. Genotype results for the loci investigated in the study, indicating whether the sire genotype
was informative (heterozygous) or non-informative (homozygous).

Electrophoresis 206
Lamb
identity
Ewe
identity
Lamb
genotype
1027 86 AB
1028 162 AA
1029 57 BB
1030 114 AA
1031 59 AB
1032 59 AA
1033 105 AA
1034 89 AA
1035 51 AB
1036 49 AD
1037 120 AA
1038 56 AB
1039 65 AC
1040 47 AB
1041 119 BB
1045 77 BB
1046 155 AA
1048 14 BB
1049 130 AA
1050 161 AA
1051 84 BB
1052 150 BB
1053 17 BB
1054 NT AA
1055 113 AB
1056 140 BB
1057 21 AB
1058 87 AA
1059 135 AB
1060 137 AA
1061 117 AC
1062 38 AB
1063 148 AB
1064 68 AB
1065 . ?
1067 116 AA
1068 58 ?
1069 64 AA

?
The genotype of the sheep could not be ascertained

Table 7. Genotype of KAP8 SL2 progeny

3.1. KAP8 (Polymorphic and informative in SL2)
Four banding patterns were identified for the KAP8 microsatellite amplimer using PCR-
SSCP typing methods, and these were named A, B, C and D (Figure 3.1). The alleles were
not sequenced. Mendelian inheritance was observed in SL2 half-sib family for KAP8 (Table
3.2). A Chi-square goodness of fit analysis to test whether the segregation of the sire alleles
differed from a 1:1 ratio confirmed normal Mendelian segregation (Table 3.3).

Figure 3. PCR-SSCP of the 124 bp amplimer of the KAP8 microsatellite showing the four alleles
identified. Amplimers were electrophoresed on a 10% non-denaturing acrylamide/bis-acrylamide gel
for 4 hours, 200 V at room temperature (20
o
C). Genotype of an individual animal is shown below each
lane. SL2 genotype (AB) is bolded, and his randomly selected half-sib progeny are shown in italics.

SL2 Genotype AB
Number of progeny inheriting allele A 17
Number of progeny inheriting allele B 12
Number of progeny genotyped same as the sire 5
Total number (n) 34
2 0.8621
P-value1 0.3532
Table 8. Segregation of sire alleles within SL2 sire-line. Chi-square goodness of fit was used for to
ascertain whether the sire alleles inherited by the progeny occurred in a 1:1 ratio within the population.
Probability values (P values) are given.
1
A P-value > 0.05 means that the allele segregation did not differ
significantly from a 1:1 ratio.

Electrophoresis 208
3.1.1. Genotype
SL1 was homozygous at the KAP8 locus based on SSCP gel patterns, and hence
uninformative. SL2 was heterozygous at the KAP8 locus, having the genotype AB.
Eleven out of 36 progeny had the genotype AB (Table 3.2), which was the same as that of
the sire. The genotype of the ewes for these lambs was subsequently determined. Five
of the ewes genotyped as AB, and the progeny of these ewes were excluded from
further statistical analysis as the allelic contribution from the sire could not be
determined.
3.1.2. Association between segregating sire alleles and wool traits
The sire alleles at the KAP8 locus showed a Mendelian pattern of inheritance and segregated
in a 1:1 ratio in the progeny of each half sib (Table 3.3). Statistical analyses within sire SL2
half-sib family showed that there were no association between the sire alleles (or gender)
and variation of wool traits.
3.1.3. Power analyses
The number of differences between alleles within sire-lines which were not statistically
significant suggested the possibility of Type II errors (failing to detect a difference
when in fact there is one). To address this issue, a power analysis was conducted for
each trait within each of the sire-lines to determine whether the sample sizes available
were adequate to detect at least 10% differences between alleles, within each sire-line,
at P<0.05 with 80% power, i.e. nper allele= (8 2 ERROR MEAN SQUAREestimate)/
(0.1 TRAIT AVERAGEacross sire-lines)
2
.
This equation was then rearranged to allowed the actual detectable difference to be
calculated for each sire-line, i.e. % detectable difference = [
(82EMS/ per allele)
n /TRAIT
AVERAGEacross sire-lines] 100. A power analysis was performed for the KAP8 data. Wool trait
measurements were only taken at 12 months for the SL2 half-sib family. There were
inadequate SL2 progeny numbers (n=29) to detect a 10% difference between sire allele
groups for yield, curvature, CVD, FDSD, staple length, brightness and yellowness (CFW
and GFW were not measured). A comparison of the smallest detectable difference between
sire-allele groups with the progeny numbers used with the observed difference between the
sire-allele groups is shown in Table 3.4.
3.2. KAP3.2 and BfMS (Polymorphic, but uninformative)
KAP3.2 and BfMS were found to be polymorphic in the progeny used in this study,
although they appeared to be homozygous for both sires used (Figures 3.2 and 3.3,
respectively). This was confirmed with cloning and sequencing amplimers derived from
sire SL1.

Sire-line Nlambs Trait
1

Trait
average
2

EMS
3

N per allele to detect at
least a 10%
difference
N lambs required to
detect a 10%
difference
SL2 29 Prickle factor 1.61 1.75 1080 2159
29 MFD 19.07 10.57 46 93
29 FDSD 3.89 67.23 7103 14207
29 CVD 20.46 91.34 349 698
29 Curvature 94.58 0.45 0 0
29 Yield 71.84 9.79 3 6
29 Staple length 73.33 2.04 1 1
29 Staple strength 31.30 12.58 21 41
29 Brightness 69.92 0.27 0 0
29 Yellowness -2.83 135.71 27195 54389
1
MFD: mean fibre diameter; FDSD: fibre diameter standard deviation; CVD: coefficient of variation of fibre diameter;
GFW: greasy fleece weight; CFW: clean fleece weight.
2
Across all progeny measured.
3
Error Mean Square-taken from
the ANOVA for each individual trait.
Table 9. Sample size required to detect at least a 10% difference between KAP8 sire allele groups in the
wool traits list for each sire-line, at P<0.05 with 80% power.

