BML 2117 BIOCHEMICAL TECHNIQUES

UNIT TOPICS OUTLINE

WEEK TOPIC
1 Buffer preparation: utilization of buffer in chemical reactions; physiological
buffer.,
2 protein analysis (total protein and albumin determinations).Denaturation of
proteins using organic solvents and selective denaturation
3 Determination of enzyme activity—enzymatic analytical methods
4 Determination of sedimentation rates,
5 centrifugation techniques, utracentrfugation
6 CAT ONE
7 Electrophoresis—types, factors affecting ,
8 Electrophoresis of protein –analytical methods
9 Protein purification, Elucidation of primary structure of proteins

10 Determination of molecular weights and preparations of solutions

11 Radioligand assays , N-terminal analysis and protein identification using mass
spectrophotometry--assignment
12 ASSAYS OF METABOLIC ACTIVITIES IN TISSUES

13 CAT TWO
14 UNIT REVISION

Assignment:
Discuss N-terminal analysis and protein identification using mass spectrophotometry (30
mks)
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BUFFER PREPARATION: UTILIZATION OF BUFFER IN CHEMICAL

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 REACTIONS; PHYSIOLOGICAL BUFFER.,

 Buffers are aqueous systems that resist changes in pH as acid or base is added. OR A
buffer is a solution that resists a significant change in pH upon addition of an acid or a
base.
 They are usually composed of a weak acid and its conjugate base.
 Biological buffers, mixture of weak acids (the proton donors) and their conjugate bases
(the proton acceptors), help maintain biomolecules in optimal ionic state of pH 7.
 EXAMPLE 1
 When an acetic acid (sodium) buffer solution is prepared from 1:1 acetic acid and
sodium acetate, for example, the buffer solution pH is approximately 4.7, and this is
where the maximum buffer action can be obtained.
 EXAMPLE 2
 Bicarbonate buffer is a mixture of carbonic acid
 (the weak acid) and the bicarbonate ion (the conjugate
 base): H2CO3 + HCO3-
 All OH- or H+ ions added to a buffer are consumed and the overall [H+] or pH is not
altered

H2CO3 + HCO3- + H+ 2H2CO3

H2CO3 + HCO3- + OH- 2HCO3- + H2O

 Buffer solutions are used as a means of keeping pH at a nearly constant value in a wide
variety of chemical applications.

 In nature, there are many systems that use buffering for pH regulation. For example, the
bicarbonate buffering system is used to regulate the pH of blood.
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 .

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 APPLICATIONS

 Buffer solutions are necessary to keep the correct pH for enzymes in many organisms to
work.

 Many enzymes work only under very precise conditions; if the pH moves outside of a
narrow range, the enzymes slow or stop working and can denature.

 In many cases denaturation can permanently disable their catalytic activity.

 A buffer of carbonic acid (H2 CO3) and bicarbonate (HCO3- is present in blood plasma,
maintaining a pH between 7.35 and 7.45.

 Industrially, buffer solutions are used in fermentation processes and in setting the correct
conditions for dyes used in colouring fabrics. They are also used in chemical analysis
[
and calibration of pH meters.

 The majority of biological samples that are used in research are made in buffers,
especially phosphate buffered saline (PBS) at pH 7.4.

SIMPLE BUFFERING AGENTS

Buffering agent pKa Useful pH range
Citric acid 3.13, 4.76, 6.40 2.1–7.4
Acetic acid 4.8 3.8–5.8

KH2PO4 7.2 6.2–8.2

CHES 9.3 8.3–10.3
Borate 9.24 8.25–10.25
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  For buffers in acid regions, the pH may be adjusted to a desired value by adding a
strong acid such as hydrochloric acid to the buffering agent. For alkaline buffers, a
strong base such as sodium hydroxide may be added.

PROTEIN ANALYSIS (TOTAL PROTEIN AND ALBUMIN DETERMINATIONS).

 Protein molecule is made up of two constituents ie ALBUMIN and GLOBULIN.

 Protein analysis is achieved through qualitative and qualitative methods.
 Under qualitative method of protein determination, the most appropriate specimen used is
urine.
 Under quantitative method of protein determination, the most appropriate specimen used
is serum/plasma

QUALITATIVE METHOD OF PROTEIN DETERMINATION

 The specimen of choice is freshly voided urine
 Urine is collected using very clean containers which are designed conveniently for both
male and female client.
 The reagents formulation comes in three forms and are commonly referred to urine
reagent strips. These forms include:(1)single protein strip (2) protein and glucose strip
(3)protein plus other analytes eg, glucose, nitrites, urobilinogen, ketones,
blood,leucocytes, ph,bilirubin. Either of these reagent strips can be used to quantitatively
determine protein in urine specimen.
 The chemicals that react with protein in the urine specimen are impregnated into the
reagent strip.

STANDARD OPERATING PROCEDURE

(i) Provide the client with a clean convenient urine container and instruct him/her to
collect urine specimen
(ii) Remove the reagent strip from the reagent container
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(iii) Dip the strip into the urine according to the instruction given by the reagent
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manufacturer

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 (iv) Remove the strip and tap it at the sides of the urine container to remove excess urine.
(v) Compare the urine strip reactions with the chart provided by the manufacture
(vi) Negative results are shown by no colour formation whilst positive results are
expressed by colour change which is read from the chart provided.

QUANTITATIVE METHOD OF PROTEIN DETERMINATION

 The most commonly used method to determine total protein in a plasma/serum specimen
in a routine laboratory is the biuret method.
 Biuret is a chemical compound with the chemical formula C2H5N3O2. It is also known as
carbamylurea
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 Biuret test

 The biuret test is a chemical test for proteins and polypeptides. It is based on the biuret
reagent, a blue solution that turns violet upon contact with proteins, or any substance with
peptide bonds.

 The test and reagent do not actually contain biuret; they are so named because both biuret
and proteins have the same response to the test.

Biuret test

The characteristic color of a positive biuret test

 The biuret reaction can be used to assess the concentration of proteins because peptide
bonds occur with the same frequency per amino acid in the peptide. The intensity of the
color, and hence the absorption at 540 nm, is directly proportional to the protein
concentration, according to the Beer-Lambert law.
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  Despite its name, the reagent does not in fact contain biuret ((H2N-CO-)2NH). The test is
named so because it also gives a positive reaction to the peptide-like bonds in the biuret
molecule.

 In this assay, the Copper (II) binds with nitrogens present in the peptides of proteins. In a
secondary reaction, the Copper (II) is reduced to Copper (I). Buffers, such as Tris and
ammonia interfere with this assay, therefore rendering this assay inappropriate for protein
samples purified from ammonium sulfate precipitation. Due to its insensitivity and little
interference by free amino acids, this assay is most useful for whole tissue samples and
other sources with high protein concentration.[4]

Biuret reagent

 The Biuret reagent is made of sodium hydroxide (NaOH) and hydrated copper(II)
sulfate, together with [[potassium Chemical Reagents</ref> Potassium sodium tartrate is
added to chelate and thus stabilize the cupric ions.

 The reaction of the cupric ions with the nitrogen atoms involved in peptide bonds leads to
the displacement of the peptide hydrogen atoms under the alkaline conditions. A tri or
tetra dentate chelation with the peptide nitrogen produces the "biuret" color. This is found
with dipeptides

 The reagent is commonly used in the biuret protein assay, a colorimetric test used to
determine protein concentration by spectroscopy at wavelength 550  nm.
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 TOTAL PROTEIN (TP)
 Total protein reagent is used to measure the concentration of total protein by a timed
endpoint biuret method.
PRINCIPLE OF THE REACTION
 In the reaction, the peptide bonds in the protein sample binds to cupric ions in an
alkaline medium to form a peptide/copper complex.

PROCEDURE

6µl of sample Is reacted with 300 µl of reagent and the change in absorbance Is
monitored at 560nm. This change Is directly proportional to the concentration of TOTAL
PROTEIN in the sample and Is used to calculate and express concentration in G/L. The
reaction took place at 37oC for four minutes.
Principle of the reaction
protein + cu2+ oh-
protein-copper complex.

(NB/ THE VOLUMES CAN VARY FROM ONE REAGENT KIT TO ANOTHER)

 ALBUMIN (ALB)
Albumin which takes the highest portion of total protein is determined quantitatively
using a common method called bromocresol green (BCG). BCG is a dye in nature.
 Albumin reagent is used to measure albumin concentration by a timed endpoint method.
Albumin combined with bromocresol green to form a coloured product. 3 µl of sample is
reacted with 300 µl of reagent and the change in absorbance is monitored at 600nm.This
change is directly proportional to the concentration of ALB in the sample and is used to
calculate and express concentration in g/l. The reaction took place at 37 oC for one and
half minutes.
 Principle of the reaction
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 Albumin +BCG Albumin / BCG complex

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 (NB/ THE VOLUMES CAN VARY FROM ONE REAGENT KIT TO ANOTHER)

GLOBULIN DETERMINATION

 There is no routine method of quantitavely determining globulin in plasma/serum

specimen in the laboratory.
 The concentration of globulin is therefore determined by subtracting albumin
concentration from the total protein concentration as expressed in the example
below:
If the total protein and albumin concentration are 80g/l and 45 g/l respectively,
then the globulin concentration would be:

80-45=35 g/l

DENATURATION OF PROTEINS USING ORGANIC SOLVENTS AND SELECTIVE

DENATURATION

DEFINITION

 Process of partial or total alteration of the native secondary, and/or tertiary, and/or
quaternary structures of proteins or nucleic acids resulting in a loss of bioactivity.

 Denaturation can occur when proteins and nucleic acids are subjected to elevated
temperature or to extremes of pH, or to non-physiological concentrations of salt,
organic solvents, urea, or other chemical agents.

 Denaturation is a process in which proteins or nucleic acids lose the quaternary

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structure, tertiary structure and secondary structure which is present in their native state,
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 by application of some external stress or compound such as a strong acid or base, a
concentrated inorganic salt, an organic solvent (e.g., alcohol or chloroform), radiation or
heat

 If proteins in a living cell are denatured, this results in disruption of cell activity and
possibly cell death. Protein denaturation is also a consequence of cell death.

