Electronic Journal of Biotechnology ISSN: 0717-3458
© 2009 by Pontificia Universidad Católica de Valparaíso -- Chile
Vol.12 No.2, Issue of April 15, 2009
Received October 16, 2008 / Accepted January 12, 2009
DOI: 10.2225/vol12-issue2-fulltext-10
TECHNICAL NOTE
Measuring β-Galactosidase activity at pH 6 with a differential pH sensor
Cristian Acevedo*#
Biotechnology Center
Universidad Técnica Federico Santa María
Av. España 1680
Valparaíso, Chile
Tel: 56 2 7184526
Fax: 56 2 7764080
E-mail: cristian.acevedo@usach.cl
Matthias H. Stach
Institute of Bioprocess Engineering
Friedrich-Alexander University of Erlangen-Nuremberg
Paul Gordan Str. 3
91052 Erlangen, Germany
Anette Amtmann
Institute of Bioprocess Engineering
Friedrich-Alexander University of Erlangen-Nuremberg
Paul Gordan Str. 3
91052 Erlangen, Germany
Manuel E. Young
Biotechnology Center
Universidad Técnica Federico Santa María
Av. España 1680
Valparaíso, Chile
Juan G. Reyes
Chemistry Institute
Pontificia Universidad Católica de Valparaíso
Av. Brasil 2950
Valparaíso, Chile
Holger Huebner
Institute of Bioprocess Engineering
Friedrich-Alexander University of Erlangen-Nuremberg
Paul Gordan Str. 3
91052 Erlangen, Germany
Rainer Buchholz
Institute of Bioprocess Engineering
Friedrich-Alexander University of Erlangen-Nuremberg
Paul Gordan Str. 3
91052 Erlangen, Germany
Financial support: FONDEF Grant (DO2I1009), MECESUP Grant (UCV0206) and Doctoral Scholarship for Cristian Acevedo (CONICYT D21050588).
Present address: #Departamento de Ciencia y Tecnología de los Alimentos, Facultad Tecnológica, Universidad de Santiago de Chile. Avenida Ecuador
3769, Estación Central, Santiago, Chile.
Keywords: β-Galactosidase, delta milli pH, differential pH.
Abbreviations: β-Gal: β-Galactosidase
β-Gal/pH6: β-Galactosidase activity at pH 6
HK: hexokinase
K: Michaelis-Menten constant
t: time
v: enzymatic velocity
VMax: maximum enzymatic velocity
*Corresponding author
This paper is available on line at http://www.ejbiotechnology.info/content/vol12/issue2/full/10/
Acevedo, C. et al.
The β-Galactosidase activity at pH 6 is used as a cellular
marker to identify senescent cell cultures. The classic
method to identify this enzymatic activity is using
cytochemical staining with X-Gal after 16 hrs. In this
work, a differential pH sensor was used to measure βGalactosidase activity at pH 6. The measurement is easy
and only takes 3 min.
Normal somatic cells invariably enter a state of irreversibly
arrested growth and altered function after a finite number of
divisions. This process, termed cellular senescence
(Hayflick’s limit), is thought to be a tumor-suppressive
mechanism and an underlying cause of aging. Normal cells
express β-Galactosidase activity at pH 4, but senescent cells
express β-Galactosidase activity at pH 6 (called senescence
associated β-Galactosidase) (Dimri et al. 1995; Severino et
al. 2000; Maier et al. 2007). The β-Galactosidase activity at
pH 6 is used as a cellular marker to identify senescent cell
cultures. The classic method to identify β-Gal/pH6 is using
cytochemical staining with X-Gal (5-bromo-4-chloro-3indolyl-β-D-galactoside)
in
cells
fixed
with
glutaraldehyde/formaldehyde solution after 16-24 hrs
(Dimri et al. 1995). But, other methods have been
developed to measurement of β-Gal/pH6 activity
(Bassaneze et al. 2008), such as the fluorescent assays
using FDG (fluorescein di-β-D-galactopyranoside) (Yang
and Hu, 2004) or MUG (4-methylumbelliferyl-β-Dgalactopyranoside) in cell extract solutions (Gary and
Kindell, 2005), because it is necessary to have quantitative
and fast methodologies.
