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8 Enzyme Activities 1791990). Both methods assay enzyme activity after electrophore-
sis, but zymography is especially intended for proteolytic en-
zyme activity staining in which gels are cast with high concen-
trations of proteolytic enzyme substrates, for example, casein,
gelatin, bovine serum albumin, collagen, and others. Samples
containing proteolytic enzymes can be subjected to gel elec-
trophoresis, but the renaturation step has to be performed if a
denaturing condition is used; then the reaction is performed un-
der conditions suitable for assaying proteolytic enzymes. The
gel is then subjected to staining and destaining, but the entire gel
background will not be destained because the gel is polymerized
with protein substrates, except in clear zones where the signifi-
cant proteolysis has occurred; the amount of staining observed
will be greatly diminished due to the loss of protein. This process
is also known as reverse staining. Otherwise, reverse zymogra-
phy is a method used to assay the proteolytic enzyme inhibitor
activity in gel. Similar to zymography, samples containing prote-
olytic enzyme inhibitor can be subjected to gel electrophoresis.
After the gel renaturation step is performed, the reaction is as-
sayed under appropriate conditions in the presence of a specific
type of proteolytic enzyme. Only a specific type of proteolytic
enzyme inhibitor will be resistant to the proteolysis, and after
staining, the active proteolytic enzyme inhibitor will appear as
protein band (Oliver et al. 1999).Other MethodsThe most commonly used assay methods are the spectrophoto-
metric, spectrofluorometric, radiometric, chromatographic, and
electrophoretic methods described above, but a variety of other
methods are utilized as well. Immunological methods make use
of the antibodies raised against the proteins (Harlow and Lane
1988). Polarographic methods make use of the change in cur-
rent related to the change in concentration of an electroactive
compound that undergoes oxidation or reduction (Vassos and
Ewing 1983). Oxygen-sensing methods make use of the change
in oxygen concentration monitored by an oxygen-specific elec-
trode (Clark 1992), and pH-stat methods use measurements of
the quantity of base or acid required to be added to maintain a
constant pH (Jacobsen et al. 1957).Selection of an Appropriate SubstrateGenerally, a low molecular mass, chromogenic substrate con-
taining one susceptible bond is preferred for use in enzymatic
reactions. A substrate containing many susceptible bonds or dif-
ferent functional groups adjacent to the susceptible bond may
affect the cleavage efficiency of the enzyme, and this can re-
sult in the appearance of several intermediate-sized products.
Thus, they may interfere with the result and make interpretation
of kinetic data difficult. Otherwise, the chromophore-containing
substrate will readily and easily support assaying methods with
absorbance measurement. Moreover, substrate specificity can
also be precisely determined when an enzyme has more than one
recognition site on both the preferred bond and the functional
groups adjacent to it. Different sized chromogenic substratescan then be used to determine an enzyme’s specificity and to
quantify its substrate preference.Unit of Enzyme ActivityThe unit of enzyme activity is usually expressed as either micro-
moles of substrate converted or product formed per unit time,
or unit of activity per milliliter under a standardized set of con-
ditions. Though any unit of enzyme activity can be used, the
Commission on Enzymes of the International Union of Bio-
chemistry and Molecular Biology (IUBMB) has recommended
that a unit of enzyme, Enzyme Unit or International Unit (U),
be used. An Enzyme Unit is defined as that amount that will
catalyze the conversion of 1 micromole of substrate per minute,
1U= 1 μmol/min, under defined conditions. The conditions
include substrate concentration, pH, temperature, buffer compo-
sition, and other parameters that may affect the sensitivity and
specificity of the reaction, and usually a continuous spectropoto-
metric method or a pH stat method is preferred. Another enzyme
unit that now is not widely used is the International System of
Units (SI unit) in which 1 katal (kat)=1 mol/sec, so 1 kat=
60 mol/min= 6 × 107 U.
In ascertaining successful purification of a specified enzyme
from an extract, it is necessary to compare the specific activity
of each step to that of the original extract; a ratio of the two
gives the fold purification. The specific activity of an enzyme
is usually expressed as units per milligram of protein when the
unit of enzyme per milliliter is divided by milligrams of protein
per milliliter, the protein concentration. The fold purification is
an index reflecting only the increase in the specific activity with
respect to the extract, not the purity of the specified enzyme.REFERENCES
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Bashford CL, Harris DA. 1987.Spectrophotometry and Spectroflu-
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Briggs GE, Haldane JBS. 1925. A note on the kinetics of enzyme
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