LAB 4 MEDICAL CLUB

Measurement of Alkaline Phosphatase Enzyme Activity

Welcome to our last genetics lab in the mid-term material, special thanks to Dr. Aya Al-smerat
for editing it! and let's get started!
v Enzymes:
§ Enzymes are biological molecules (mainly) proteins that speed up the rate of a
biochemical reaction by decreasing the activation energy, and not consumed during
that reaction.
§ Most of chemical reactions in biological processes are governed by enzymes, most of
enzymes are proteins.

v Energy Changes Occurring During the Reaction:
§ All chemical reaction have an energy barrier separating the
reactants and the products.

§ This barrier is called the free energy of activation and it’s
the energy difference between the reactants and the high
energy intermediate occurring during the formation of the
products.

§ Transition state: where the high-energy intermediate is formed during the conversion
of reactants to products.
§ There must be sufficient energy to overcome the energy barrier (transition state) for
the reaction to occur.
Lower energy of activation ---> faster rate of the reaction.
v How enzyme work?
Like we said above, the rate of the reaction depends on the activation energy.
Enzymes increase the rate by decreasing the activation energy, as u can see in the figure.

Note that enzymes DON’T change the energy of the reactant
or the products, they ONLY lower the activation energy.
Therefore it doesn’t affect ∆G or whether the reaction is
spontaneous or not.

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v Factors affecting enzyme activity thus reaction velocity:

1. Substrate concentration:
the rate of an enzymatic catalyzed reaction increases when we
increase the substrate conc. Until it reaches the maximum
velocity then the velocity will remain constant at which the
active sites of enzymes will be saturated.

After reaching the maximum velocity, adding more substrate will have no added effect.

2. Temperature:
the reaction velocity increases with temperature until a peak
velocity is reached (optimum temperature) at which increasing
the temperature will start denaturation the enzymes thus
decreasing the velocity of the reaction.
Increasing the temperature will increase the number of molecules having sufficient energy to
overcome the energy barrier.

3. pH level: pH at which maximal enzyme activity is achieved is different for different
enzymes.
E.g. the enzyme pepsin is maximally active at pH 2 while other enzymes
require higher pH level to work and are denatured in acidic environment,
as u can see in the figure.


v Co-Factors:
§ In many cases enzymes need additional molecules to function and these molecules are
known as cofactors.
§ Cofactors: small non-protein helper or accessory molecules that are necessary to activate
the enzyme.
§ Cofactors can be inorganic (Mg+2, Cu+2…) or organic coenzymes such as vitamins (B-
complex).

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v Enzyme inhibitors:
§ Enzyme inhibitor: is any substance that can decrease the velocity of an enzyme
catalyzed reaction.
§ Inhibitors can be reversible which bind non-covalently or irreversible which bind
covalently to the enzyme.
§ Two types of reversible inhibition, competitive or non-competitive.

§ the compititive inhibition occures when the inhibitor binds
to the same site that the substrate bind (active site).
so it competes with the substrate.
§ Non-compititive inhibition occures when the inhibitor and
the substrate bind at different sites.

As u can see in the figure the effect of competitive inhibitors is
reversed when we increase the substrate conc. and the rate of
the reaction will increase until it reaches Vmax observed in the
absence of an inhibitor, while in non-competitive inhibitor
increasing the substrate won’t increase the rate that much, which
means the non-competitive inhibitors decrease Vmax.

v Measuring enzyme activity:

§ Enzyme assays are laboratory methods for measuring enzymatic activity.
§ There are several methods to measure the enzyme activity including
spectrophotometric method.
§ In spectrophotometric assay change in the intensity of the light absorbed by the
reaction solution is measured.

§ Enzyme activity is measured by international unit.
§ One unit, U or IU is the amount of enzyme that catalyzes the conversion of 1 micromole
of substrate into product per minute under specified conditions of the assay method,
and the specific condition will usually be the optimum conditions including the
substrate concentration, temperature and pH.

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v Measuring Alkaline phosphatase enzyme activity:
§ Alkaline phosphatases are group of enzymes that split off a terminal phosphate group
from an organic ester and as the name indicate they are most effective in alkaline
environment.
§ Alkaline phosphatases are expressed in many tissues of the body, particularly hepatic,
renal and skeletal tissues.
§ ALP serum level activity increased in bone and liver diseases.

v Principle of the assay: (follow the figure while reading)
ALP catalyze the hydrolysis the p-nitrophenylphosphate (substrate) and it’s colorless and it’ll
be hydrolyzed to p-nitrophenol and inorganic phosphate (products) and this reaction occurs in
the presence of Mg+2 as a cofactor.
After the reaction (the product), p-nitrophenol has a color which is yellow and its maximum
absorbance is 405nm.
We measure the enzyme activity by monitoring the product formation, measure A, increase in
A is proportional to enzyme activity. (A= absorbance).





405nm

v Lab work (practical part):
• Equipment: we need fixed volume micropipette with 2 different sizes 100 microL and 1000
microL. We also need test tube to contain the reaction solution.
• Materials:
1. Reagent that contains diethanolamine buffer with 9.8pH to
provide alkaline medium for the reaction, it also contains
MgSO4 to provide the cofactor (Mg+2 ions) and the substrate
p-nitrophenylphosphate.
2. Sample contains ALP enzyme.

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v Procedure: 1 2
1. Using fixed volume micropipette Take 1 ml (1000
microliter) of the reagent.
2. Transfer it to the test tube.
3. Take 100 microliter of the sample.
4. Add it to the test tube (to the reagent).
5. Wait for 1 minute, then measure the absorbance of the
solution at 405nm. 3 4
6. Take empty plastic cuvette and place in the sample
compartment of the spectrophotometer.
7. Press zero for zero adjustment (against air).
8. Transfer the solution from the test tube to a plastic
cuvette and place it in the sample compartment to
measure the absorbance. 6 7
9. as u can see the absorbance is 1.386
A1 (after 1 minute)=1.386
10.Wait for 3 minutes, then measure the absorbance of the
solution at 405nm
11.Measure the absorbance,
A2 (after 3 minutes from A1) =1.466 8 9
note that A2 was taken after 4 minutes from the
beginning of the reaction and after 3 minutes from A1.
12.Calculate ALP Activity:

10 11

- ΔA= A2-A1
- ΔT=T2-T1 = 3
- DF= total volume/sample volume, 1100/100= 11.
- Lp= Light path = 1cm
- Millimolar absorptivity of p-nitrophenol = 18.75.

Done by: Moath Al-Shaikh. ‫ اﻟﮭدف ﻣرﺻود واﻟرﺷﺎش ﺟﺎھز‬
If you have any questions don’t hesitate to ask moath.