On the other hand, the differential pH measurement
technique is an analytical tool to detect and determine all
sorts of metabolites in biological samples. Much faster to
be performed and developed for a certain purpose than
HPLC protocols, and much cheaper than most enzymatic
Figure 1. Differential pH response of Hexokinase activity at pH 4 and 5. Hexokinase activities were measured at three concentrations
of glucose (0, 20 and 40 mM), at different levels of pH, using 2 units of hexokinase (2.0 U = 10 µL x 0.2 U/µL). Not significant responses
were determined at pH 4 and 5.
2
Measuring β-Galactosidase activity at pH 6 with a differential pH sensor
test kits. Instead of determining the amount of metabolites,
one can use this method to determine the amount of an
enzyme of interest by adding the substrate in excess.
The differential pH technique is based on the possibility to
correlate the pH variation, induced by the change in the
concentration of H+ or OH- by specific enzymatic
reactions. The technique measures differences in the order
of 10-3 units of pH using two micro glass electrodes of high
sensitivity, reason why the study and definition of the
system are essential for the standardization of new
procedures. This technique has been used to measure
substrates and enzymatic activities in biological systems
with high precision and quickly (Compagnone et al. 1995;
Luzzana and Giardino, 1999; Gast and Pingoud, 2001;
Luzzana et al. 2001; Tagarelli et al. 2004).
In this technical note, we developed a new technique for
measuring β-Galactosidase activity at pH 6 using a
differential pH sensor, which could be used in the future in
cell culture technology and tissue engineering applications,
in fast and quantitative way.
MATERIALS AND METHODS
The enzymatic system used consists of two reactions:
Figure 2. β-Galactosidase activity at pH 6 (β-Gal/pH6).
(A) Differential pH response of β-Gal system at pH 6 using 1.00, 0.50 and 0.25 units of β-Galactosidase (in triplicate).
(B) Purified kinetics of β-Gal system (means of sample-kinetics less means of blank-kinetics in absolute value). The linear section is
called slope. Black blocks 1.00 units, Gray blocks 0.50 units and white blocks 0.25 units.
(C) Plot type Lineweaver-Burk of β-Gal/pH6 activity (p < 0.05; ANOVA).
3
Acevedo, C. et al.
[Eq. 1]
[Eq. 2]
Since pKA values of ATP differ from the pKA values of
glucose-6P and ADP, the pH changes in the course of the
reaction (Luzzana et al. 2001). Our hypothesis is that the
pH changes will be a function of the amount of glucose,
providing an indirect method for the determination of the βGalactosidase activity according to Michaelis-Menten
kinetics.
The Hexokinase solution was prepared at 0.2 U/µL and the
β-Galactosidase standard solution at 0.1 U/µL. Both
enzymes were purchased from Sigma-Aldrich. The
reaction-buffer was prepared with citric-phosphate buffer
(20 mM), ATP (15 mM), MgCl2 (2 mM) and NaCl (150
mM). The pH of reaction-buffer was checked and adjusted
to 6.00 previously to use.
The differential pH device used was the CL-10 plus (mode
Init. String “CS4000”) (Eurochem, Italy). The kinetics in
the differential pH electrodes was monitored for 200 sec at
37ºC. In the reaction chamber, 10 µL of lactose solution
(substrate in excess: 100 mg/mL) with 10 µL of
Hexokinase (10 µL x 0.2 U/µL = 2 U) were added. The
measurement was started and immediately after the
activation of the enzyme-pump, 10 µL of β-Galactosidase
solution were injected into the reaction chamber (10 µL x
0.1 U/µL = 1 U). To measure the blank, lactose solution
was replaced by pure water. The sample-kinetics (with
substrate in excess) and blank-kinetics (without lactose)
were measured per triplicate.
The optimum pH of Hexokinase is 7.5 (Sols et al. 1958). In
our experience, the differential pH sensor can detect the
Hexokinase activity at this pH. However, to verify the
response in an acid pH, the Hexokinase activity was
checked adding 10 µL of glucose solution (0, 20 and 40
mM) in the reaction chamber using reaction-buffers
adjusted to pH 4.00, 5.00 and 6.00. The measurement was
started and 10 µL of Hexokinase solution were injected
immediately after the activation of the enzyme-pump.
