‫ﺑﺴﻢ ﷲ اﻟﺮﺣﻤﻦ اﻟﺮﺣﯿﻢ‬

Measurement of
Enzyme Activity
Bioc. 231

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 Measurement of Enzyme Activity
• Because enzymes are usually present in very small
quantities in biological fluids and often difficult to
isolate from similar compounds, a convenient method
of enzyme quantification is measurement of catalytic
activities. Activity is then related to concentration.
• Common methods might photometrically measure:
- an increase in product concentration,
- a decrease in substrate concentration,
- a decrease in coenzyme concentration, or
- an increase in the concentration of an altered
coenzyme.

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Measurement of Enzyme Activity

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 Measurement of Enzyme Activity
• One of two general methods may be used to
measure the extent of an enzymatic reaction:
1. Stopped assays.
2. Continuous assays (kinetic assays).

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 Measurement of Enzyme Activity
§ Stopped assays involve stopping the reaction after a fixed
time, then measuring how much product has been formed.

• Methods for stopping the reaction include those which

denature the enzyme, such as strong acid, alkali or detergent;
heat; or treatments with irreversible inhibitors such as heavy
metal ions. In some cases, the enzyme can be stopped by
addition of a complexing agent such as EDTA, which removes
metal ions essential for activity; even chilling on ice may be
sufficient.

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 Measurement of Enzyme Activity
Stopped assays

It is important that stopped assays are checked at least once

with varying times of incubation, to ensure that the rate is linear
through the period selected for the standard method.

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 Measurement of Enzyme Activity
§ Continuous assays: the progress of the reaction is followed as it
occurs.

• In continuous-monitoring, multiple measurements (usually of

absorbance change) are made during the reaction, either at specific
time intervals (usually every 30 to 60 seconds) or continuously by a
continuous-recording spectrophotometer.

• Continuous assays are much more convenient in that the result is

seen immediately.

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 Measurement of Enzyme Activity
§ Continuous assays:

§ The simplest continuous assay is one in which the action of the

enzyme itself can be followed by changes in absorbance,
fluorescence, viscosity, pH, or one of several other possible physical
parameters.
• In many examples of hydrolase assays, an artificial substrate which
releases a colored or fluorescent product is used.

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 Detection Methods
§ The detection techniques include:
• Colorimetric,
• Spectrophotometric,
• Chemiluminescence,
• Chromatography,
• Radiochemical,
• Fluorescence and UV absorbance,
• Electrochemical techniques.

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 The International Unit (IU)
• When enzymes are quantified relative to their
activity rather than a direct measurement of
concentration, the units used to report
enzyme levels are activity units.
• The definition for the activity unit must
consider variables that may alter results (e.g.
pH, temperature, substrate).

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 The International Unit (IU)
• The EC defined the international unit (IU) as the
amount of enzyme that catalyze the reaction of 1µmol
of substrate per minute under specific conditions of
temperature, pH, substrate, and activators.
• Enzyme concentration is usually expressed in units per
liter (IU/L).
• The unit of enzyme activity recognized by the
International System of Units (SI) is the katal (mol/s).
The mole is the unit for substrate concentration, and
the unit of time is the second. Enzyme concentration is
then expressed as ketals per liter (kat/L) (1.0 IU = 17
nkat).

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 References
• Clinical Chemistry by Bishop M, Fody E and Schoeff L.
• Scopes R. (2002): Enzyme Activity and Assay. Encyclopedia of life sciences.
Macmillan Publishers Ltd, Nature Publishing Group.
• Huang X, et al., (2006): Aspartate aminotransferase (AST/GOT) and alanine
aminotransferase (ALT/GPT) detection techniques. Sensors (Basel). 6(7):
756–782.

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