Sire-line Nlambs
Age
(months)
Trait
1

Trait
average
2

Smallest
detectable
difference (%)
3

Difference
observed between
alleles (%)
SL2 29 12 Prickle factor 1.77 86.3 29.5
29 12 MFD 19.21 17.9 3.2
29 12 FDSD 3.91 221.3 1.9
29 12 CVD 20.45 49.1 -0.4
29 12 Curvature 97.04 0.7 6.4
29 12 Yield 72.69 4.6 1.1
29 12 Staple length 33.56 2.0 4.3
29 12 Staple strength 69.06 11.9 22.6
29 12 Brightness -2.97 0.8 -2.1
29 12 Yellowness 72.60 -433.1 7.1
1
MFD: mean fibre diameter; FDSD: fibre diameter standard deviation; CVD: coefficient of variation of fibre diameter.
2
Across all progeny measured.
3
At 80%.
Table 10. A comparison of the smallest detectable difference between KAP8 sire-allele groups with the
progeny numbers used and the observed difference between the sire-allele groups means for each wool
trait measured.
3.3. KAP6.1, KAP7 and KRT2.10 (Non-polymorphic in SL1 and SL2)
Polymorphism could not be detected at the KAP6.1, KAP7 and KRT2.10 loci in any of the
animals used in this study. KAP 7 was sequenced, and nucleotide sequences from SL1 KAP7
amplimer (GenBank accession number AY791846) was aligned with the published KAP7
gene by Kuczek and Rogers (1987); GenBank accession number X05638) which shows two
unique sequences (Figure 3.4).

Electrophoresis 210

Figure 4. PCR-SSCP analysis of the 424 bp amplimer of the KAP3.2 gene showing the three alleles
identified (A, B and C). The genotype of an individual animal is shown below each lane.

Figure 5. PCR-SSCP analysis of the 200 bp amplimer of the BfMS microsatellite showing the three
alleles identified (A, B and C) using a half-sib test family. The genotype of an individual animal is
shown below each lane. Sires SL1 and SL2 genotypes are bolded.


Figure 6. Alignment of the KAP7 gene sequence cloned from sire MV144-58-00 (Accession number
AY791846) with Kuczek and Rogers (1987) published KAP7 gene (Accession number X05638). Upstream
and downstream primers are underlined and the start and stop codons are bolded). Dashes represent
same nucleotides to the nucleotide above and dots represent nucleotides missing in the other sequence.

Electrophoresis 212
Sex Average
(cM)
Female
(cM)
Male
(cM)
Locus code Marker Marker description or associated gene
101.9 84.3 119.3 \BM4129 BM4129 Sequence tagged site
104.1 86.6 122.0 \UCDO31 UCD031 RAPD Marker
107.1 89.0 125.2 \MCM58 MCM58 Microsatellite
111.3 91.7 130.5 \BL41 BL41 VANGL1
111.3 91.7 132.2 \BM723 BM723 STS
111.3 91.7 132.2 \BM723 BM723A STS
113.8 92.9 133.9 \OARAE57 AE57 Microsatellite
123.4 105.2 142.2 \MCMA6 MCMA6AH ?
124.5 106.8 142.2 \MCMA6L MCMA6AL ?
124.5 106.8 142.2 \BMS482 BMS482 Sequence tagged site
124.5 106.8 142.2 \CSSM054 CSSM54 Phosphoglycerate dehydrogenase
126.0 107.9 143.2 PRPF3 BMS963 PRPF3 protein
126.0 108.9 143.2 ARNT RME23
Aryl hydrocarbon receptor nuclear
translocator
126.0 108.9 143.2 THH TRHY Trichohyalin
127.3 111.1 144.3 \RM065 RM65 Dinucleotide repeat
132.0 115.3 149.1 ~CSAP033E CSAP33E Microsatellite
134.9 120.0 150.4 IGSF9 KIA1355 Immunoglobulin superfamily 9
135.7 121.5 150.4 ATP1A2 INRA6 ATPase
137.0 121.5 152.9 ADAMTS4 ADAMST4 ADAM metallopeptidase
139.8 122.7 156.8 \URB006 URB006 Sequence tagged site
143.6 127.6 160.4 \BM6438 BM6438 Sequence tagged site
143.6 127.6 160.4 OLIG2 OLIG2 Oligodendrocyte transcription factor 2
144.8 127.6 162.2 \SRCRS23H SRCR23H ?
144.8 127.6 162.2 \TGLA49 TGLA49 Microsatellite
144.8 127.6 162.2 \DVEPC88 DVEPC88 Neu associated kinase
145.3 127.6 163.1 KRTAP7-1 KAP7HAP Keratin associated protein 7.1
145.3 127.6 163.1 KRTAP7-1 KAP7_B Keratin associated protein 7.1
145.3 127.6 163.1 KRTAP8-1 KAP8 Keratin associated protein 8.1
145.3 127.6 163.1 KRTAP7-1 KAP7_M Keratin associated protein 7.1
145.3 127.6 163.1 KRTAP11-1 1-105 Keratin associated protein 11.1
145.4 127.6 163.2 KRTAP6-1 KAP6 Keratin associated protein 6.1
145.8 127.6 164.0 GRIK1 GRIK1 Glutamate receptor, ionotropic, kainite 1
149.6 131.5 168.1 APP APPO10 Amyloid beta (A4) precursor protein
150.5 131.5 170.2 \DVEPC117 DVEP117 Sequence tagged site
150.5 131.5 170.2 \DVEPC117 DVEPC96 Sequence tagged site
153.2 132.8 173.1 \DVEPC128 DVEP128 Neural cell adhesion molecule 2
157.1 138.0 176.7 \RM095 RM095 Dinucleotide repeat
158.1 138.0 177.6 \MAF64 MAF64 Dinucleotide repeat
169.2 150.1 188.2 \ILSTS004 ILSTS04 Sequence tagged site
171.1 152.6 188.2 \DVEPC54 DVEPC54 Microsatellite
174.4 154.8 194.2 \MCMA8 MCMA8 Sequence tagged site
176.0 154.8 197.2 \MNS94 MNS94A Microsatellite
193.1 169.9 216.0 \CSSM004 CSSM04 Microsatellite
200.0 177.1 223.6 \UCDO46 UCD046 ?
Figure 7. Linkage map for part of ovine chromosome 1 (modified from
http://rubens.its.unimelb.edu.au/~jillm/jill.htm). The bolded genetic markers were investigated in this
study.

