Enzymes are biological catalysts that speed up chemical reactions without being consumed. Their activity can be measured by determining the amount of substrate converted to product per unit time. There are two main types of enzyme assays: continuous assays that measure reaction rates over time, and discontinuous assays that take samples at intervals to measure substrate/product levels. Common techniques to measure enzyme activity include spectrophotometry, fluorescence spectroscopy, chromatography, and radiometric methods. Spectrophotometry is often used to examine light absorption of substrates and products in the ultraviolet-visible range.

1. MeasureMent of enZYMe
aCtIVItY

2. What is an EnzymE?
Enzymes are powerful biological catalyst which speed up a
reaction but are not consumed in the reaction.
The catalysis process takes place at the active site.
Enzymes are extremely selective with their reactants, or
substrates, and the type of chemical reactions they are
involved with.

3. EnzymE activity
The enzyme activity can be defined as the number of
moles of substrate converted per unit time.
Enzyme activity = moles of substrate converted
per unit time
= rate × reaction volume.
The SI unit is the katal, 1 katal = 1 mol s−1
A more practical and commonly used value is 1 enzyme
unit (U) = 1 μmol min−1

4. tyPEs OF EnzymE assays
CONTINUOUS ASSAYS
• With continuous assays,
one can measure the
linearity of the assay which
can be used to conduct a
fixed-timed assay
• A few methods are
spectrophotoometric,
fluorometric, calorimetric
and chemi-luminescent.
DISCONTINUOUS ASSAYS
• Discontinuous assays are
when samples are taken
from an enzyme reaction at
intervals and the amount of
product production or
substrate consumption is
measured in these samples.
• The discontinuous assays
are radiometric and
chromatographic.

5. MeasureMent Of
enzyMatic activity By
spectrOscOpy
• Spectrophotometry is the measureable analysis
technique using the electromagnetic spectra. It deals
with the ranges of wavelengths such as near
ultraviolet, near infrared and visible light
• Activity of the enzyme, the following spectroscopic
techniques are used: Fluorescence spectroscopy,
UV/VIS Spectroscopy, Spectrophotometric Assays,
and Infrared spectroscopy.

6. fluOrescence
spectrOscOpy
• Fluorescence spectroscopy reveals the existence of
ES complexes and what they are made of.
• A compound is exposed to UV-light which excites
certain molecules and causes them to emit light at a
lower wavelength, which is in the visible light range.
• The fluorescence of the substrate is measured and
compared to the fluorescence of the product, and in
the difference of the two measurements, enzymatic
activity is measured

7. infrared spectrOscOpy
• In enzyme-substrate complexes, there are well-organized
binding modes, which is quantifiable using
infrared methods.
• In analyzing infrared data, it is possible to identify
binding modes and heterogeneity of ES complexes.

8. ultraviOlet-visiBle
spectrOscOpy
• It is a commonly used spectrophotometric assay that
examines photons in the UV-visible region.
• It is mainly used to determine the amount of a
highly-conjugated organic compound or enzyme
contained in a specific solution.

9. • It is complimentary to fluorescence spectroscopy, as
fluorescence spectroscopy deals with transitions
from the excited state to the ground state,
ultraviolet-visible spectroscopy deals with transitions
from the ground state to the excited state.

10. • The device, spectrophotometer, measures the
transmittance of light through a sample
The equation used to calculate the transmittance is
A=-log(%T)
Here, A= absorbance
T= I/Io
I=the intensity of light passing
through the sample
Io=the initial intensity of the light
before it is transmitted
through the sample

12. RadiometRic assay
• It measures the incorporation of radioactivity into
substrates or its release from substrates.
• The radioactive isotopes most frequently used in
these assays are 14C, 32P, 35S and 125I.
• These assays are both extremely sensitive and
specific.
• They are often the only way of measuring a specific
reaction in crude extracts (the complex mixtures of
enzymes produced when you lyse cells).

13. ChromatographiC assay
• It measures product formation by separating the
reaction mixture into its components
by chromatography.
• This is usually done by high-performance liquid
chromatography (HPLC), but can also use the simpler
technique of thin layer chromatography.

14. • Although this approach can need a lot of material, its
sensitivity can be increased by labelling the
substrates/products with a radioactive or fluorescent
tag, by switching protocols to improved
chromatographic instruments (e.g. ultra-high
pressure liquid chromatography) that operate at
pump pressure a few-fold higher than HPLC
instruments.