 Denatured proteins can exhibit a wide range of characteristics, from conformational

change and loss of solubility to aggregation due to the exposure of hydrophobic groups.

 Protein folding is key to whether a globular protein or a membrane protein can do its job
correctly. It must be folded into the right shape to function.

 But hydrogen bonds, which play a big part in folding, are rather weak, and it doesn't take
much heat, acidity, varying salt concentrations, or other stress to break some and form
others, denaturing the protein. This is one reason why tight homeostasis is
physiologically necessary in many life forms.

 This concept is unrelated to denatured alcohol, which is alcohol that has been mixed with
additives to make it unsuitable for human consumption.

 An enzyme loses its catalytic activity when it is denaturized.

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 THE EFFECTS OF TEMPERATURE ON ENZYME ACTIVITY

Denaturation

Top - increasing temperature increases the rate of reaction).

Middle - the fraction of folded and functional enzyme decreases above its denaturation
temperature. Bottom - consequently, an enzyme's optimal rate of reaction is at an intermediate
temperature.

Protein denaturation

Denatured proteins can exhibit a wide range of characteristics, from loss of solubility to protein
aggregation.
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 Functional proteins have four levels of structural organization:
1) Primary Structure : the linear structure of amino acids in the polypeptide chain
2) Secondary Structure : hydrogen bonds between peptide group chains in an alpha helix or beta
sheet
3) Tertiary Structure : three-dimensional structure of alpha helixes and beta helixes folded
4) Quaternary Structure : three-dimensional structure of multiple polypeptides and how they fit
together

 When a protein is denatured, secondary and tertiary structures are altered but the peptide
bonds of the primary structure between the amino acids are left intact.

 Since all structural levels of the protein determine its function, the protein can no
longer perform its function once it has been denatured.

LOSS OF FUNCTION

 Most biological substrates lose their biological function when denatured. For example,
enzymes lose their activity, because the substrates can no longer bind to the active site,
and because amino acid residues involved in stabilizing substrates' transition states are no
longer positioned to be able to do so.

 The denaturing process and the associated loss of activity can be measured using
techniques such as dual polarization interferometry

REVERSIBILITY AND IRREVERSIBILITY

 In very few cases, denaturation is reversible (the proteins can regain their native state
when the denaturing influence is removed).
 This process can be called renaturation.
 This understanding has led to the notion that all the information needed for proteins to
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assume their native state was encoded in the primary structure of the protein, and hence
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 in the DNA that codes for the protein, the so-called "Anfinsen's thermodynamic
hypothesis

AMINO ACID STRUCTURE

Figure 1 BELOW shows the general structure of an a-amino acid

 Proteins are the most abundant organic molecules in animals, playing important roles in
all aspects of cell structure and function.
 Proteins are biopolymers of acids, so named because the amino group is bonded to the
carbon atom, next to the carbonyl group.
 The physical and chemical properties of a protein are determined by its constituent amino
acids.
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 The individual amino acid subunits are joined by amide linkages called peptide bonds.
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 Essential Amino Acids
 Humans can synthesize about half of the amino acids needed to make proteins.
 Other amino acids, called the essential amino acids, must be provided in the diet.
 The ten essential amino acids, starred in Table 24-2, are the following: arginine (Arg)
valine (Val) methionine (Met) leucine (Leu) threonine (Thr) phenylalanine (Phe)
histidine (His) isoleucine (Ile) lysine (Lys) tryptophan (Trp)
 Proteins that provide all the essential amino acids in about the right proportions for
human nutrition are called complete proteins.
 Examples of complete proteins are those in meat, fish, milk, and eggs. About 50 g of
complete protein per day is adequate for adult humans.
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 DETERMINATION OF SEDIMENTATION RATES
MODIFIED WESTERGREN METHOD
 The most commonly used method today for determination of sedimentation rates of blood
cells is a modified Westergren method.
 EDTA-anticoagulated blood is diluted 4 to1 with sodium citrate or sodium chloride
solutions and placed in a 200-mm column(20 cm tall tube) with an internal diameter of
2.55 mm or greater.
 Favorable attributes of the modified Westergren method include:
(1) convenience of using EDTA blood, as for other hematology testing,
(2) highly elevated ESRs can be detected due to the tall column height,
(3) this method is recommended by the International Council for Standardization
in Hematology and the Clinical and Laboratory Standards Institute.

STANDARD OPERATING PROCEDURE FOR MODIFIED WESTERGREN METHOD

--using a volumetric pipette, suck 4 mls EDTA whole blood and put it in a clean test tube.
---Into the above tube, add I ml of either 0.1 M sodium citrate or Normal saline solution
and mix appropriately.
---Transfer the above mixture to the westergren cup and fill up to the mark
----take the long tube and insert into the westergren cup containing the diluted blood
---press until the blood mixture reaches the 200 mm mark on the tube
---leave the set up standing on a rack for 1hr
--read the sedimentation rate after 1 hr (read the length covered by the clear solution)
--results are given as X mm/hr
WINTROBE METHOD
 Another commonly used method today for determination of sedimentation rates of blood
cells is the Wintrobe method.
 Originally, the Wintrobe method utilized oxalate anticoagulated whole blood placed
into a 100-mm column. Today “Wintrobe” generally means only that a shorter column of
100mm(10cm) tall tube) with an internal diameter of 1.5-2 mm ) is used as an alternative
of the Westergren method.
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 EDTA (ethylenediamine tetra acetic acid) or citrated whole blood are often used.
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  The Wintrobe method is considered to have increased sensitivity for detecting mildly
elevated ESRs, but marked elevations are hard to detect due to the short column height.

STANDARD OPERATING PROCEDURE FOR WINTROBE METHOD

 --using a pastuer pipette with a long tip that can reach the bottom of the wintrobe
tube, suck enough blood and insert the pipette into the wintrobe tube until the tip of
the pipette touches the bottom of the wintrobe tube.
 Deliver the EDTA whole blood as you withdraw the pipette from the wintrobe tube
up the 0 mm mark on the wintrobe tube.
 ---leave the set up standing on a rack for 1hr
 --read the sedimentation rate after 1 hr (read the length covered by the clear
solution)
 --results are given as X mm/hr

LIMITATIONS OF THE ABOVE MENTIONED TEST METHODS

 Long analytical time ie 60 minutes sedimentation time
 Method of set up the tests is tedious
 requirements for relatively large specimen volumes (1.0-2.0 mL).

PHYSIOLOGICAL STATES OF INCREASED ESR:

After meals, After hot baths, During menstruation, After physical exercises ,Increases with age.

PATHOLOGICAL STATES OF INCREASED ESR:

Infectious diseases, Neoplasmatic, invasive tumours, Anaemias, Chronic and acute diseases of
liver, Connective tissue diseases

REFERENCE VALUES FOR OF ESR IN LITERATURE

Infants, children (1 – 2 mm/hr), Adult males (0 –10 mm/h). Adult females ((0 –15 mm/h).
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 ENZYMATIC REACTIONS

Introduction------Clinical enzymology is the application of the science of enzymes to the

diagnosis and treatment of pathological disorders. Enzymes are proteins with catalytic properties
due to their powers of specific activation of their substrates. This definition indicates the
characteristic properties of enzymes which in turn govern the principles of methods of enzymes
analysis.

Enzyme nomenclature---Due to the number and different types of enzymatic reactions

available, it became necessary to have a definitive and standardized system of identifying
enzymes.The responsibility of naming enzymes has been achieved by the Enzyme Commission
(EC) of the International Union of Biochemistry(IUB)

Two names are given to each enzyme: (1)—systematic name that clearly describes the nature of
reaction catalyzed, (2)—practical name which is suitable for everyday use.

The unique numerical designation for each enzyme consists of four numbers separated by
periods. The number is prefixed by the letters EC, denoting Enzyme Commission eg EC
2.2.8.11.

The first three numbers indicates the class, subclass, and sub-subclass to which the enzyme has
been assigned. The last number is the specific serial number given to each enzyme within its sub-
class.

It is a common and convenient practice in clinical chemistry to use capital letter abbreviations
for the names of certain enzymes, such as ALT for alanine aminotransferase (EC 2.61.2).
Example of others, AST for aspartate aminotransferase, LD for lactatedehydrogenase and CK for
creatine kinase.

DEFINATION OF AN ENZYME-----
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Enzymes are catalysts in nature that is control a reaction by increasing the rate of a particular
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reaction without itself being consumed or permanently altered; at the end of a catalysed reaction

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 the catalyst appears unchanged in form and in quantity, whereas the main reaction materials
have undergone transformation into new products.

Enzymes are protein catalysts of biological origin and virtually all the chemical reactions that
take place in living matter are catalysed by specific enzymes.

Enzyme act through the formation of an enzyme-substrate complex, ES, in which a molecule of
substrate is bound to the specialized substate –binding region (the active centre) of the enzyme
molecule. The binding process transforms the substrate molecule to an activated state, and the
energy required for this transformation is provided by free energy of binding of S and E. At the
end of the reaction, the ES complexone breaks down to give the reaction products(P) and free
enzyme(E).

E + S = ES→P +E

The rate of enzymatic catalysed reactions are governed by :-

(A) Enzyme concentration---- ie the rate of reaction is generally proportional to the

concentration of enzyme present in the system and this is the basis for the quantitative
determination of enzymes by measurement of reaction rates
(B) Substrate concentration----If the enzyme concentration is fixed and the substrate
concentration is varied, the rate of reaction is almost directly proportional to the
concentration of substrate at low values of the latter ie the reaction is essentially first
order with respect to substrate concentration.
(C) Ph -----the rate of enzyme catalysed reactions typically shows a marked dependence on
Ph. Many of the enzyme in blood plasma show maximum activity in vitro somewhere in
the ph range from 7 to 8 (nb/ in vivo the ph of blood is 7-35-7.45). The pronounced
effects of ph on enzymes reactions emphasize the need always to control this variable by
means of adequate buffer solutions.
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(D) Temperature—The rate of an enzymatic reaction increases as the temperature at which

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the reaction is taking place increases. The choice of temperature for the assay of enzymes

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 of clinical importance is (30-37) degree centigrades. During the enzymatic reactions the
changes should be +/- 0.1 degree centigrade.Otherwise high temperatures are known to
denature the enzyme molecules.

