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EXPERIMENT 2.

1
P27- P29

Enzyme Activity Determination

Assay the activity of alanine
aminotransferase (ALT) in serum
(Mohun’s Method)
Introduction
1. Enzymes are synthesized by the cells of all
living organisms. All enzymes are proteins.
2. They act like catalysts and accelerate the
substrate which are metabolic reactions
that life depends on .
3. Their catalytic activity depends on the
precise conformational structure in the
folded polypeptide chains.
4. Enzyme activity is the ability of an enzyme
to catalyse a specific reaction and a
measure of quantity of enzyme present .
5. Reaction rate can be measured as
disappearance of reactant or accumulation
of product per unit time under some
condition such as identified reactant and
product.
6. A unit of enzyme activity is the amount of enzyme
activity which will catalyse the transformation of 1
micromole of the substrate per minute under
standard conditions. The unit has the symbol “U”.
7. The enzymes of cellular metabolism are located
within the tissue cells and are present there at
high concentrations. When tissues break down or
their membranes leak, the level of these enzymes
in the plasma rises. So the present of these
enzyme in plasma has been shown to be of
diagnostic significance, their catalytic activities
may serve as qualitative or quantitative indexes of
tissue damage.
8. The plasma concentration of most enzymes
remains fairly constant in the case of a normal
individual. It will be altered if there is:
a) change of synthesis of enzymes within the cell;
b) cellular damage;
c) change in the size of enzymes forming tissue ;
d) an alteration in the rate of inactivation and disposal of
enzymes;
e) an obstruction to a normal pathway of enzyme
excretion.
9. The serum nonfunctional enzyme
determinations are particularly helpful in
clinical medicine, it may be useful to:

a) assess the severity of the organ damage;
b) differentiate a particular type of disease;
c) follow the trend of the disease;
d) determine post operative risk.
10. Normally the serum transaminase levers
are low but after extensive tissue destruction
these enzymes are liberated into the serum.

Liver tissue is rich in Aspartate Transferase
(AST) and alanine aminotransferase (ALT),
but contains more of ALT than of AST.
Therefore measurement of the serum levels
(activity) of ALT is to ascertain the potential
for liver cell damage.
Principle
The quantitative of serum ALT reflect the
damage of liver cell. Activities of serum ALT
were determined colorimetrically according to
Mohun (1957).
ALT can catalyze the transamination:
ALT
L-Alanine+α-ketoglutarate↔Pyruvate+ Glutamic acid
Then, 2,4-dinitro-phenyldrazine is added for
stopping the reaction and marron compounds
are formed which response to α -ketoacid.
The absorbance at 520nm of the product
formed from pyruvate is bigger than from
α -ketoglutatrate. So we can detect the
activity of ALT by spectrophotometry.
In this experiment, one Unit of ALT
activity is defined as the amount of
enzyme needed to produce 2.5μg of
pyruvate per ml serum after it is
incubated with the substrate at 37oC,
pH 7.4 for 30 min.
Procedure
4 tests tubes are used and the operation is done
according to the following 4 tables.
(1)
Test Test Blank Standard Standard Blank
(1) (2) (3) (4)
Substrate buffer 0.5 0 0.5 0.5

Put the tubes into water bath at 37°C for 5 min
(2)
(1) (2) (3) (4)
Serum 0.1 0.1 0 0
Pyruvate(200μg/ml) 0 0 0.1 0
Phosphate buffer 0 0 0 0.1

Mix the tubes sufficiently, and put them into water bath at 37°c for 30 min
(3)
(1) (2) (3) (4)
2,4-dinitro-phenylhydrazine 0.5 0.5 0.5 0.5
Substrate buffer 0 0.5 0 0

Mix the tubes, and put them into water bath at
37°c for 20 min
(4)
(1) (2) (3) (4)

0.4mol/L NaOH 5.0 5.0 5.0 5.0

Mix the tubes, after 20 min at room temperature, the
absorbance is read within 30 min at wavelength 520
nm on condition that absorbance is adjusted to zero
with distilled water.
Calculation
A1-A2 20 1
ALT enzyme activity = X X
(Mohun’s Unit ) A3-A4 2.5 0.1

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