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.

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.

ABSORBANCE

◦ I - intensity of light at a specified wavelength λ passing through a sample.

◦ I0 - intensity of the light before it enters the sample.Relation between concentration and absorbance:

◦ ɛ → extinction – proportionality constant relating absorbance to concentration.

◦ c – concentration.

010logIIA

cIA

Ac

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.

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.

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.

RADIOMETRY • Requirement of labelled substrates and counting

instruments.• Substrates can be labelled with 14C, 3H, 32P, 35S, 125I.• 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

pH statStationary 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.

DIRECT DISCONTINUOUS ASSAYp-nitrophenol in

alkaline condition – highly electronegative.

Colorless in acidic condition and yellow in alkaline condition.

Yellow color measured at 405nm.

INDIRECT ASSAYS• Further treatment of reaction

mixture – produce a measurable product or increase sensitivity of assay procedure.

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.

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 +CO2 + AMP + light

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.

Examples • Hexokinase.– Coupling of the

formation of glucose-6-phosphate to the reduction of NADP+ in the presence of G6P dehydrogenase.

GlucoseATP, Mg2+

ADP, Mg2+

Glucose 6-phosphateNADP+

NADPH + H+

6-Phosphogluconolactone

G6PDEHYDROGENASE

HEXOKINASE

Fructose 6-phosphatePHOSPHOFRUCTOKINASE Fructose 1,6-bisphosphate

ATP ADP

Pyruvate Phosphoenolpyruvate

NADH + H+

NAD+

PYRUVATE KINASE

LDH

Lactate

Phosphofructokinase

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+

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

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.

Cycling coupled assay• Alcohol dehydrogenase

Ethanol

NAD+ NADH + H+

Acetaldehyde

LactaldehydePropanediol

Forward coupled assay• Malate dehydrogenase.

Malate + NAD+ MDH Oxaloacetate + NADH + H+

Acetyl CoA

CoACITRATE SYNTHASE

Citrate

References • Enzyme Assays by Robert Eisenthal.• Photometric assays – Robert A. John.• Principles of enzyme assays and

kinetic studies – Keith F. Tipton.