CO ENZYMES----these are smaller molecules than enzymes proteins molecules. Co-enzymes,

act as specific substrates in two substrate reaction and their effect on the rate of reaction follows
Michaelis-Menten pattern of dependence on substrate concentration. Example NAD +, NADH,
NADP+. These co-enzymes are bound to the enzymes during a enzymatic reactions and the
reaction would not take place in their absence.

Progress Of Enzymatic Reactions And The Measurements Of Reaction Rates.

Since the rate of an enzyme-catalyzed reaction is directly proportional to the amount of active
enzyme present in the system, determination of the rate of reaction under defined and controlled
conditions provides a very sensitive and specific method for the measurement of enzyme in
specimens such as serum.

The progress of conversion of the substrate into products in the presence of an enzyme can be
followed by measuring the decreasing concentration of substrate or the increasing concentration
of products. These principles are found among assays of clinically important enzymes.

-All determinations of reaction rate involve the measurement of the amount of change produced
in a defined time interval ie all measurements of reaction rate are kinetic measurements.

-At the moment when the enzyme and substrate are mixed, the rate of the reaction is zero.
Typically, the rate then rises rapidly to a maximum value, which remains constant for a period of
time. During the period of constant reaction rate, which may short or long in different
circumstances, the rate depends only on the enzyme concentration and is completely
independent of substrate concentration. The reaction is said to follow zero-order kinetics ie
the rate is proportional to the zero power of substrate concentration. As more and more
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substrate is consumed, the reaction rate declines and enters a phase of first –order dependence
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on substrate concentration. Other factors that contribute to the decline in the reaction include

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 accumulation of products that may be inhibitory, the growing importance of the reverse reaction,
and even enzyme denaturation.
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 MOLECULAR WEIGHTS

MOLECULAR MASS

 Molecular mass or molecular weight is the mass of a molecule. It is calculated as the

sum of the atomic weights of each constituent element multiplied by the number of atoms
of that element in the molecular formula.

 The molecular mass of small to medium size molecules, measured by mass spectrometry,
determines stoichiometry.

 For large molecules such as proteins, methods based on viscosity and light-scattering can
be used to determine molecular mass when crystallographic data are not available.

Definitions

 Both atomic and molecular masses are usually obtained relative to the mass of the isotope
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C (carbon 12), which by definition is equal to 12. For example, the molecular weight of
methane, whose molecular formula is CH4, is calculated as follows:

Atomic mass
C 12.011
H 1.00794
Mwt (13.02g

Determine the mwt of: cuso4, NaCl, NaOH,MgCl2, ---exercise

Involve the students in calculating molecular weights of compounds and how they are used to
make solutions in the laboratory

CONSIDERATIONS
Eg sodium chloride,

HOW DO YOU PREPARE THE FOLLOWING SOLUTION

NB/USE DISTILLED AS THE DILUENT
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1 litre of 1M copper sulphate

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 HOW DO YOU PREPARE THE FOLLOWING SOLUTION
NB/USE DISTILLED AS THE DILUENT

1.5L of 0.25M sodium hydroxide

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 CELLULAR METABOLISM ASSAYS

CELLULAR METABOLISM ASSAYS

Easily measure metabolic activity

 Metabolism refers to the catabolic and anabolic biochemical reactions inside of the cell.

 It is the collection of biological reactions by which living cells process nutrient molecules
and maintain a living state.

 Metabolism involves complex sequences of chemical reactions called metabolic

pathways.

QUANTITATIVE ASSAY OF ENZYMES IN TISSUES

 There are two main groups of conditions in which the determination of enzyme activities

in tissues is necessary or desirable. They are:

(a)primary defects of enzyme synthesis and

(b)alterations of enzyme activity secondary to damage of the cell.

 Since the time of Garrod, hereditary metabolic disorders have been recognized to be due

to a defect of one or perhaps more enzymes, which may have far- reaching metabolic

consequences and produce multiple biochemical and other abnormalities.

 This communication is confined to enzyme assays using tissues from organs such as

liver, kidney, or gastro- intestinal mucosa and including the leucocytes of the blood.

 It excludes the examination of blood plasma, the enzymes of which are, of course, not

synthesized there, but accumulate by leakage from the organs it bathes or from

degradation of red and white blood cells.

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  A general review of methods of enzyme assay is not attempted, but the principles of the

methods used for individual enzyme assays are*emphasized. Selection has been based on

the special factors which must be taken into account in this field, eg, the limited amount

of material available so that the sensitivity of a method is important, and the fact that

tissues contain a mixture of enzymes as well as many different substrates. The methods

of assay available for pure enzymes are not always applicable to a crude tissue

homogenate.

 The organ most commonly involved in hereditary metabolic disorders is the liver, which

is therefore the tissue most frequently biopsied. A specimen can be obtained either by

percutaneous needle aspiration or by open biopsy.It is always desirable to examine as

many enzymes as possible of the metabolic sequence in which the block is thought to

occur.

ENZYMATIC REACTIONS

Introduction------Clinical enzymology is the application of the science of enzymes to the

diagnosis and treatment of pathological disorders.

Enzymes are proteins with catalytic properties due to their powers of specific

activation of their substrates.

This definition indicates the characteristic properties of enzymes which in turn

govern the principles of methods of enzymes analysis.

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 Enzyme nomenclature---

Due to the number and different types of enzymatic reactions available, it

became necessary to have a definitive and standardized system of identifying

enzymes.

The responsibility of naming enzymes has been achieved by the Enzyme

Commission (EC) of the International Union of Biochemistry(IUB)

Two names are given to each enzyme: (1)—systematic name that clearly

describes the nature of reaction catalyzed, (2)—practical name which is

suitable for everyday use.

The unique numerical designation for each enzyme consists of four numbers

separated by periods.

The number is prefixed by the letters EC, denoting Enzyme Commission eg EC

2.2.8.11. The first three numbers indicates the class, subclass, and sub-

subclass to which the enzyme has been assigned. The last number is the

specific serial number given to each enzyme within its sub-class.

It is a common and convenient practice in biochemistry to use capital letter

abbreviations for the names of certain enzymes, such as ALT for alanine
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 aminotransferase (EC 2.61.2). Example of others, AST for aspartate

aminotransferase, LD for lactate dehydrogenase and CK for creatine kinase.

---DEFINATION OF AN ENZYME-----

Enzymes are catalysts in nature that is control a reaction by increasing the rate of

a particular reaction without itself being consumed or permanently altered; at the

end of a catalysed reaction the catalyst appears unchanged in form and in

quantity, whereas the main reaction materials have undergone transformation into

new products.

Enzymes are protein catalysts of biological origin and virtually all the chemical

reactions that take place in living matter are catalysed by specific enzymes.

Enzyme act through the formation of an enzyme-substrate complex, ES, in which

a molecule of substrate is bound to the specialized substrate –binding region (the

active centre) of the enzyme molecule.

The binding process transforms the substrate molecule to an activated state, and

the energy required for this transformation is provided by free energy of binding

of S and E. At the end of the reaction, the ES complexone breaks down to give
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the reaction products(P) and free enzyme(E).

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 E + S = ES→P +E

The rate of enzymatic catalysed reactions are governed by :-

(E) Enzyme concentration---- ie the rate of reaction is generally proportional to the

concentration of enzyme present in the system and this is the basis for the quantitative

determination of enzymes by measurement of reaction rates

(F) Substrate concentration----If the enzyme concentration is fixed and the substrate

concentration is varied, the rate of reaction is almost directly proportional to the

concentration of substrate at low values of the latter ie the reaction is essentially first

order with respect to substrate concentration.

(G) Ph -----the rate of enzyme catalysed reactions typically shows a marked dependence on

Ph. Many of the enzyme in blood plasma show maximum activity in vitro somewhere

in the ph range from 7 to 8 (nb/ in vivo the ph of blood is 7-35-7.45). The pronounced

effects of ph on enzymes reactions emphasize the need always to control this variable by

means of adequate buffer solutions.

(H) Temperature—The rate of an enzymatic reaction increases as the temperature at

which the reaction is taking place increases. The choice of temperature for the assay of

enzymes of clinical importance is (30-37) degree centigrades. During the enzymatic

reactions the changes should be +/- 0.1 degree centigrade.Otherwise high temperatures
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are known to denature the enzyme molecules.

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 COENZYMES----

Coenzymes are small molecules. They cannot by themselves catalyze a reaction but they can

help enzymes to do so. In technical terms, coenzymes are organic non protein molecules that

bind with the protein molecule (apoenzyme) to form the active enzyme (holoenzyme).

A cofactor is a non-protein chemical compound or metallic ion that is required for an enzyme's

activity as a catalyst, a substance that increases the rate of a chemical reaction. Cofactors can be

considered "helper molecules" that assist in biochemical transformations.

EXAMPLE OF COENZYMES

 NAD+(, nicotinamide adenine dinucleotide in its oxidized state), NADH (nicotinamide

adenine dinucleotide in its reduced state), NADP (nicotinamide adenine dinucleotide 2'-

phosphate).These co-enzymes are bound to the enzymes during a enzymatic reactions and

the reaction would not take place in their absence.

 Vitamin B-3, is the precursor for the nicotinamide coenzymes, nicotinamide adenine

dinucleotide, or NAD, and nicotinamide adenine dinucleotide 2'-phosphate, or NADP,

29

which carry electrons between different proteins.

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  Cofactors serve the same purpose as coenzymes, as they regulate, control, and adjust

how fast these chemical reactions would respond and take effect in our body.

 The big difference is that coenzymes are organic substances, while cofactors are

inorganic.

inorganic cofactors, such as the metal ions Mg2+, Cu+, Mn2+, or iron-sulfur clusters.

Progress Of Enzymatic Reactions And The Measurements Of Reaction Rates.

Since the rate of an enzyme-catalyzed reaction is diretly proportional to the amount of active

enzyme present in the system, determination of the rate of reaction under defined and controlled

conditions provides a very sensitive and specific method for the measurement of enzyme in

specimens such as serum.