Sex Average
(cM)
Female
(cM)
Male
(cM)
Locus Code Marker Marker description or associated gene
149.6 151.4 148.8 \BM827 BM827 Microsatellite
152.5 151.4 153.4 \MCM141 MCM141A ?
153.1 151.4 154.3 \OARSHP2 SHP2 Microsatellite
163.1 155.2 170.4 \BP1 BP1 Blood pressure QTL1
163.1 155.2 170.4 \DU469297 DU469297 ?
165.6 160.5 170.4 \EPCDV025 EPCDV25 ?
167.1 166.2 170.4 KITLG SCF KIT Ligand
168.7 166.6 172.4 \UCDO13 UCD013 ?
170.7 166.6 174.3 KERA KERA Keratocan
170.7 166.6 174.3 LUM CSAP19E Lumican
177.8 172.1 182.4 \AGLA293 AGLA293 Microsatellite
179.4 174.9 183.5 ~CSAP017E CSAP17E Microsatellite
179.4 174.9 183.5 \OARFCB5 FCB5 Dinucleotide repeat
179.4 174.9 183.5 GLYCAM1 GLYCAM1
Glycosylation dependant cell adhesion
molecule
179.4 174.9 183.5 \OARHH38 HH38 Microsatellite
180.0 176.2 183.5 RARG RARG Retinoic acid receptor 8
182.9 178.7 186.5 KRTHB* KRT2.10 Keratin
183.9 181.2 186.5 KRTHB* KRT2.13 Keratin
183.9 181.2 186.5 \BMC1009 BMC1009
Similar to intermediate filament type II
keratin
186.3 181.2 190.9 \CABB011 CABB11 Genomic survey sequence
188.2 185.3 190.9 \CSSM034 CSSM34 Microsatellite
188.2 185.3 190.9 HDAC7A KD103 Histone deacetylase 7A
188.2 185.3 190.9 \UCDO52 UCD052 ?
195.5 188.6 201.0 \BL4 BL4 Bell-like homeodomain protein 4
197.0 190.4 202.8 LYZ LYZ Lysozyme
198.6 191.8 204.6 \CSRD2125 CSRD125 ?
199.1 191.8 205.5 IFNG KP6 Interferon gamma
199.1 191.8 205.5 IFNG IFNG Interferon gamma
199.1 191.8 205.5 IFNG IFNGHAP Interferon gamma
202.2 195.4 207.8 \BMS1617 BMS1617 STS
204.1 196.7 210.6 \OARVH34 VH34 Microsatellite
206.2 197.7 213.7 \BR2936 BR2936 Sequence tagged site
207.0 197.7 215.5 \OARVH130 VH130 Microsatellite
207.0 197.7 215.5 \MAF23 MAF23 Microsatellite
209.0 199.0 218.1 \OARCP43 CP43 Microsatellite
214.7 208.6 219.8 \RM154 RM154 Tandem repeat region
218.5 211.3 223.7 IGF1 IGF1 Insulin like growth factor
218.5 211.3 223.7 IGF1 IGF1.B Insulin like growth factor
218.5 211.3 223.7 IGF1 IGF1HAP Insulin like growth factor
218.5 211.3 223.7 IGF1 CSAP40E Insulin like growth factor
223.9 215.4 231.6 \CSRD2111 CSRD111 ?
224.4 215.4 232.5 ~CSAP009E CSAP09E ?
Figure 8. Linkage map for part of ovine chromosome 3 (modified from
http://rubens.its.unimelb.edu.au/~jillm/jill.htm). The bolded genetic markers were investigated in this
study.

Electrophoresis 214
4. Discussion
Four alleles, designated A, B, C and D were identified at the KAP8 (CA)n repeat
microsatellite locus using PCR-SSCP in this study. The microsatellite at the KAP8 locus was
included in the study because this region is highly polymorphic, with 15 alleles previously
reported (Wood et al., 1992) using denaturing polyacrylamide gel electrophoresis, while
Parsons et al. (1994a) detected four allelic fragments (123, 125, 133 and 139 bp) at the same
locus using the methods by Wood et al. (1992) in a Merino half-sib family. Only SL2 was
heterozygous at the KAP8 microsatellite in this study. SL1 was homozygous, despite the
reported highly polymorphism in this locus (Wood et al., 1992). The method used to detect
polymorphism in this study differed to that of (Wood et al., 1992), which used denaturing
polyacrylamide gel electrophoresis. In this study, PCR-SSCP was used because this
technique is simple, sensitive, relatively inexpensive and routinely used in the laboratory
where the research was carried out. It is possible that if the original technique was
employed, more alleles may have been observed at this locus.
Neither of the SL2 alleles were associated with variation in the wool traits that were
measured (data not shown). The possibility of this locus having an affect on wool traits
cannot be ruled out however, because only two alleles (that were the genotype of SL2) were
analyzed, and that the sample numbers used in the study were relatively small (n = 29).
Power analysis results (Table 9.5) showed that the observed differences between the sire
allele groups were smaller than the smallest detectable difference for MFD, FDSD, CVD,
curvature, yield, staple length, brightness and yellowness and therefore the possibility of
making a Type II error (i.e. not detecting an association when there was one) is likely.
Variation in MFD has previously been significantly associated with alleles at the KAP8 locus
(Parson et al., 1994a). The authors did not describe the alleles associated, and no sequence
data was presented. Though alleles at the KAP8 microsatellite locus were not sequenced in
this study, it is possible that SL2s alleles were different from those associated with
differences in average MFD by Parsons et al., (1994a).
Three alleles, designated A, B and C were identified at the KAP3.2 locus. However, both sire
lines were homozygous, and thus uninformative. McLaren et al. (1997) identified two alleles
at the KAP3.2 locus using PCR-SSCP methods. KAP3.2 (together with KAP1.1, KAP1.3 and
K33) have been mapped to ovine chromosome 1 (Figure 3.5). Variations in all of the three
genes (KAP1.1, KAP1.3 and K33) have been previously associated with variation in wool
traits (Itenge et al., 2009; Itenge et al., 2010; Rogers et al., 1994b). It is therefore suggested that
sires that are heterozygous get investigated in further studies. Three alleles, designated A, B
and C were identified at the BfMS microsatellite. Bot et al. (2003) reported eight alleles at the
BfMS locus. Two of these alleles were significantly associated with CFW and GFW.
However, both sire lines were homozygous, and thus uninformative at the BfMS locus.
Polymorphism could not be detected at the KAP6.1, KAP7 and KRT2.10 loci in this study,
although all of these genes have been reported to be polymorphic in the literature (Parsons

