Enzyme assays

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 a
λ
sample.
◦I
0
- intensity of the light before it enters the sample.
Relation between concentration and absorbance:
◦ extinction – proportionality constant relating absorbance to
ɛ →
concentration.
◦c – concentration.
÷
÷
ø
ö
ç
ç
è
æ
-=
0
10
log
I
I
A
cIA=Î
Î
=
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
↔
•λ
exci
= 325nm, λ
emi
= 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 +O
2
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, Mg
2+
ADP, Mg
2+
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 A
340
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 + CO
2
CO
2
+ 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.