The progress of convertion of the substrate into products in the presence of an enzyme can be

followed by measuring the decreasing concentration of substrate or the increasing concentration

of products. These principles are found among assays of clinically important enzymes.

-All determinations of reaction rate involve the measurement of the amount of change produced

in a defined time interval ie all measurements of reaction rate are kinetic measurements.

-At the moment when the enzyme and substrate are mixed , the rate of the reaction is zero.
30

Typically, the rate then rises rapidly to a maximum value , which remains constantfor a period of
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time. During the period of constant reaction rate, which may short or long in different

30
 circumstances, the rate depends only on the enzyme concentration and is completely

independent of substrate concentration. The reaction is said to follow zero-order kinetics ie

the rate is proportional to the zero power of substrate concentration. As more and more

substrate is consumed, the reaction rate declines and enters a phase of first –order dependence

on substrate concentration. Other factors that contribute to the decline in the reaction include

accumulation of products that may be inhibitory, the growing importance of the reverse reaction,

and even enzyme denaturation.

RELATIONSHIP BETWEEN INTRACELLULAR FLUID (ICF) AND

EXTRACELLULAR FLUID (ECF)

 Inside the cell of any living thing is where the metabolic activities processes take place.

 The different metabolic activities are under the control of enzymes which catalyze

different types of enzymatic reaction.

 These enzymatic reactions are what makes the cell to be alive.

 The content of the ICF are contained within the ICF by the cell membrane which is

known to be very rigid

 In a normal circumstances the cell membrane will only allow substances /nutrients to

pass through it but not to come out .

 The only time the contents of the cell ICF can come out is when the rigidity of the cell

membrane has been compromised by an external agent such as: bacteria, viruses or
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trauma.
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  ECF happens to be the reservoir of the many substances which are required by the

metabolic processes taking place within the ICF

THEREFORE THE TWO TYPES OF FLUIDS ARE SEPERATED BY THE RIGID

CELL MEMBRANE.

ELECTROPHORESIS

-Electrophoresis refers to migration of charged solutes or particles in a liquid medium under the

influence of an electric field (electric current).

- The widely practiced type of electrophoresis is the zone electrophoresis which refers to the

migration of charged macromolecules in a porous supporting medium such as cellulose

acetate paper or agarose gel film.

- The end product of zone electrophoresis is an electrophoretogram, which is a display of

protein zones, each one sharply separated from neighbouring zones on the electrophoretic

support material..

-Solutes analyzed by this technique in clinical chemistry laboratory are mainly macro-molecular

in size and colloidal in nature, they include:- protein in serum, urine, csf and other biological

fluids as well as protein in rbcs and tissues

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 THEORY OF ELECTROPHORESIS

- Chemical species carrying an electric charge by virtue of ionization will move either to the

cathode or to the anode in an electrophoresis system, depending on the kind of charge on the

molecule

- The kind of solutes separated by use of this technique are ampholyte and are said amphoteric

in nature ie they can either be positively or negatively charged depending on the ph of the

solution they are contained in. These solutes have an iso-electric point (PI) ie a point where

the solute has no charge. These types of solutes can also be referred to as zwitterions.

TYPES OF ELECTROPHORESIS

There are six types of electrophoresis namely:-

-(1)Paper electrphoresis(PE), (2) Agarose Gel Electrophoresis (AGE), (3)Cellulose Acetate

Electrophoesis (CAE),(4) Polyacrylamide Gel electrophoresis (PAGE), (5)Starch Gel

Electrophoresis (SGE), (6) Isoelectric Focusing(IEF).

-Among the above mentioned types of electrophoresis the most widely used in clinical chemistry

laboratories for routine separation of solutes (particles) are agarose gel and cellulose acetate

electrophoresis.

-(1)Paper electrphoresis(PE)

-This was once widely used in clinical laboratories for the separation of serum proteins but has

been replaced by cellulose acetate or agarose gel electrophoresis.Significant disadvantage of the

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use of paper electrophoresis include the long separation time (14-16hrs), excessive

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 background(tailing),and decreased resolution.The advantages of paper electrophoresis were, high

tensile strength, low cost, and ease of handling.

(2) Agarose Gel Electrophoresis (AGE)

-This method has been successively applied to the analysis of serum proteins, haemoglobin

variants, lactate dehydrogenase isoenzymes and lipoproteins fractions.

-This medium parallels cellulose acetate interms of versatility, convenience and applicability to

routine clinical demands.

-Agarose gel has a low affinity for protein and has very little effect on migration rate . Since it is

clear after drying, it permits excellent densitometric examination.

-Usually 0.5-1.0 g of agarose /dl of buffer provides a gel with the desired strength and with good

migration properties.

-There are two application methods of agarose gel electrophoresis:-

(A)Dissolving serum into warm agarose and applying it directly into precut or precast wells on

the agarose gel medium.The precut method has an advantage in that it causes an uneven surface

at the sample application points and an artificial peak in densitometry. The precast method is less

convenient but it has an advantage in that the agarose –sample solution solidifies to become part

of the agarose support.After application, the gel is introduced into the electrophoretic tant for

electrophoretic process which takes 30-90 minutes

(B) The other alternative method uses a thin plastic template that has small slots corresponding
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to sample application points.The template is placed upon the agarose surface and 5 ml samples

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 are introduced to each slot. The serum is allowed to diffuse into the agarose for 5 minutes, the

excess is removed by blotting, and the template is then removed from the agarose surface and

placed onto the electrophoretic tank for electrophoresis process which takes 30-90 minutes

(3)Cellulose Acetate Electrophoesis (CAE)

-When hydroxyl (OH -) groups of cellulose are reacted with acetic anhydride, cellulose acetate is

formed.This acetylated cellulose forms the raw material for cellulose acetate membranes.The

membranes commercially available contain about 80% air spaces in the form of pockets within

the interlocking cellulose acetate fibres. The membranes as purchased are dry, opaque, brittle

films that crack easily if not handled gently.When the film is placed in the buffer, the air spaces

fill with liquid and the film becomes quite pliable(flexible). Characteristics of specific

membranes vary with the extent of acetylation, the prewashing procedure employed by the

manufacturers, and the additives used, as well as the pore size and thickness of the membrane.

Cellulose acetate that has been especially prepared to reduce electroendosmosis is also

commercially available.

NB/The procedure is given below as a representative of electrphoresis process.

4) Polyacrylamide Gel electrophoresis (PAGE)

-Protein electrophoresis on paper, cellulose acetate or some agarose gels yields only five to seven

zones owing to diffusion during the electrophoretic procedures. Polyacrylamide , or starch gel ,

electrophoresis commonly yields 20 or more fractions and is therefore widely used to study
35

individual serum proteins, particularly genetic variants and isoenzymes.

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 --PAGE technique employs layers of gel that differ in composition and pore size. Because of the

discontinuities of the electrophoretic matrix and the discoid shape of the separated zones of

protein, this method is sometimes reffered to as disc electrophoresis.

- Polyacrylamide Gel is thermalstable, transparent, strong, and relatively chemically inert, it can

be made in a wide range of pore sizes to optimize particular separations.This gels are uncharged,

thus eliminating electroendosmosis.It is also used in isoelectric focusing method.

(5)Starch Gel Electrophoresis (SGE)

-SGE, like PAGE seperates macromolecular ions on the basis of both surface charge and

molecular size. Partially hydrolyzed starch is used , since native starch does not gel. Starch gel

may be used in a horizontal process or with migration taking place in the vertical direction.

Proper preparation of gel is relatively difficult and requires considerable skill. The starch

concentration is 10-16 g/dl and the ph of the buffer varies according to the specific seperation

(6) Isoelectric Focusing(IEF).

-Isoelectric focusing is a further advance in the electrophoretic separation of proteins into

discrete bands. The medium provides a stable ph gradient by distributing carrier ampholytes

along its axis. The compounds to be separated (eg proteins ) migrate to the zone in the medium

where the ph is equal to the isoelectric point (pI) of each compound.In that zone, the charge on

the protein becomes zero and migration ceases.With IEF , separated protein zones are very sharp

because the pI of a protein is confined to a narrow pH range and because diffusion of the protein
36

is counteracted by acquisition of charge as it moves away from its (pI) position.

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 - Several support media have been used for IEF including polyacrylamide, agarose and cellulose

acetate. PAGE-IEF is widely used in analytical work.

PRINCIPLE OF SERUM PROTEIN ELECTROPHORESIS

-Protein molecules in an alkaline medium (PH 8.6) are negatively charged and when subjected to

an electric current will migrate towards the anode (positively charged side) of the electrophoretic

system. The migration will progress within a supporting medium, for a standard period of time.

The protein molecule will be separated into its various charged particles namely : albumin,

alpha 1-, alpha 2-,beta- and gamma globulins

FACTORS THAT CONTROL THE MIGRATION OF PROTEIN MOLECULES

(1)Net electrical charge of the molecule----molecules with more net electrical charge will

migrate faster than the ones with less. The net charge is directly proportional to the

electrophoretic mobilityof the protein molecules.

(2) Size and shape of the molecules-----protein molecule migration is inversely proportional to

the size and shape of the protein molecule. Increase in the size of protein molecule will decrease

the migration .Irregular shaped protein molecules will migrate slower than regular shaped protein

molecules.

(3) Viscosity of the medium (electrophoretic buffer)---- migration of protein molecule will be

faster in medium of low viscosity than that of high viscosity.

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 (4) Strength of the electrical field--- The current applied should be enough to cause the

migration of the charged protein molecules.. In a low electrical field the migration will be less

(5) Temperature of operation-----The temperature in the electrophoretic tank should be enough

not to cause evaporation of the buffer.

(6) Buffer properties (supporting medium properties) ---the buffer used in electrophoresis has

two-fold purpose: a) they carry the applied current, b) they fix the Ph at which the

electrophoresis is carried out (ie they determine the electrical charge on the solute).