et al., 1993; McLaren et al., 1997). The reported polymorphism in KRT2.10 (two alleles) and
KAP7 (four alleles) was identified using PCR-RFLP (McLaren et al., 1997) whereas the
polymorphism within KAP6.1 (two alleles) was revealed with PCR-SSCP of AluI-digested
PCR amplimers (McLaren et al., 1997). Parsons et al. (1993) reported a diallelic
polymorphism using BamHI PCR-RFLP to give alleles designated A1 (24.5 kb) and A2 (14.1
kb). However, no sequence data was presented. Since only two KAP6.1 alleles have
previously been reported, thus it was accepted that SL1 was homozygous at this locus
without further sequencing although this locus could still be polymorphic which only
sequencing would reveal. The KAP6.1 amplimers were also subjected to a variety of PCR-
SSCP conditions in an effort to detect sequence variation. Digestion of the amplimer with
AluI or BamHI as per McLaren et al. (1997) was not performed, however, and it is possible
that this may have revealed variation at KAP6.1. KRT2.10 has been mapped to ovine
chromosome three and two alleles have been reported at the KRT2.10 locus using a BsrDI
PCR-RFLP (McLaren et al, 1997). Genes coding for the KRT proteins are highly conserved
during evolution (Powell, 1996; Marshall and Gillespie, 1982), and do not have much
variation within them. Therefore, it was easy to accept that the KRT2.10 locus (with only
two alleles) was likely to be homozygous. The fact that the KRT proteins are highly
conserved during evolution (Powell, 1996; Marshall and Gillespie, 1982) suggests that genes
coding for these proteins are intolerant to major changes and that they are very important to
the integrity of the wool fibre.
5. Conclusion
Loci that were polymorphic, but uninformative in this study (KAP3.2, BFMS) )need to be
investigated further. Sires that are heterozygous at these loci need to be identified and used
in half-sib analysis. Other loci that map to the same chromosome regions as the keratin
genes investigated in this study are also worth of investigating in the future as potential
gene markers for wool quality traits. On chromosome 1, future genes of interest include
KAP11.1 and genes coding for trichohyalin (a very important wool follicle protein) (refer to
Figure 3.5). On chromosome 3, loci of interest include KRT2.13, BMC1009 (Similar to
intermediate filament type II keratin), RARG (Retinoic acid receptor 8) and IGF1 (insulin
like growth factor) (refer to Figure 3.6). It is worth noting that previous studies by Damak et
al. (1996) have shown positive effects of IGF1 on wool traits. Transgenic sheep produced
by pronuclear microinjection with a mouse ultra-high-sulphur keratin promoter linked
to an ovine IGF1 resulted in significant increase of CFW and bulk in transgenic sheep
compared to non-transgenics, although MFD did not show significant differences (Damak et
al.,1996).
There are other genes that have not been positioned on the linkage map that may be
potential gene markers for wool quality traits. Some of these have already been associated
with wool quality traits. These include the retinoic acid receptor (RAR) (Nadeau et al.,
1992), homeobox proteins (HOX2) (Nadeau et al., 1992) and growth hormone (Hediger et al.,

Electrophoresis 216
1990). Retinoic acid induces expression of genes such as homeobox and KRTs and there is a
possibility that retinoic acid is involved in the regulation of KAPs, given its genomic
position on chromosome 11 (Parsons et al, 1994c). Growth hormone has been positioned on
chromosome 11 through in situ hybridization (Hediger et al., 1992). Furthermore, there have
been numerous reports with variable effects of growth hormone on wool characteristics. For
example, Ferguson (1954) and Johnson et al. (1985) observed significant increase in GFW
during the injections of growth hormone. In contrast, no effect of recombinant growth
hormone on CFW was found in a study by Zainur et al. (1989). Wheatley et al. (1966) found
that growth hormone suppressed wool growth and that there was accelerated wool growth
after withdrawal of growth hormone. Polymorphism at the genes encoding growth
hormone have been reported (Valinsky et al., 1990; Wallis et al., 1998; Sami et al., 1999), and
different alleles of growth hormone may affect wool growth in different ways.
5.1. Genetic markers versus genetic engineering
The search for genetic markers affecting wool quality traits is very different to genetic
engineering (GE) and transgenesis. While GE involves the manipulation or modification of
genetic composition of an organism, and transgenesis requires the development and use of
transgenic animals, the former detects changes within the genetic make-up of an organism,
but does not alter it. Marker-assisted selection may therefore be better preferred within the
wider non-scientific community, than the use of transgenic sheep to produce superior
wool traits. Transgenesis in sheep is also still in its infancy, and successful transgenesis rates
are very low (less than 13%) (Powell et al., 1994). This makes marker-assisted-selection a
more efficient, relatively cheaper and easier technique to improve wool quality traits than
sheep transgenesis. The debate on GE will most likely continue and intensify especially
where animals are involved. However, marker-assisted technology in livestock offers a
powerful "green" alternative to gene manipulation.
5.2. Advantages of marker-assisted selection
Genetic markers are not affected by environmental noise and would allow sheep breeders to
select animals with improved wool characteristics at an early age and cull the non-desirable
lambs. This would speed up the process of genetic selection and decrease the generation
interval. There is therefore a potential to select superior animals very early in life and not
have to wait for an animal to reach its adult life to demonstrate that it has superior wool
quality. This has the advantage of overcoming the limitation of blind selection, and
increase the accuracy and efficiency of selection and result in a more profitable wool
industry with direct benefits of cost to the consumer.
Author details
Department of Animal Science, Faculty of Agriculture and Natural Resources, University of
Namibia, Namibia

