Laboratory method for measuring enzyme activity. Vital for study of enzyme kinetics and enzyme inhibition. Measurement of enzyme activity – follow the change in concentration of substrate or product – measure reaction rate.

1. ENZYME ASSAYS

2. • Laboratory method for measuring enzyme
activity.
• Vital for study of enzyme kinetics and enzyme
inhibition.
• Measurement of enzyme activity – follow the
change in concentration of substrate or
product – measure reaction rate.

4. DIRECT CONTINUOUS ASSAYS
• Difference in properties of substrate and product –
measured directly.
• Continuous observation of the progress curve – most
preferred.
• Change in
– Absorbance. - Fluorescence.
– pH. - Optical rotation.
– Enthalpy. - Viscosity.
– Volume of reaction mixture.

5. ABSORBANCE
◦ I - intensity of light at a specified wavelength passing through aλ
sample.
◦ I0 - intensity of the light before it enters the sample.
 Relation between concentration and absorbance:
◦ extinction – proportionality constant relating absorbance toɛ →
concentration.
◦ c – concentration.






−=
0
10log
I
I
A
cIA =∈
∈
=
A
c

6. TURBIDIMETRY
• Light scattering not absorbance.
• Action of enzymes on turbid polymer solutions.
• Difficult to standardize – difficult to reproduce
results.
• E.g.: bacterial lysozyme assay – on dried bacterial
cells – measured at 450nm.
• Unit: one unit of activity – initial rate of change in
absorbance of 0.001 per minute when the volume in
the cuvette is 2.6ml, pH- 6.24 at 25˚C.

7. FLUORESCENCE
• Result of electronic transition – converts the
absorbing molecule to an excited state.
• Fluorescent molecule emits part of absorbed
energy as light – lower energy but higher
wavelength.
• More sensitive than absorbance assays.

8. FLUORIMETRY
• Fluorospectrophotometer – more specific than
spectrophotometer.
• Disadvantage: fluorescing molecules quench
in solution.
• E.g: anthranilate synthase
Chorismate + L-glutamine anthranilate + pyruvate↔
• λexci = 325nm, λemi = 400nm.

9. RADIOMETRY
• Requirement of labelled substrates and counting
instruments.
• Substrates can be labelled with 14
C, 3
H, 32
P, 35
S, 125
I.
• E.g: galactosyl transferase.
UDP-galactose* + glucosamine UDP + lactosamine*
• Stop the reaction by adding EDTA.
↓
Pass through ion exchange column – separate substrate and
products.
↓
Product collected – check radioactivity by scintillation counter.
GT, Mn+2

10. pH stat
Stationary pH.
Used to monitor progress of chemical reaction in
which protons are liberated or taken up.
Achieved by measuring the amount of acid or base
required to be added to maintain constant pH.

11. DIRECT DISCONTINUOUS ASSAY
p-nitrophenol in
alkaline condition
– highly
electronegative.
Colorless in acidic
condition and
yellow in alkaline
condition.
Yellow color
measured at
405nm.

12. INDIRECT ASSAYS
• Further treatment of reaction mixture –
produce a measurable product or increase
sensitivity of assay procedure.

13. CONTINUOUS ASSAYS
• Manipulation necessary to detect product formation – allows
continuous observation of the change.
• Less prone to errors from sample manipulation in
discontinuous assays
• Reagents required for color development or measurement of
activity included in the reaction mixture.
• E.g.: carnitine acyl transferase.
Acyl CoA + carnitine acyl carnitine + CoASH↔
CoASH + 5,5’-dithiobis-2-nitrobenzoate 4-nitrothiolate anion→
(DTNB - reagent)
• λmax = 412nm.

14. DISCONTINUOUS ASSAYS
• Also called sampling assay.
• Stopping reaction - after a fixed time.
• Treating the reaction mixture to separate the product for
analysis or produce a measurable change in properties of
substrates or product.
• Separate product for analysis (radiochemical assay)
– No modification made on the substrate/product can be considered→
as a direct assay.
• Produce change in properties of one substrate/product can→
be measured.
– Formation of ATP can be determined by measuring light intensity in
the presence of luciferase.
ATP + luciferin +O2 oxyluciferin + PPi +CO→ 2 + AMP + light

15. Coupled assays
• Use of one or more additional enzymes to
catalyse a reaction of one of the products to
yield a compound that can be directly
detected.
• Additional enzyme – coupled enzymes.

16. Examples
• Hexokinase.
– Coupling of the
formation of
glucose-6-
phosphate to the
reduction of
NADP+
in the
presence of G6P
dehydrogenase.
Glucose
ATP, Mg2+
ADP, Mg2+
Glucose 6-phosphate
NADP+
NADPH + H+
6-Phosphogluconolactone
G6P
DEHYDROGENASE
HEXOKINASE

17. Fructose 6-phosphate PHOSPHOFRUCTOKINASE Fructose 1,6-bisphosphate
ATP ADP
Pyruvate Phosphoenolpyruvate
NADH + H+
NAD+
PYRUVATE
KINASE
LDH
Lactate
Phosphofructokinase

18. Coupled continuous assay
1. Aspartate amino transferase (serum glutamate
oxaloacetate transaminase)
• AST/SGOT – 30˚C, pH 7-8 in 80mM tris.→
• Change in A340 measured.
Aspartate + α-ketoglutarate oxaloacetate + glutamate↔
Oxaloacetate + NADH + H+
malate + NAD↔ +

19. 2. Alanine aminotransferase (serum glutamate pyruvate
transaminase)
• ALT/SGPT.
Alanine + α-ketoglutarate pyruvate + glutamate↔
Pyruvate + NADH + H+
lactate + NAD↔ +
3. Decarboxylase.
Lysine cadaverine + CO2
CO2 + PEP oxaloacetate
Oxaloacetate + NADH + H+
malate + NAD+
Lysine decarboxylase
Wheat PEP carboxylase
MDH

20. Validity of results
• Reaction step should not be rate limiting.
• Velocity of the reaction increases till coupling
enzyme reaches the rate of the first enzyme.
• Coupling enzyme – high Km for the enzyme
and low Km for substrate.

21. Cycling coupled assay
• Alcohol dehydrogenase
Ethanol
NAD+
NADH + H+
Acetaldehyde
LactaldehydePropanediol

22. Forward coupled assay
• Malate dehydrogenase.
Malate + NAD+ MDH Oxaloacetate + NADH + H+
Acetyl CoA
CoA
CITRATE SYNTHASE
Citrate

23. References
• Enzyme Assays by Robert Eisenthal.
• Photometric assays – Robert A. John.
• Principles of enzyme assays and kinetic
studies – Keith F. Tipton.