- The ionic strength of the buffer determines the thickness of the ionic cloud surrounding the

charged molecule and thus also the rate of migration and the sharpness of the zones. Increasing

the ionic strength by increasing the concentration of the buffer ions causes an increase in the

ionic cloud around the charged molecule, and the molecule becomes more hindered in its

movement. Buffers of high ionic strength yield sharper separations of bands, but the benefits of

sharper resolutions are diminished by the Joule (heat) effect that leads to denaturation of heat-

labile proteins.

- The most widely used buffer systems in electrophoretic procedures are barbital buffers (ionic

strength=0.025-0.075) and tris buffers (ionic strength=0.03-0.12).The advantage with these

buffers is that the base of both is monovalent therefore any association between buffer and

protein is minimal.
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 PROCEDURE FOR PROTEIN ELECTROPHORESIS

(a) materials required:

-sample (serum,urine,plasma,or csf)

- electrophoretic apparatus including the power pack(supply)

- electrophoretic supporting medium(cellulose acetate paper or agarose gel)

-buffer solution (barbital (sodium barbital) or tris buffers

- stain eg ponseau S, methylene blue or silver nitrate

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 AN OVER VIEW OF AN ELECTROPHORETIC TANK
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 METHODOLOGY OF ELECTROPHORESIS USING CELLULOSE ACETATE PAPER

AS AN EXAMPLE

(1) A reasonable size of hydrated support material (cellulose acetate paper) is placed on the

electrophoresis chamber. It acts as a bridge between the anode and the cathode chambers of the

electrophoretic tank. The hydrating medium is the buffer to be used in the procedure. Note that

the excess buffer is removed by placing the cellulose acetate paper (hydrated) on a piece of filter

paper.

(2) The two chambers of the electrophoretic tank are filled with the buffer.

(3) filter papers are used as contacts of the buffer (PH 8.6) and the cellulose acetate paper on the

bridge.

(4) a capillary tube is used to place approximately 5 ul of sample on the cathode side of cellulose

acetate paper.

(5) A constant voltage of 100 V is set and a current of 4 m A is applied. The total current will
41

depend on the number of cellulose paper placed on the bridge.

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(6) Electrophoressis is conducted for a determined length of time ie( 20 min-1 hr)

41
 (7) A cover is placed on the electrophoretic tank to prevent evaporation of the buffer and drying

of the cellulose paper

(8) After 1 HR , the current is stoped and paper removed from the tank and placed in a staining

trough containing the staining reagent (ponseau S ) for 5 minutes.

(8) After 5 minutes the stained paper is placed on a 2nd trough containing 3 % acetic acid solution

for 5 minutes to remove excess stain.

(9) After 5 minutes the paper is placed in another trough containing another shade of 3% acetic

acid until time desired for reading

NB/ The advantages of this procedure (CAE) are the speed of separation ( 20 min-1 hr) and the

stability of the cleared membranes when stored for long periods.

INTERPRETATION.

- The result of protein electrophoresis is an electrophoretogram. In a normal serum , five protein

bands are expressed. The expression from the fastest to the slowest is as follows: albumin, alpha

1 globulin, alpha 2 globulin, beta globulin and gamma globulin.

- Another important factor to consider in the interpretation is the intensity of the stain which is

directly proportional to the amount of specific protein.

- Densitometry is another method used for interpretation. It involves the use of a densitometer

which give the location and intensity of the zones (bands) as successive peaks on a recorder
42

chart. In some densitometer the area under the peak is automatically integrated
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 - Another method of interpretation is the ELUTION method. This involves cutting of the specific

bands and eluting the stain using suitable solvents such as basic buffers or alcoholic solutions.

-Appearance of a normal electrophoretogram

Densitometry

Zones (bands) as successive peaks on a recorder chart

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  THINGS TO NOTE

(1) urine from patients suspected to have multiple myeloma can be used as a specimen for

electrophoresis. In such case , the urine must be centrifuged and the deposits used for inoculation

into the support medium. A positive report is indicated by the appearance of only one

electrophoretic band called M band or MYELOMA BAND.(presences of bence jones protein

detection by brandshaw test)

(2) IF plasma is used instead of serum, a sixth band appears and this is the fibrinogen band.

(3) it IS A GOOD LABORATORY practice TO INCLUDE A CONTROL SERUM IN EVERY

ANALYSIS

LIMITATIONS AND ERRORS IN ROUTINE ELECTROPHORESIS

Limitations and errors in routine electrophoresis

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 Wick flow

-Heat generated causes evaporation of solvent from the electrophoretic support

-Drying effect causes buffer to rise from both tank compartments

-Flow of buffer from both directions affects protein migration

Electroendosmosis
 -Some electrophoretic support media take the negative charge due to adsorption of OH-
when in contact with water

 -Since the ions are fixed on the surface of the electrophoretic support they are rendered
immobile relative to the other ions in solution

 -Positive ions in solution cluster about the fixed negative charge ions sites forming an
ionic cloud of mostly positive ions

 -application of electric current causes the positive ions in ionic cloud to migrate towards
the negative side of the system thus affecting the flow of the intended negatively charged
solutes

 -electroendosmosis strong medium ( eg cellulose acetate paper and agarose gel ) and
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with minimal electroendosmosis(starch,polyacrylamide gel ,highly purified agarose)

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 Buffers storage

growth of micro-organisms when stored at room temparatures and effect of electrolysis of water
during electrophoresis

-Amount of stain solution

Recommended volume approximately 100ml for 387cm2 of cellulose acetate or agarose film,
tightly closed to avoid evaporation

Sample application
Amount applied must be optimal and small enough to avoid overloading

SEPARATION TECHNIQUES

CENTRIFUGATION

CENTRIFUGE
-This is an important piece of equipment in the clinical laboratory. It is primarily used for
separating particles from solutions by centrifugal force. It can also be defined as a device to
accelerate gravitational seperatrion of substances differing significantly in their masses.
Therefore in clinical laboratory centrifugation is used:
(i) To separate particles from solutions in which they are suspended. Examples of this
application are (a) separating cells from blood to provide cell-free plasma (liquid part of anti
coagulated blood) or serum (liquid part of coagulated blood or clotted blood) for analysis; (b)
concentrating cellular elements and other components of biological fluids for microscopic
examination or chemical analysis; (c) eliminating chemically precipitated protein from an
analytic specimen; (d) separating protein –bound or antibody –bound ligand from free ligand in
immunochemical and other assays.
(ii) To separate two liquid phases of different densities (e.g. an aqueous phase from an organic
solvent) or to separate lipid components from plasma or serum , or from each other.
46

-Despite years of experience with centrifuges, there is no specific recommendation for the
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relative centrifugal force (RCF) or the time used for centrifugation of blood specimens. Some

46
 organizations (eg NCCLS) proposes an RCF of 1000-1200 x g for 10+/- 5 min, but clearly
considerable deviations from the recommendation allow adequate separation. No standards have
been developed for centrifugation of other specimens , such as serum to which a protein
precipitant has been added.
- IN CENTRIFUGATION, IT IS VERY IMPORTANT TO OBSERVE BALANCING OF
THE MATERIALS TO BE CENTRIFUGED SO AS TO AVOID CAUSING MECHANICAL
DAMAGE OF THE ROTOR AND OTHER COMPONENTS OF THE CENTRIFUGE.

CENTRIFUGATION

 Centrifugation is a technique used for the separation of particles using a centrifugal field.
 The particles are suspended in liquid medium and placed in a centrifuge tube.  The tube is
then placed in a rotor and spun at a definitive speed. Rotation of the rotor about a central
axis generates a centrifugal force upon the particles in the suspension.
Two forces counteract the centrifugal force acting on the suspended particles:

 Buoyant force: This is the force with which the particles must displace the liquid media
into which they sediment.
 Frictional force: This is the force generated by the particles as they migrate through the
solution.

 Particles move away from the axis of rotation in a centrifugal field only when the
centrifugal force exceeds the counteracting buoyant and frictional forces resulting in
sedimentation of the particles at a constant rate.

 Particles which differ in density, size or shape sediment at different rates. The rate of
sedimentation depends upon:
1.       The applied centrifugal field
2.       Density and radius of the particle.
3.       Density and viscosity of the suspending medium.
 The larger the size and the larger the density of the particles, the faster they separate from the
mixture.

 Chemists and biologists may increase the effective gravitational force on a test tube so as
to more rapidly and completely cause the precipitate (pellet) to gather on the bottom of
the tube. The remaining solution (supernatant) may be discarded with a pipette.
 By applying a larger effective gravitational force to the mixture, like a centrifuge does,
the separation of the particles is accelerated. This is ideal in industrial and lab settings
because particles that would naturally separate over a long period of time can be
separated in much less time
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 The rate of centrifugation is specified by the angular velocity usually expressed as

revolutions per minute (RPM), or acceleration expressed as g.
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  The conversion factor between RPM and g depends on the radius of the centrifuge rotor.
The particles' settling velocity in centrifugation is a function of their size and shape,
centrifugal acceleration, the volume fraction of solids present, the density difference
between the particle and the liquid, and the viscosity

TYPES OF CENTRIFUGES BASED ON PATTERN OF SEPERATION

Centrifuges may be classified generally into three types: horizontal-head (or swinging-
bucket),fixed-angle (angle-head), and ultracentrifuges.
(a) Horizontal-head (swinging-bucket) centrifuges------allow the tubes placed in the cups of the
rotor to assume a horizontal plane when the rotor is in motion and a vertical position when it is at
rest. During centrifugation, particles travel in a constant manner along the tube while the tube is
at right angle to the shaft of the centrifuge .Thus , the sediment is distributed uniformly against
the bottom of the tube. The surface of the sediment is flat (parallel to the shaft of the centrifuge)
and remains so, with a column of liquid on top of it when the rotor stops.
(b) Angle –head rotor centrifuge----------tubes in this centrifuge are held at a fixed angle from
25 to 45 degrees to the vertical axis of the rotation. As in horizontal –head centrifuge, particles
are driven outward horizontally but strike the side of the tube so that the sediments packs at the
side and bottom of the tube and the surface of the sediment is parallel to the shaft of the
centrifuge. As the rotor slows down and then stops, gravity may cause the sediments to slide
down the tube, generally a poorly packed pellet is formed.
The shape of a fixed angle –head rotor allows more rapid sedimentation of small particles than is
usually possible with horizontal-head rotor. Fixed angle rotor can be run at a higher speed than
swinging –bucket rotors., which offer considerable resistance to rotation and generate heat as a
result of air friction.The sedimentation of large particles require low speed , so the horizontal-
head rotor is adequate for the for the separation of rbcs from plasma or of a protein precipitate
from a supernatant.