Acknowledgement
I thank the Almighty God for everything in my life. I wish to thank my Honours
supervisor, Prof. Jim Reynoldson from Murdoch University and my PhD supervisors
(Prof. Jon G. H. Hickford from Lincoln University and Dr. Rachel Forrest from Eastern
Institute of Technology). I am very grateful for the AUS-AID and NZAID scholarships
that I received from the Australian and New Zealand governments, respectively. I am also
very grateful for the Staff Development Fellowship award that I received from the
University of Namibia.
6. References
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genome scan for QTL affecting fleece and wool traits in Merino sheep. Wool Technology
and Sheep Breeding. 49:88-9.
Benavides, M. V. and Maher, A. P. (2000) Quantitative genetic studies on wool yellowing
in Corriedale sheep. II. Clean wool colour and wool production traits: Genetic
parameter estimates and economic returns. Australian Journal of Agricultural Research.
51:191-196.
Bot, J., Karlsson, L. J. E., Greef, J. and Witt, C. (2003) Association of the MHC with
production traits i 50:349-358.
Damak, S., Su, H., Jay, N. P., and Bullock, D. W. (1996) Improved Wool Production
in Transgenic Sheep Expressing Insulin-like Growth Factor 1. Biotechnology. 14:185
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Electrophoresis 220
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Chapter 12
Temporal Expression of Isozymes, Alozymes
and Metabolic Markers at the Early Ontogeny
of Prochilodus argenteus (Characidae
Prochilodontidae) from So Francisco Basin,
Trs Marias, Minas Gerais, Brazil
Flavia Simone Munin, Maria Regina de Aquino-Silva,
Maria Luiza Barcellos Schwantes, Vera Maria Fonseca de Almeida-Val,
Arno Rudi Schwantes and Yoshimi Sato
http://dx.doi.org/10.5772/45949
1. Introduction
It is well-known phenomenon that all changes in a population depend on reproduction,
growth and mortality. Analysis of differential expression of genes which encode enzymes
has made it possible to relate developmental changes at the molecular level to the general
physiological changes whit accompany differentiation. According to [1], the specific protein
expression during the different stages of development indicates the gene activity that can be
started or turned off during embryogenesis. These properties make the multiple forms
particularly interesting for the beginning ontogenetic gene regulation. Many enzymes exist
as isozymes, and these isozymes are often differentially expressed during embryogenesis
[2]. Thus, the enzymatic studies, including isozymes and allozymes must be informative
about genes activity and regulation during the early development. The electrophoretic and
kinetics studies can be employed to investigate when genes are started during development,
and how these enzymes are increased or reduced during ontogeny.
The Trs Marias hydroelectric station was built in 1960s in the main canal of the So
Francisco River in Minas Gerais state. According to [3] it has been observed that several
migratory fish collected at downstream region closet to the dam, are smaller in size and
have immature gonads during the spawning season. A distinct condition is observed 30 Km
downstream from the dam, where these animals generally are normal-sized and have

