Enzyme Assay Methods

Enzyme Assay Methods

• The assay is the act of measuring how fast a given
(unknown) amount of enzyme will convert
substrate to product (the act of measuring a
velocity).

• An assay requires to determine the concentration

of a product or substrate at a given time after
starting the reaction.

• Different enzymes require different estimation

methods depending on the type of reaction
catalyzed, the nature of S and P or coenzyme.
 Fluorescence Spectrophotometric
method method

Polarimetric Enzyme Assay Sampling

method Methods method

Manometric
Electrode method
method
Spectrophotometric methods
• Many substrates and products of enzyme reactions absorb
light either in the visible region or in the U.V. region.

• Mostly the spectra of S and P are not the same.

▫ The conversion of one into another is followed by a
considerable change of absorption and by measuring this
change the progress of the reaction can be followed
quantitatively.

• The enzyme is allowed to react with substrate and the

decrease in the conc. of substrate or the increase of
product produced will be followed spectrophotometrically
Advantages
• Easy
• Simple
• Sensitive
• Require small sample
• Whole progress curve can be followed
quantitatively
Cases in which product absorb but not
the substrate
• This will include enzymes catalyze the addition of
groups to double bonds or the reverse reaction.
Absorbs
at 300nm Fumarate hydratase
Fumarate Malate

These is easily
followed

Xanthine oxidase Absorbs

Hypoxanthine Urate at 290nm
The coenzyme undergoes change in absorption on
reduction or oxidation by S (oxidizing enzymes)

NAD+ NADH
Oxidized form Reduced form Absorbs
at 340nm

NADP+ NADPH

FAD+ FADH2 Much less absorption at

450nm

High absorption at 450nm

 Lactate Dehydrogenase
O O
− (LDH) O O−
+ +
C NADH + H NAD C
C O HC OH
CH3 CH3
pyruvate lactate

NADH: Absorbs at 340nm

NAD+ : No absorption at 340nm

In decrease in abs/min can be used to measure rate

of LDH activity
 NADPH: Absorbs at 340nm
NADP+ : No absorption at 340nm
Coupled
• When the enzyme reaction being studied cannot be
made to give an absorption change

• Spectrophotometric method will be used by adding

another purified enzyme which acts on the product
of the first reaction to cause such a change

• This help to follow the first enzymatic reaction.

Coupled

NADH: Absorbs at 340nm

NAD+ : No absorption at 340nm
 Both ATP and ADP absorb
at 260nm

In reaction can be followed by measuring

the increase in absorbance at 340 nm
(specific for NADH+ H+ but not NAD+)
Fluorescence Method: (Fluorimetric
method)
• Fluorescence is when a molecule emits light of one
wavelength after absorbing light of a different
wavelength.
• Uses a fluorometer
• Flavin compounds : fluoresce strongly in the
oxidized form, and lose their fluorescence on
reduction
NAD(P)H, FADH2 (Reduced form):
No fluorescence
NAD, NADP (oxidized form):
Blue fluorescence
Fluorescence Method: (Fluorimetric
method)

• In this can be used for following enzymatic

oxidation reduction reaction
• Oxidation: decrease in fluorescence
• Reduction reactions: an increase in fluorescence
Advantages
• Highly sensitive - much more sensitive than
spectrophotometric assays
• Detection in small quantities
• non-dangerous
• sensitive to environment
Manometric Method
• Use manometer
• These are convenient and accurate methods for
following reactions in which one of the
component is a gas.
• For the study of:
▫ Oxidases (O2 uptake)
▫ Decarboxylase (CO2 output)
Electrode Method
• To follow reactions which involve the
production of acids.
▫ Use glass or platinum electrode.
▫ In this method pH meter is used to measure
change in H+ conc. During enzyme
reactions. (i.e. measure change in pH as the
reaction proceeds).
 Radiometer pH meter

• pH is kept constant by addition of alkali. The

rate of additional alkali gives the reaction
velocity and does not depend on the amount of
buffer
• Automatic apparatus
• Convenient
• Keeps pH constant by addition of acid or alkali
• And plots a curve of amount added against
time.
• With this apparatus progress cures of many
enzyme reactions can be obtained automatically
Polarimetric method
• Use polarimeter
• For isomerases that convert one isomer to
another
• Or that convert optically active to inactive or vice
versa
• It can be used if both S and P are optically active
but different in specific rotation

D-glucose L-glucose

Sucrose D-glucose + D-fructose

Sampling method
• Many enzyme reactions are followed by
withdrawing samples at intervals and
estimating the substrate or product by
chemical methods in different enzymes require
different estimation methods.
▫ Fiske and SabbaRow method:
It is for inorganic phosphate. It can be used
for phosphatase, phosphorylase, nucleotides
and all enzymes involving ATP or ADP
including some kinase and synthetase.
For Carbohydrates: Reducing Sugars

• Used for study of enzymes acting on carbohydrates (since

the breaking of a glycosidic link produces a reducing
group).

Breaking to glycosidic bond reducing group

Cu+2

Cu+2 reduction
(colour change)
α-amylase
Starch + H2O glucose (reducing sugar)
Cu+2

Cu reduction (coloured product)

*Intensity of colour α E activity
 ALT
ala + α-Kg Glutamate

Pyruvate

2,4 DNPH
2,4 dinitrophenyl
Hydrazone NaOH
(Brown colour) (alkaline pH)

ALT: Alanine aminotransferase

2,4 DNPH: 2,4 dinitrophenyl hydrazine