TYPES OF CENTRFUGATION BASED ON CAPACITY AND SPEED

 MICROCENTRIFUGES
Microcentrifuges are used to process small volumes of biological molecules, cells, or
nuclei. Microcentrifuge tubes generally hold 0.5 - 2.0 mL of liquid, and are spun at
maximum angular speeds of 12,000–13,000 rpm.
 HIGH-SPEED CENTRIFUGES

High-speed or superspeed centrifuges can handle larger sample volumes, from a few tens of
48

millilitres to several litres.

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 Additionally, larger centrifuges can also reach higher angular velocities (around 30,000 rpm).
The rotors may come with different adapters to hold various sizes of test tubes, bottles, or
microtiter plates.

(c)ULTRACENTRIFUGES-------these are high speed centrifuges that use mainly fixed-angle

rotors. The most common application of an ultracentrifuge in clinical laboratory is the separation
of lipoproteins, including ribosomes, proteins, and viruses.

Ultracentrifuges can also be used in the study of membrane fractionation. This occurs because
ultracentrifuges can reach maximum angular velocities in excess of 70,000 rpm

COMPONENTS OF A CENTRIFUGE
-All centrifuges contain a rotor (centrifuge head), drive shaft, and motor.
- Most centrifuges also have a switch, timer, speed control, tachometer, and a brake.
- Some are equipped with a refrigerator to reduce the temperature within the chamber
-Some may have audible or visible alarms to indicate malfunctions such as an imbalance of the
rotor.
- Centrifuges are fitted with a cover to reduce aerosol formation

PRINCIPLES OF CENTRIFUGATION
The speed of centrifugation , measured in revolution per minute (rpm), does not describe the
force required to separate two phases in a centrifuge. The correct term is RELATIVE
CENTRIFUGAL FORCE (RCF), also called relative centrifugal field. Units are expressed as
number of times greater than gravity (eg 5000 x g) .
-Maximum relative centrifugal force is calculated as follows:
RCF=1.118 X 10 -5 X r X n2
Where 1.118 X 10 -5 is an empirical factor,
r= horizontal distance (radius in cm) from the centre of rotation to the bottom of the
tubein the rotor cavity or bucket during centrifugation
n= speed of rotation of the rotor in rpm.
Care and maintenance
49

(1) loads should be balanced before centrifuging

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(2) rubber cushions should be checked whether they are kin place before spinning.

49
 (3) the lid should always be closed when a centrifuge is in operation.
(4) should be cleaned regularly
(5) incase of a mechanical problem consult a biomedical engineer.
(6)Standard operating procedures of the system should always be displayed for efficient
management of the system.

TYPES OF CENTRFUGATION

 MICROCENTRIFUGES

Microcentrifuges are used to process small volumes of biological molecules, cells, or nuclei.
Microcentrifuge tubes generally hold 0.5 - 2.0 mL of liquid, and are spun at maximum angular
speeds of 12,000–13,000 rpm. Microcentrifuges are small enough to fit on a table-top and have
rotors that can quickly change speeds. They may or may not have a refrigeration function.

 HIGH-SPEED CENTRIFUGES

High-speed or superspeed centrifuges can handle larger sample volumes, from a few tens of
millilitres to several litres. Additionally, larger centrifuges can also reach higher angular
velocities (around 30,000 rpm). The rotors may come with different adapters to hold various
sizes of

 FRACTIONATION PROCESS

General method of fractionation: Cell sample is stored in a suspension which is:

1. Buffered - neutral pH, preventing damage to the structure of proteins including enzymes
(which could affect ionic bonds)
2. Isotonic (of equal water potential) - this prevents water gain or loss by the organelles
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3. Cool - reducing the overall activity of enzyme released later in the procedure
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 Cells are homogenised in a blender and filtered to remove debris

50
  The homogenised sample is placed in an ultracentrifuge and spun in low speed - nuclei
settle out, forming a pellet
 The supernatant (suspension containing remaining organelles) is spun at a higher speed -
chloroplasts settle out
 The supernatant is spun at a higher speed still - mitochondria and lysosomes settle out
 The supernatant is spun at an even higher speed - ribosomes, membranes settle out

 The ribosomes, membranes and Golgi complexes can be separated by another technique
called density gradient centrifugation.

 ULTRACENTRIFUGATIONS

 Ultracentrifugation makes use of high centrifugal force for studying properties of

biological particles.

 Compared to microcentrifuges or high-speed centrifuges, ultracentrifuges can

isolate much smaller particles, including ribosomes, proteins, and viruses.

 Ultracentrifuges can also be used in the study of membrane fractionation. This

occurs because ultracentrifuges can reach maximum angular velocities in excess
of 70,000 rpm. Additionally, while microcentrifuges and supercentrifuges
separate particles in batches (limited volumes of samples must be handled
manually in test tubes or bottles), ultracentrifuges can separate molecules in batch
or continuous flow systems.

 In addition to purification, analytical ultracentrifugation (AUC) can be used for

determination of the properties of macromolecules such as shape, mass,
composition, and conformation.

 Samples are centrifuged with a high-density solution such as sucrose, caesium

chloride, or iodixanol.

 The high-density solution may be at a uniform concentration throughout the test

tube ("cushion") or a varying concentration ("gradient").

 Molecular properties can be modeled through sedimentation velocity analysis or

sedimentation equilibrium analysis.

 During the run, the particle or molecules will migrate through the test tube at
different speeds depending on their physical properties and the properties of the
solution, and eventually form a pellet at the bottom of the tube, or bands at
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various heights.
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  An ultracentrifuge spins at a faster rate. This enables to it to separate
macromolecules (proteins, nucleic acids etc) which a normal centrifuge can't do
and measure sedimentation rate of particles.
 The ultracentrifuge is a centrifuge optimized for spinning a rotor at very high
speeds, capable of generating acceleration as high as 1 000 000 g (approx. 9 800
km/s²).[1] There are two kinds of ultracentrifuges, the preparative and the
analytical ultracentrifuge. Both classes of instruments find important uses in
molecular biology, biochemistry, and polymer science.

Preparative ultracentrifuge

 Preparative ultracentrifuges are available with a wide variety of rotors suitable for
a great range of experiments. Most rotors are designed to hold tubes that contain
the samples.
 Swinging bucket rotors allow the tubes to hang on hinges so the tubes reorient to
the horizontal as the rotor initially accelerates.
 Fixed angle rotors are made of a single block of material and hold the tubes in
cavities bored at a predetermined angle.
 Zonal rotors are designed to contain a large volume of sample in a single central
cavity rather than in tubes. Some zonal rotors are capable of dynamic loading and
unloading of samples while the rotor is spinning at high speed.
 Preparative rotors are used in biology for pelleting of fine particulate fractions,
such as cellular organelles (mitochondria, microsomes, ribosomes) and viruses.
They can also be used for gradient separations, in which the tubes are filled from
top to bottom with an increasing concentration of a dense substance in solution.
Sucrose gradients are typically used for separation of cellular organelles.
Gradients of caesium salts are used for separation of nucleic acids. After the
sample has spun at high speed for sufficient time to produce the separation, the
rotor is allowed to come to a smooth stop and the gradient is gently pumped out of
each tube to isolate the separated components.


 DENSITY GRADIENT CENTRIFUGATION

 Density gradient centrifugation Is considered one of the more efficient methods of

separating suspended particles.

 Density gradient centrifugation can be used both as a separation technique and as a

method of measuring the densities of particles or molecules in a mixture.
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  A tube, after being centrifuged by this method, has particles in order of density based on
height. The object or particle of interest will reside in the position within the tube
corresponding to its density

 DIFFERENTIAL CENTRIFUGATION
 Differential Centrifugation is a type of centrifugation in which one selectively spins
down components of a mixture by a series of increasing centrifugation forces.
 This method is commonly used to separate organelles and membranes found in cells.
 Organelles generally differ from each other in density in size, making the use of
differential centrifugation, and centrifugation in general, possible.
 The organelles can then be identified by testing for indicators that are unique to the
specific organelles

Xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx

RADIOLIGAND

A radioligand is a radioactive biochemical substance (in particular, a ligand that is

radiolabeled) that is used for diagnosis or for research-oriented study of the receptor systems of
the body.

In a neuroimaging application the radioligand is injected into the pertinent tissue, or

infused into the bloodstream. It binds to its receptor. When the radioactive isotope in the
ligand decays it can be measured by positron emission tomography (PET) or single photon
emission computed tomography (SPECT).

In in vivo systems it is often used to quantify the binding of a test molecule to the binding site of
radioligand.

The higher the affinity of the molecule the more radioligand is displaced from the binding site
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and the increasing radioactive decay can be measured by scintillography.

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This assay is commonly used to calculate binding constant of molecules to receptors.

53
 The transport of the radioligand is described by receptor kinetics.

Radioligands are credited with making possible the study of biomolecular behaviour, a
previously mysterious area of research that had evaded researchers. With this capacity
radioligand techniques enabled researchers to identify receptor devices within cells.