Electrophoresis 222
developed gonads. These facts reveal that conditions on this region are less favorable to they
reproduction. There are two possible factors, among others caused by hydroelectric station,
as a lower water temperature and oxygenation.
The Prochilodontidade family is composed by iliophagous-migratory fish, that swimming
upstream to deposit their eggs every year in the end of dry season. Prochilodus argenteus, is
an endemic fish specie, that migrate to the water spring for the spawning. It is called
curimbat, and today is endangered specie of fish from So Francisco basin.
2. Objective
The general objectives are to verify the correspondence among enzymatic levels, early
ontogeny and physiological activities, as well as the correspondence between spatial and
temporal enzymatic activities and their metabolic role, through electrophoretic and
spectrophotometric studies, verifying the moment of gene activation, monomorphism or
polymorphism of the enzymatic systems, synchrony or asynchrony of paternal and maternal
genes, what kind of metabolism (glycolytic or aerobic) is predominant and changes in
enzymes activities that occurring during the early ontogeny of P. argenteus.
3. Material and methods
- Five adults and mature couples of P. argenteus were induced to reproduce through a
hypophysation process. After the gametes extrusion, fertilized eggs or larvae were
collected at 0, 4, 8, 12, 16, 20, 36, 60, 87, and, 135 hours post fertilization (h.p.f.) and
immediately iced. Temperature, solved oxygen and pH of water measured in each
incubation tank, whit a Horiba U10 instrument. After the reproduction, the adults were
sacrificed and, muscle, heart, and liver were collected and iced.
- Were performed electrophoretic analysis for alcoholic dehydrogenase (ADH, EC
1.1.1.1), glucose phosphate dehydrogenase (GPI, EC 5.3.1.9), glucose 6 phosphate
dehydrogenase (G6PDH, EC 1.1.1.49), isocitrate dehydrogenase NADP-dependent
(IDHP, EC 1.1.1.41), lactate dehydrogenase (LDH, EC 1.1.1.27); malate dehydrogenase
(MDH, EC 1.1.1.37), malic enzyme NADP-dependent (ME, EC 1.1.1.38), 6-
phosphogluconate dehydrogenase (6PGDH, EC. 1.1.1.44), and enzymes activities
analysis for lactate dehydrogenase (LDH, EC1.1.1.27), malate dehydrogenase (MDH,
EC1.1.1.37), piruvate kinase (PK, EC 2.7.40), and citrate synthase (CS, EC 4.1.3.7).
- A small piece of each tissue for adults or a few eggs or larvae was/were homogenized
(w/v) in 50mM phosphate potassium buffer (pH 7.0), using a manual homogenizer and
centrifuged at 27000g for 30 min at 4
o
C in a Sorvall RC5B centrifuge. The resulting crude
extracts were used for electrophoretic analysis. Electrophoreses were carried out
employing a horizontal gels containing 13% (pH 8.7) (for GPI, G6PDH, LDH and, 6PGDH)
or 14% (pH 6.9)(for ADH, IDHP, sMDH, mMDH, ME ) corn starch prepared according to
[4]. A voltage gradient of 5V/cm was applied for 12-14 h at 4
o
C. After electrophoreses, the
gels were sliced lengthwise and the lower halves incubated in a specific staining solution.
The histochemical solutions used were described by [5] and modified by us, for G6PDH,
Temporal Expression of Isozymes, Alozymes and Metabolic Markers at the Early Ontogeny of Prochilodus
argenteus (Characidae Prochilodontidae) from So Francisco Basin, Trs Marias, Minas Gerais, Brazil 223
6PGDH, IDHP, LDH and ME. For ADH and MDH, we used the solution described by [6]
with modifications and, for GPI [7]. Nomenclature for the gene loci and iso/allozymes was
taken from [8]. Alleles were designated by number with *100 representing the most
frequent allele. Subsequent numbers refer to their relative mobility.
- The activities enzymes analyses were performed using revised proceedings by [9]. Was
used a GENESYS 2 spectrophotometer at 25C for activities measures. After each assay
the total protein was measured by Bradford method, using a wavelength 595 nm. The
graphics were performed in Excel and Origin 6.0 by Windows program, were calculated
median and stand deviation and the statistics Students Test.
4. Results
Conditions of temperature, solved oxygen and pH of the incubation water are in table 1.
Offsprings Temperature (
o
C) Solved oxygen (mg/ l) pH
1 (couple 1) 25.2 4.24 6.15
2 (couple 2) 25.1 4.27 6.12
3 (couple 3) 25.1 4.43 6.04
Table 1. Temperature, solved oxygen and pH measured of the incubation water in nursery tank.
5. Electrophoresis
Zimograms with phenotypes patterns detected from the couples of P. argenteus used for
reproduction are in table 2 and figure 1.
Couple 1 Couple 2 Couple 3
Male1 Female1 Male 2 Female 2 Male 3 Female 3
GPI-A* *100 *100 *100 *100 *100 *100
GPI-B* *-100/50 *-100 *-100/50 *-150/50 *-150 *50
G6PDH* *100 *100 *100 *100 *100 *100
IDHP-A* *100 *100 *100 *100 *100 *100
IDHP-B* *100 *100 *100 *100 *100 *100
LDH-A* *100 *29/100 *29/100 *29/100 *29/100 *29/100
LDH-B* *100 *100 *100 *100 *100 *100
MMDH* *100 *100 *100 *100 *100 *100
SMDH-A* *100 *100 *100 *100 *100 *100
SMDH-B* *100 *100 *100 *100 *100 *100
ME-1* *100 *100 *100 *100 *100 *100
ME-2* *100 *100 *100 *100 *100 *124
6PGDH* *100 *88/100 *100 *100 *88 *100
Table 2. Phenotypes detected in zimograms from the 3 couples of P. argenteus used for reproduction.

Electrophoresis 224

Figure 1. Electrophoretic patterns detected in couple 1 and offspring: A- GPI; B- G6PDH; C- IDHP; D-
LDH; E- MDH; F- ME and G- 6PGDH. m - muscle; h - heart; l - liver.
The zimograms showed variant loci: GPI-B* (*-100, *200 and, *-300), 6PGDH* (*88 and *118),
ME-1* (*124) and, LDH-A* (*16 and *-16). Another systems: G6PDH, IDHP and, MDH
showed no variation. For the G6PDH*, IDHP-A*, B*, s-MDH-A*, B*, m-MDH* and ME-1* and
ME-2* loci was not possible to detect the asynchrony or synchrony of gene expression,
because the paternal and maternal phenotypes were identical or both had 1 common allele
in heterozygotes (table 2).
6. Enzymes activities
The low high ratios L/H are presented in Figure 2. There were detected decreasing of these
ratios during the development, what indicated decreasing of B subunits and/or A subunits
synthesis. These subunits A synthesis were expected, because the muscle predominant
subunit is type A, the product of LDH-A* was detected late, 36 hours post fertilization, in
heteropolymeric form.

Figure 2. Low/high LDH ratios in different developing phases of offsprings of 3 couples of
P. argenteus.
The obtained ratios between the glycolytic enzyme LDH and MDH from different phases of
offspring developing from each pair, as well the medians calculated by the 3 couples
together offspring showed the greater oxidative metabolism/malate aspartate shuttle until
87 hours post fertilization are in Figure 3.

Figure 3. LDH/MDH ratios in different developing phases of offsprings of 3 couples of
P. argenteus.
0
1
2
3
4
0 4 8 12 16 20 36 63 87
Hours post fertilization
L
D
H

L
/
H

offspring 1
offspring 2
offspring 3
0
1
2
3
4
0 4 8 12 16 20 36 63 87
L
D
H
/
M
D
H
offspring 1
offspring 2
offspring 3

Electrophoresis 226
Differently of the observed for the LDH/MDH ratios, the LDH/CS observed ratios, for the 3
progenies, as well as the ratios obtained by the median of each enzyme for the 3 issues from
the 3 couples presented all the values greater than one, and the greatest values in the stages
between 12 and 16 hours post fertilization, decreasing after that (Figure 4). The PK/CS ratios
and its medians observed were not great when compared with LDH/CS ratios, showing the
maximum values at 16 hours post fertilization (Figure 5). The median value obtained for the
4 enzymes activities, showed (Figure 6).

Figure 4. LDH/CS ratios in different developing phases of offsprings of 3 couples of P. argenteus.