In order to validate the differential pH technique in cells,
the β-Galactosidase solution was replaced for a cell extract
solution (Nasim and Trembath, 2005). Three cell extracts
were obtained from three cultures incubated in T75 flask:
cell line of 3T6 fibroblasts, human primary culture from
early-passage (< 3) and long-term human primary culture.
The extracts obtained (100 µL) were dissolved in 400 µL of
reaction buffers (pH 6) and measure in the differential pH
sensor. The early-passage culture showed a fast growth
(doubling time < 30 hrs), indicating a non-senescence state.
The long-term culture was considered those that showed
arrest of cell growth. After roughly 50 population doublings
the primary culture reached the arrest of the growth
(evaluated as no change in cell counting after one week).
As a criterion, it is accepted that human fibroblasts are
senescent after 50 duplications. Our experience and
previous work (Acevedo et al. 2009) indicate that this state
is close to 90% of senescence evaluated by X-Gal staining.
RESULTS AND DISCUSSION
The system did not show activity at pH inferior to six. But,
the system presented activity to pH 6. This is because the
Hexokinase activity in that pH ranges - minor to six - is
very low (Sols et al. 1958). The enzymatic activity of
Hexokinase in the differential pH sensor was checked,
confirming this one (Figure 1). Thus, it is not possible to
determinate β-Galactosidase activity at other pH values
using this enzymatic system, but those reactions are not
important to determine β-Gal/pH6 activity. Nevertheless,
this presents a limitation for the extension of methods at
others pH values, for example, at physiological pH of
lysosome (pH 4) (Dimri et al. 1995), when the normal βGalactosidase is active.
At pH 6, the delta-milli-pH kinetics (change of milli-pH by
time) was always linear in the presence of lactose, and the
slopes are increasing with the β-Galactosidase activity
(Figure 2A). But, the system without substrate (lactose)
also showed a weak signal, maybe by unspecific reactions.
The unspecific reactions always were minor to the specific
reactions. To remove the unspecific reaction of the assay,
the blank (without substrate) was subtracted from samplekinetics, showing a new purified kinetics with two sections
(Figure 2B). The final section of the purified delta-milli-pH
kinetics was linear and depends on the β-Galactosidase
activity. The final sections (called slope in Figure 2B) are
controlled by β-Galactosidase activity in the sample,
because this enzyme produces glucose at constant velocity
(v1), which is transformed by Hexokinase into glucose-6P
(v2), changing the pH of the system:
[Eq. 3]
[Eq. 4]
In the first section of the kinetics, the delta-milli-pH slope
is not linear because the glucose concentration increases
with the time (v1 > v2). Then, the glucose kinetics is
described by:
[Eq. 5]
4
Measuring β-Galactosidase activity at pH 6 with a differential pH sensor
But, in the linear section (when the slope is constant), a
steady-state is established because v1 = v2, and then, the
glucose concentration is constant:
[Eq. 6]
Using the results shown in Figure 2B and the relationship
indicated in equation 6, an inverse plot of type LineweaverBurk was made showing a linear dependency of β-Gal/pH6
activity with the slope of the purified kinetics (samplekinetics less blank-kinetics). The linear fit was significant
(p < 0.05; ANOVA), with a Pearson correlation coefficient
of 0.999 (Figure 2C).
In order to validate this technique, the β-Galactosidase
solutions were replaced for three cell extracts obtained from
a cell line, early-passage primary culture and long-term
primary culture. The differential pH responses of cell line
and early-passage primary culture were not significant (p >
0.05). But, the long-term primary culture showed a
significant change in differential pH response (p < 0.05).
Those results are correct, because cell lines and young cells
not expresses a senescence phenotype, but long-term cell
cultures after 50 duplications have large amount of
senescence cells (> 90%) expressing β-Galactosidase
activity at pH 6 (Dimri et al. 1995).
CONCLUDING REMARKS
In conclusion, this simple method can be used to determine
enzymatic activity of β-Galactosidase at pH 6 in diluted
samples, such as samples of cell extract or other kind of
solutions, in fast and quantitative way, because the
measurement is easy and the time per measurement only
takes 3 min.
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