Radioactive isotopes commonly used

 Tritium, 3H
 Carbon-14, 14C
 Sulfur-35, 35S
 Iodine-131, 131I
 Fluorine-18, 18F
 Technetium-99m, 99mTc
 Copper-64, 64

LOWRY PROTEIN ASSAY

 The Lowry protein assay is a biochemical assay for determining the total level of protein
in a solution. The total protein concentration is exhibited by a color change of the sample
solution in proportion to protein concentration, which can then be measured using
colorimetric techniques. It is named for the biochemist Oliver H. Lowry who developed
the reagent in the 1940s. His 1951 paper describing the technique is the most-highly cited
paper ever in the scientific literature, cited over 300,000 times.
 The method combines the reactions of copper ions with the peptide bonds under alkaline
conditions (the Biuret test) with the oxidation of aromatic protein residues.
 The Lowry method is based on the reaction of Cu+, produced by the oxidation of peptide
bonds, with Folin–Ciocalteu reagent (a mixture of phosphotungstic acid and
phosphomolybdic acid in the Folin–Ciocalteu reaction).
 The reaction mechanism is not well understood, but involves reduction of the Folin–
Ciocalteu reagent and oxidation of aromatic residues (mainly tryptophan, also tyrosine).
Experiments have shown that cysteine is also reactive to the reagent.
 Therefore, cysteine residues in protein probably also contribute to the absorbance seen in
the Lowry Assay.
 The concentration of the reduced Folin reagent is measured by absorbance at 750 nm. As
a result, the total concentration of protein in the sample can be deduced from the
concentration of Trp and Tyr residues that reduce the Folin–Ciocalteu reagent.

PROTEIN STRUCTURE
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Increasingly, drug developers are looking to large molecules and particularly proteins as a
therapeutic option. Formulation of a protein drug product can be quite a challenge, but without
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 a good understanding of the nature of protein structure and the conformational characteristics of
the specific protein being formulated, the results can be ruinous. This technical brief aims to give
the reader a quick overview of protein structure. It will also cover briefly how protein structure
can be affected during formulation and some of the analytical methods which can be used both to
determine the structure and analyze the stability of the protein.

The term structure when used in relation to proteins, takes on a much more complex meaning
than it does for small molecules. Proteins are macromolecules and have four different levels of
structure – primary, secondary, tertiary and quaternary.

Primary Structure

There are 20 different standard L-α-amino acids used by cells for protein construction.

Amino acids, as their name indicates, contain both a basic amino group and an acidic carboxyl
group. This difunctionality allows the individual amino acids to join together in long chains by
forming peptide bonds: amide bonds between the -NH2 of one amino acid and the -COOH of
another. Sequences with fewer than 50 amino acids are generally referred to as peptides, while
the terms protein or polypeptide are used for longer sequences.

A protein can be made up of one or more polypeptide molecules. The end of the peptide or
protein sequence with a free carboxyl group is called the carboxy-terminus or C-terminus. The
terms amino-terminus or N-terminus describe the end of the sequence with a free α-amino group.

The amino acids differ in structure by the substituent on their side chains. These side chains
confer different chemical, physical and structural properties to the final peptide or protein. The
structures of the 20 amino acids commonly found in proteins are shown in Figure 1. Each amino
acid has both a one-letter and three-letter abbreviation. These abbreviations are commonly used
to simplify the written sequence of a peptide or protein.

Depending on the side-chain substituent, an amino acid can be classified as being acidic, basic
or neutral.

Although 20 amino acids are required for synthesis of various proteins found in humans,
we can synthesize only 10. The remaining 10 are called essential amino acids and must be
obtained in the diet.

The amino acid sequence of a protein is encoded in DNA. Proteins are synthesized by a series
of steps called transcription (the use of a DNA strand to make a complimentary messenger RNA
strand - mRNA) and translation (the mRNA sequence is used as a template to guide the synthesis
of the chain of amino acids which make up the protein).
55

Often, post-translational modifications, such as glycosylation or phosphorylation, occur which

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are necessary for the biological function of the protein. While the amino acid sequence makes up

55
 the primary structure of the protein, the chemical/biological properties of the protein are very
much dependent on the three-dimensional or tertiary structure.

Secondary Structure
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 Stretches or strands of proteins or peptides have distinct characteristic local structural
conformations or secondary structure, dependent on hydrogen bonding. The two main types of
secondary structure are the α-helix and the ß-sheet.

The α-helix is a right-handed coiled strand. The side-chain substituents of the amino acid groups
in an α-helix extend to the outside. Hydrogen bonds form between the oxygen of the C=O of
each peptide bond in the strand and the hydrogen of the N-H group of the peptide bond four
amino acids below it in the helix. The hydrogen bonds make this structure especially stable. The
side-chain substituents of the amino acids fit in beside the N-H groups.

The hydrogen bonding in a ß-sheet is between strands (inter-strand) rather than within strands
(intra-strand). The sheet conformation consists of pairs of strands lying side-by-side. The
carbonyl oxygens in one strand hydrogen bond with the amino hydrogens of the adjacent strand.
The two strands can be either parallel or anti-parallel depending on whether the strand directions
(N-terminus to C-terminus) are the same or opposite. The anti-parallel ß-sheet is more stable due
to the more well-aligned hydrogen bonds.

Tertiary Structure

The overall three-dimensional shape of an entire protein molecule is the tertiary structure. The
protein molecule will bend and twist in such a way as to achieve maximum stability or lowest
energy state. Although the three-dimensional shape of a protein may seem irregular and random,
it is fashioned by many stabilizing forces due to bonding interactions between the side-chain
groups of the amino acids.

Under physiologic conditions, the hydrophobic side-chains of neutral, non-polar amino acids
such as phenylalanine or isoleucine tend to be buried on the interior of the protein molecule
thereby shielding them from the aqueous medium. The alkyl groups of alanine, valine, leucine
and isoleucine often form hydrophobic interactions between one-another, while aromatic groups
such as those of phenylalanine and tryosine often stack together. Acidic or basic amino acid side-
chains will generally be exposed on the surface of the protein as they are hydrophilic.

The formation of disulfide bridges by oxidation of the sulfhydryl groups on cysteine is an

important aspect of the stabilization of protein tertiary structure, allowing different parts of the
protein chain to be held together covalently. Additionally, hydrogen bonds may form between
different side-chain groups. As with disulfide bridges, these hydrogen bonds can bring together
two parts of a chain that are some distance away in terms of sequence. Salt bridges, ionic
interactions between positively and negatively charged sites on amino acid side chains, also help
to stabilize the tertiary structure of a protein.

Quaternary Structure
57

Many proteins are made up of multiple polypeptide chains, often referred to as protein subunits.
Page

These subunits may be the same (as in a homodimer) or different (as in a heterodimer). The

57
 quaternary structure refers to how these protein subunits interact with each other and arrange
themselves to form a larger aggregate protein complex. The final shape of the protein complex is
once again stabilized by various interactions, including hydrogen-bonding, disulfide-bridges and
salt bridges. The four levels of protein structure are shown in Figure 2.

Protein Stability

Due to the nature of the weak interactions controlling the three-dimensional structure, proteins
are very sensitive molecules. The term native state is used to describe the protein in its most
stable natural conformation in situ. This native state can be disrupted by a number of external
stress factors including temperature, pH, removal of water, presence of hydrophobic surfaces,
presence of metal ions and high shear. The loss of secondary, tertiary or quaternary structure due
to exposure to a stress factor is called denaturation. Denaturation results in unfolding of the
protein into a random or misfolded shape.

A denatured protein can have quite a different activity profile than the protein in its native form,
usually losing biological function. In addition to becoming denatured, proteins can also form
aggregates under certain stress conditions. Aggregates are often produced during the
manufacturing process and are typically undesirable, largely due to the possibility of them
causing adverse immune responses when administered.

In addition to these physical forms of protein degradation, it is also important to be aware of the
possible pathways of protein chemical degradation. These include oxidation, deamidation,
peptide-bond hydrolysis, disulfide-bond reshuffling and cross-linking. The methods used in the
processing and the formulation of proteins, including any lyophilization step, must be carefully
examined to prevent degradation and to increase the stability of the protein biopharmaceutical
both in storage and during drug delivery.

Protein Structure Analysis

The complexities of protein structure make the elucidation of a complete protein structure
extremely difficult even with the most advanced analytical equipment. An amino acid analyzer
can be used to determine which amino acids are present and the molar ratios of each. The
sequence of the protein can then be analyzed by means of peptide mapping and the use of Edman
degradation or mass spectroscopy. This process is routine for peptides and small proteins, but
becomes more complex for large multimeric proteins.

Peptide mapping generally entails treatment of the protein with different protease enzymes in
order to chop up the sequence into smaller peptides at specific cleavage sites. Two commonly
used enzymes are trypsin and chymotrypsin. Mass spectroscopy has become an invaluable tool
for the analysis of enzyme digested proteins, by means of peptide fingerprinting methods and
58

database searching. Edman degradation involves the cleavage, separation and identification of
one amino acid at a time from a short peptide, starting from the N-terminus.
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58
 One method used to characterize the secondary structure of a protein is circular dichroism
spectroscopy (CD). The different types of secondary structure, α-helix, ß-sheet and random coil,
all have characteristic circular dichroism spectra in the far-uv region of the spectrum (190-250
nm). These spectra can be used to approximate the fraction of the entire protein made up of each
type of structure.

A more complete, high-resolution analysis of the three-dimensional structure of a protein is

carried out using X-ray crystallography or nuclear magnetic resonance (NMR) analysis. To
determine the three-dimensional structure of a protein by X-ray diffraction, a large, well-ordered
single crystal is required. X-ray diffraction allows measurement of the short distances between
atoms and yields a three-dimensional electron density map, which can be used to build a model
of the protein structure.

The use of NMR to determine the three-dimensional structure of a protein has some advantages
over X-ray diffraction in that it can be carried out in solution and thus the protein is free of the
constraints of the crystal lattice. The two-dimensional NMR techniques generally used are
NOESY, which measures the distances between atoms through space, and COESY, which
measures distances through bonds.