Figure 5. PK/CS ratios in different developing phases of offsprings of 3 couples of P. argenteus.
0
10
20
30
40
50
60
70
80
0 4 8 12 16 20 36 63 87
L
D
H
/
C
S
offspring 1
offspring 2
offspring 3
0
2
4
6
8
10
12
14
16
18
20
0 4 8 12 16 20 36 63 87
P
K
/
C
S
offspring 1
offspring 2
offspring 3

Figure 6. Quantitative glycolytics and e oxidatives average enzymes alterations in the early ontogeny of
the offsprings of the 3 couples of P.argenteus, and H= hatching; 36 hours: starting LDH-A*.
7. Discussion
Temporal iso/allozymes expression during early ontogeny of P. argenteus.
The investigation of gene expression in vertebrates embryos indicated that developing
program is determined in part by the transcripts of matter genes during the egg
maturation and the other part by the expression of the embryos genes after the
fertilization [10].
The later GPI* heteropolimeric detection at 16 hours post fertilization, indicates the both A
and B subunits were simultaneously synthesized in many cells of embryos. These B
homodimers were firstly detected, late at 36 hours post fertilization, like [11] also detected
late expression for this locus in L. cyanellus, between 25 and 38 h.p.f.. According these
authors, during the development of the L. cyanellus, the A homodimer levels were constant
before the B subunits levels have been increased. According to [12] P. scrofa, detected the B
homodimer activity after 12.5 hours after fertilization. According [13], A/B ratios change due
the miotonic differentiation. This hypothesis can be accepted since the first skeletal muscle
contractions were visible in L. cyanellus, P. scrofa and P. argenteus simultaneously at early
subunits B expression.

Electrophoresis 228
The G6PDH from adults and their offspring presented one monomorphic locus liver
restricted. In ontogenetic studies the single band was detected 36 hours post fertilization,
revealing the late expression, like was observed by [11] in L. cyanellus at between 11 and 14
h.p.f.
The IDHP (IDH NADP
+
- linked) catalyzing the carboxylation of 2-ketoglutarate, is not a
strictly a Krebs cycle enzyme. Nevertheless, it is sometimes utilized by organisms to
generate NADPH for fat biosynthesis. Under this conditions the carbon source for the
reaction is usually glutamate, which is transaminated 2-ketoglutarate, the immediate
substrate for the cell simultaneously generates isocitrate and thus helps to augment the pool
of Krebs cycle intermediates (anaplerotic reactions) [14]. On the adult animals studied the
expression of the IDHP loci was bidirectional divergent due the IDHP-A* is skeletal and
heart muscle predominant and the IDHP-B* is liver restricted. The IDHP-A* locus had an
early expression while the IDHP-B* had a late expression at 36 h.p.f.
On this study the LDH was product from two loci: the polymorphic LDH-A* in skeletal
muscle with two variant alleles *-16 and *29 and, the monomorphic LDH-B* predominant
in liver and heart. In the electrophoretic studies realized by the 3 offspring, the B subunit
was the only one band observed in all stages of developing in the same level. This early
expression of the B subunit was described for other teleosts as well as for other
vertebrates [15; 16; 17]. The LDH-A* loci product, the A subunit had been detected 36
h.p.f. in heteropolimeric form. Author [15] detected this locus activity at 34 h.p.f. it can be
related to firstly muscle larvae contractions and has an important biochemical role,
at anaerobic energetic modulation, that could be one requisite to muscle cells
differentiation.
The sMDH-A* liver predominant, and muscle predominant sMDH-B* were non
monomorphics and did not presented the typical temporal divergence, probably reflecting
the heart loci expression, like detected by author [18] in P. scrofa. In L. cyanellus, the
homodimer sMDH-A* and heterodimer sMDH-B were detected at the same time, early of
development. B homodimer was visible at 28-31 h.p.f. [11].
There are two forms from vertebrates malic enzyme (ME), the mitochondrial (less anodic
mME) and citosolic (more anodic sME) [19]. In this work we called the ME-2 for mME and
ME-1for sME . The ME-2 detected in muscle and heart appeared at 36 h.p.f. and the ME-1
liver-restricted was detected at 63 h.p.f. These enzymes provide the Krebs cycle with
metabolic intermediates and play an important role in muscular gluconeogenesis in situ [20].
In the adult electrophoretic analysis the ME-1* isoform product was detected only in liver,
and the ME-2* in heart and skeletal muscle, bidirectional divergent expression pattern.
During the ontogeny both loci showed later expression: the ME-2* at 36 hours post
fertilization and the ME-1* at 63 hours post fertilization.
The 6PGDH liver-restricted enzyme, showed a new variant allele, the *88. And only one
band for this locus was detected at 16 hours post fertilization. Thus there are two loci
detected at 16 hours post fertilization: the GPI-B* muscle predominant and 6PGDH* liver
restricted, from glycolytic and 6-phosphogluconate pathways respectively. On this
moment the larvae movements were stronger inside of the egg, two hours before the
hatching. Were detected, at the early stage of development, enzymes from glycolysis
represented by GPI and LDH, the Krebs cycle by IDHP, mMDH, sMDH, gluconeogenesis,
and lipogenesis shuttle malate-aspartate in the mitochondria by sMDH and ME activities.
The Krebs cycle, it is important to mention, not only provides the catabolism of energy
compounds, but also serves in the formation of equivalent reducing required to other
metabolic pathways in the -ketoglutarate generation of anabolic ways precursors like as
oxaloacetate, (precursors in the amino acids formation) and citrate (which can be diverted
to the fatty acids synthesis). Only ME, whose principal function is the provision of
equivalent to reducing the fatty acids synthesis did not take any of his two loci expressed
since the beginning of development.
The ADH and SOD, enzymes related to detoxification, studied in adults, showed no
embryonic activity.
With 16 (6PGDH) and 36 (G6PDH) h.p.f. has been detected by the activity of the 6-
fosfogluconate, important NADPH generator which is used in the synthesis of fatty acids,
and pentose for the synthesis of nucleic acids. Author [21] analyzing the initial ontogeny of
the Erymizon sucetta, there ova and in the early stages of their development, high levels of
G6PDH and 6-PGDH suggesting that much of the glucose available free at that time, be
directed towards the pathway of 6-fosfogluconate.
In P. argenteus, the asynchrony from paternal alleles were detected within 2 loci only at
crossings where the parents had not alleles present in mothers, GPI-B* and 6PGDH* and
maternal alleles in the GPI-B* during P. scrofa ontogeny asynchrony during activation of
paternal alleles for GPI-B* was also detected [12]. Beyond this locus, [1, 15, 18] show
asynchrony paternal in: LDH-A*, sMDH-A* and B* and GPI- A* in P. scrofa. Author [22]
found, in studies of early ontogenetic development of zebra fish, Danio rerio, that the
expression of the mRNA of the way ADH3 of alcohol dehydrogenase is of maternal origin,
there asynchrony of expression between maternal and paternal genes.
This some late expression loci could be a result of its regulation on molecular and cellular
levels as proposed by [10] and [23]. The molecular events that lead to patterns of ontogenetic
early enzyme and isozyme expression are under genetic control, direct or indirect, and
represent the gene regulation in its broadest sense [24, 25]. Although this gene regulation
occurs in the embryo, many genes appear to be cast from the first moments of the early
ontogeny, ensuring that the basic enzymatic machinery is in operation in the most critical
phases of the development process, in which the body just gets energy from the egg reserve
and formed all the structures needed for their survival in the subsequent phases of life.
Apparently the asynchrony for father genes expression is more common that the mother
because the egg or ovum brings a wealth of content cytoplasmic, and organelles. According