Protein Structure Stability Analysis

Many different techniques can be used to determine the stability of a protein. For the analysis of
unfolding of a protein, spectroscopic methods such as fluorescence, UV, infrared and CD can be
used. Thermodynamic methods such as differential scanning calorimetry (DSC) can be useful in
determining the effect of temperature on protein stability. Comparative peptide-mapping (usually
using LC/MS) is an extremely valuable tool in determining chemical changes in a protein such as
oxidation or deamidation. HPLC is also an invaluable means of analyzing the purity of a protein.
Other analytical methods such as SDS-PAGE, iso-electric focusing and capillary electrophoresis
can also be used to determine protein stability, and a suitable bioassay should be used to
determine the potency of a protein biopharmaceutical. The state of aggregation can be
determined by following “particle” size and arrayed instruments are now available to follow this
over time under various conditions.
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59
 The variety of
methods for
determining protein
stability again
emphasizes the
complexity of the
nature of protein
structure and the
importance of
maintaining that
structure for a
successful
biopharmaceutical
product
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 References

1. 1. Protein Structure, Stability and Folding, Methods in Molecular Biology, Vol. 168,
Edited by Kenneth P. Murphy
2. 2. Protein Stability and Folding, Theory and Practice, Methods in Molecular Biology,
Vol. 40, Edited by Bret A. Shirley
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 Ligand binding assay

Ligand binding assays (LBA) is an assay, or an analytic procedure, whose procedure or method
relies on the binding of ligand molecules to receptors, antibodies or other macromolecules. A
detection method is used to determine the presence and extent of the ligand-receptor complexes
formed, and this is usually determined electrochemically or through a fluorescence detection
method.This type of analytic test can be used to test for the presence of target molecules in a
sample that are known to bind to the receptor.

There are numerous types of ligand binding assays, both radioactive and non-radioactive.As
such, ligand binding assays are a superset of radiobinding assays, which are the conceptual
inverse of radioimmunoassays (RIA). Some newer types are called "mix-and-measure" assays
because they do not require separation of bound ligands.[5]

Ligand binding assays are used primarily in pharmacology for various demands. Specifically,
despite the human body’s endogenous receptors, hormones, and other neurotransmitters,
pharmacologists utilize assays in order to create drugs that are selective, or mimic, the
endogenously found cellular components. On the other hand, such techniques are also available
to create receptor antagonists in order to prevent further cascades. Such advances provide
researchers with the ability not only to quantify hormones and hormone receptors, but also to
contribute important pharmacological information in drug development and treatment plans.

History

Historically, ligand binding assay techniques were used extensively to quantify hormone or
hormone receptor concentrations in plasma or in tissue. The ligand-binding assay methodology
quantified the concentration of the hormone in the test material by comparing the effects of the
test sample to the results of varying amounts of known protein (ligand).

The foundations for which ligand binding assay have been built are a result of Karl Landsteiner,
in 1945, and his work on immunization of animals through the production of antibodies for
certain proteins.[9] Landsteiner’s work demonstrated that immunoassay technology allowed
researchers to analyze at the molecular level. The first successful ligand binding assay was
reported in 1960 by Rosalyn Sussman Yalow and Solomon Berson.[9] They investigated the
binding interaction for insulin and an insulin-specific antibody, in addition to developing the first
radioimmunoassay (RIA) for insulin. These discoveries provided precious information regarding
both the sensitivity and specificity of protein hormones found within blood-based fluids.[9] Yalow
and Berson received the Nobel Prize in Medicine as a result of their advancements. Through the
development of RIA technology, researchers have been able to move beyond the use of
radioactivity, and instead, use liquid- and solid-phase, competitive, and immunoradiometric
assays.[9] As a direct result of these monumental findings, researchers have continued the
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advancement of ligand binding assays in many facets in the fields of biology, chemistry, and the
like.
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 Applications for Ligand Binding Assays

Ligand binding assays provide a measure of the interactions that occur between two molecules,
such as protein-bindings, as well as the degree of affinity (weak, strong, or no connection) for
which the reactants bind together.[10] Essential aspects of binding assays include, but are not
limited to, the concentration level of reactants or products (see radioactive section), maintaining
the equilibrium constant of reactants throughout the assay, and the reliability and validity of
linked reactions.[10] Although binding assays are simple, they fail to provide information on
whether or not the compound being tested affects the target's function.[11]

Radioactive Ligand Binding Assays

Radioligands are used to measure the ligand binding to receptors and should ideally have high
affinity, low non-specific binding, high specific activity to detect low receptor densities, and
receptor specificity.[7]

Levels of radioactivity for a radioligand (per mole) are referred to as the specific activity (SA),
which is measured in Ci/mmol.[12] The actual concentration of a radioligand is determined by the
specific stock mix for which the radioligand originated (from the manufactures.)[12] The
following equation determines the actual concentration:

Non-radioactive Ligand Binding Assays

Despite the different techniques used for non-radioactive assays, they require that ligands exhibit
similar binding characteristics to its radioactive equivalent. Thus, results in both non-radioactive
and radioactive assays will remain consistent.[5] One of the largest differences between
radioactive and non-radioactive ligand assays are in regards of dangers to human health.
Radioactive assays are harmful in that they produce radioactive waste; whereas, non-radioactive
ligand assays utilize a different method to avoid producing toxic waste. These methods include,
but are not limited to, fluorescence polarization (FP), fluorescence resonance energy transfer
(FRET), and surface plasmon resonance (SPR). In order to measure process of ligand-receptor
binding, most non-radioactive methods require that labeling avoids interfering with molecular
interactions.[5]

Liquid Phase Ligand Binding Assays

Real-Time Polymerase Chain Reaction (qPCR)

Real-time polymerase chain reaction.

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 Immunoprecipitation

The liquid phase ligand binding assay of Immunoprecipitation (IP) is a method that is used to
purify or enrich a specific protein, or a group of proteins, using an antibody from a complex
mixture. The extract of disrupted tissue or cells is mixed with an antibody against the antigen of
interest, which produces the antigen-antibody complex.[14] When antigen concentration is low,
the antigen-antibody complex precipitation can take hours or even days and becomes hard to
isolate the small amount of precipitate formed.[14]

The enzyme-linked immunosorbent assay (ELISA) or Western blotting are two different ways
that the purified antigen (or multiple antigens) can be obtained and analyzed. This method
involves purifying an antigen through the aid of an attached antibody on a solid (beaded)
support, such as agarose resin.[15] The immobilized protein complex can be accomplished either
in a single step or successively.[15]

IP can also be used in conjunction with biosynthetic radioisotope labeling. Using this technique
combination, one can determine if a specific antigen is synthesized by a tissue or by a cell.[14]

Solid Phase Ligand Binding Assays

Multiwell plates-set

Multiwell plates are multiple petri dishes incorporated into one container, with the number of
individual wells ranging from 6 to over 1536. Multiwell Plate Assays are convenient for
handling necessary dosages and replicates.[16] There are a wide range of plate types that have a
standardized footprint, supporting equipment, and measurement systems.[16] Electrodes can be
integrated into the bottom of the plates to capture information as a result of the binding assays.[9]
The binding reagents become immobilized on the electrode surface and then can be analyzed.[9]

The multiwell plates are manufactured to allow researchers to create and manipulate different
types of assays (i.e., bioassays, immunoassays, etc.) within each multiwell plate.[16] Due to the
variability in multiwell plate formatting, it is not uncommon for artifacts to arise. Artifacts are
due to the different environments found within the different wells on the plate, especially near
the edges and center of the wells. Such effects are known as well effects, edge effects, and plate
effects. Thus, emphasizing the necessity to position assay designs in the correct manner both
within, and between, each plate.[16]

The use of multiwell plates are common when measuring in vitro biological assay activity, or
measuring immunoreactivity through immunoassays.[16] Artifacts can be avoided by maintaining
plate uniformity by applying the same dose of the specific medium in each well, in addition to
maintaining atmospheric pressure and temperature rates in order to reduce humidity.[16]
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 On-Bead Ligand Binding assays

On-Bead Ligand Binding assays are isolation methods for basic proteins, DNA/RNA or other
biomolecules located in undefined suspensions and can be used in multiple biochromatographic
applications. Bioaffine ligands are covalently bound to silica beads with terminal negatively
charged silanol groups or polystyrene beads and are used for isolation and purification of basic
proteins or adsorption of biomolecules. After binding the separation is performed by
centrifugation (density separation) or by magnetic field attraction (for magnetic particles only).
The beads can be washed to provide purity of the isolated molecule before dissolving it by ion
exchange methods. Direct analyzation methods based on enzymatic/fluorescent detection (e.g.
HRP, fluorescent dye) can be used for on-bead determination or quantification of bound
biomolecules. [17][18][19]

On-Column Ligand Binding Assays

Filter Assays

Filter assays are a solid phase ligand binding assay that use filters to measure the affinity
between two molecules. In a filter binding assay, the filters are used to trap cell membranes by
sucking the medium through them.[8] This rapid method occurs at a fast speed in which filtration
and a recovery can be achieved for the found fraction.[20] Washing filters with a buffer removes
residual unbound ligands and any other ligands present that are capable of being washed away
from the binding sites.[8] The receptor-ligand complexes present while the filter is being washed
will not dissociate significantly because they will be completely trapped by the filters.[8]
Characteristics of the filter are important for each job being done. A thicker filter is useful to get
a more complete recovery of small membrane pieces, but may require a longer wash time.[8] It is
recommended to pretreat the filters to help trap negatively charged membrane pieces.[8] Soaking
the filter in a solution that would give the filter a positive surface charge would attract the
negatively charged membrane fragments.[8]

Binding Specificity

The effects of a drug are a result of their binding selectivity with macromolecule properties of an
organism, or the affinity with which different ligands bind to a substrate.[21] More specifically,
the specificity and selectivity of a ligand to its respective receptor provides researchers the
opportunity to isolate and produce specific drug effects through the manipulation of ligand
concentrations and receptor densities.[21] Hormones and neurotransmitters are essential
endogenous regulatory ligands that affect physiological receptors within an organism.[21] Drugs
that act upon these receptors are incredibly selective in order to produce required responses from
signaling molecules.[21]

Specific binding refers to the binding of a ligand to a receptor, and it is possible that there is
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more than one specific binding site for one ligand.[22] Non specific binding refers to the binding
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of a ligand to something other than its designated receptor such as various other receptors, or
different types of transporters in the cell membrane.[22] For example, various antagonists can bind

65
 to multiple types receptors. In the case of muscarinic antagonists, they can also bind to histamine
receptors.[22] Such binding patterns are technically considered specific, as the destination of the
ligand is specific to multiple receptors. However, researchers may not be focused on such
behaviors compared to other binding factors.[22] Nevertheless, nonspecific binding behavior is
very important information to acquire. These estimates are measured by examining how a ligand
binds to a receptor while simultaneously reacting to a substitute agent (antagonist) that will
prevent specific binding to occur.[22]
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