Electrophoresis 230
[26] cases of asynchrony are rare in offspring from couples of the same species, and can be
caused by genetic changes in regulatory loci.
8. Enzymatic activities
According to [27], fat acids, glycogen and adenilic nucleotides (ATP, ADP e AMP) are the
more important energetic substrates from mature egg.
The LDH low/high ratios calculated in the initial phases of the early ontogeny of P. argenteus
(of 0 the 135 hours after the fertilization), average L/H values of each phase of the initial
ontogeny of the 3 couples sample that in the interval of 0 to the 12 hours after fertilization
occurred the biggest inhibition of the LDH and, therefore, the biggest amount of B subunits.
After this period, occurs the reduction of this inhibition and reasons L/H, produced,
probably, for the synthesis of subunits, whose detection occurs in electrophoresis 36 hours
after the fertilization.
The analyses of the reasons between indicating enzymes of anaerobic metabolism and the
MDH must be seen with exceptions when we try to measure the level of aerobic
metabolism, a time that this enzyme is involved also in the malate-aspartate shuttle and,
at least part of its activity, it must be related with this function. When we used laccolitic
enzyme in the reason is the LDH, average values shows predominance of the oxidative
metabolism / malate aspartate shuttle up to 87 hours after the fertilization, with its higher
value with 8 hours after the fertilization when it is differentiated head extremity and the
tail extremity. After this phase, considering the average reasons, an increase of the
glycolytic metabolism occurred, and in the morphogenetic development, the movements
if they intensify inside of the egg. When the glycolytic enzyme of the reason was the PK,
in all phases and in the averages of these ratios we observed predominance of the
oxidative metabolism and/ or malate-aspartate shuttle. The biggest reason was detected
36-87 hours after the fertilization when was detected an increase of the glycolytic
metabolism.
The observed values in the offspring of the 3 couples of P. argenteus, for LDH/CS and
average of these (even so not homogeneous, what it can have been consequence of the crude
extract use), had disclosed predominance of the anaerobic metabolism in all the analyzed
phases, being this, bigger between 12 and 16 hours after fertilization decaying after that. The
values of PK/CS and average of these not so high how much of LDH/CS, they after show to
its maximum (anaerobic metabolism) 16 hours post fertilization.
The water of the nursery used for the P. argenteus development, contained on average 4.3
mg oxygen/liter, more than what the water where the adult fish had been collected,
therefore the water that comes of the barrage passes for aeration process before arriving at
the nurseries. Thus, the higher enzymatic activity, in all the phases of the initial ontogeny
of the 3 couples of P. argenteus, kept in these conditions, was of the LDH, characterizing
the predominance of the anaerobic metabolism. The second more active enzyme was the
MDH what it would characterize high activity of the function malate-aspartate shuttle.
The aerobic metabolism, characterized for the activity of the CS, lowest of the 4 analyzed,
it was remained low. Soon after the hatching, the activities of 4 enzymes had suffered
increase what it would be in accordance with the active movement of the larvae soon after
hatching according to [28] with increase of the carbohydrates metabolism described for
[29].
Studies carried through with the teleost Misgurnus fossils [30] show the dependence, during
its initial development, of the use of the stored glycogen, as energy source. If it will be
possible to surpass these results for other species of teleosts, this would be the only
substratum of glycolysis in the embryos. According to [25], glycolysis and the
phosphogluconate way are the more important energy sources for the biosynthetic activities
and maintenance of the embryo morphology. The glucose is, doubtlessly, important in the
period between the fertilization and after-hatching, as indicated for the high levels of B
subunits from LDH and A subunits from GPI, as well as the high activity of the LDH,
during this period and for the appearance of the delayed subunits, in P.argenteus, P.scrofa [1,
12, 15, 18] and in E. sucetta [21].
The subunits B predominance since the first phases of the initial ontogeny of P.argenteus,
kinetically adjusted to the aerobic metabolism, in the lactate oxidase function, would supply,
it would supply to private the shuttle function malate-aspartate of the MDH, enzyme with
the highest activity detected here.
The adults of this species are migratory in time of piracema (November to February) and the
So Francisco basin river rough relief and the fish have to use of essentially anaerobic
metabolism (it has pulled out) to cross these obstacles.
Author details
Flavia Simone Munin
University of So Paulo, Pirassununga, So Paulo, Brazil
Maria Regina de Aquino-Silva
University of Paraba Valley UNIVAP, So Jos dos Campos, So Paulo, Brazil
Maria Luiza Barcellos Schwantes
Federal University of So Carlos, So Carlos, So Paulo, Brazil
Vera Maria Fonseca de Almeida-Val
National Institute for Amazon Research INPA, Manaus, Amazonas, Brazil
Arno Rudi Schwantes
Federal University of So Carlos, So Carlos, So Paulo, Brazil
Yoshimi Sato
Hydrobiology and Pisciculture Station

of CODEVASF, Trs Marias, Minas Gerais, Brazil

Electrophoresis 232
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Electrophoresis 234
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