7 Enzyme Activities 171greatly diminished due to the loss of protein. This
process is also known as reverse staining. Other-
wise, reverse zymography 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 elec-
trophoresis. After the gel renaturation step is per-
formed, the reaction is assayed under appropriate
conditions in the presence of a specific type of pro-
teolytic 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 Methods
The most commonly used assay methods are the
spectrophotometric, spectrofluorometric, radiomet-
ric, 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 current related to the change in con-
centration of an electroactive compound that under-
goes oxidation or reduction (Vassos and Ewing
1983). Oxygen-sensing methods make use of the
change in oxygen concentration monitored by an
oxygen-specific electrode (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 ANAPPROPRIATESUBSTRATE
Generally a low molecular mass, chromogenic sub-
strate containing one susceptible bond is preferred
for use in enzymatic reactions. A substrate contain-
ing many susceptible bonds or different functional
groups adjacent to the susceptible bond may affect
the cleavage efficiency of the enzyme, and this can
result in the appearance of several intermediate
sized products. Thus, they may interfere with the
result and make interpretation of kinetic data diffi-
cult. Otherwise, the chromophore-containing sub-
strate will readily and easily support assaying meth-
ods with absorbance measurement. Moreover,
substrate specificity can also be precisely deter-
mined when an enzyme has more than one recogni-
tion site on both the preferred bond and the func-
tional groups adjacent to it. Different sized chro-
mogenic substrates can then be used to determine an
enzyme’s specificity and to quantify its substrate
preference.UNIT OFENZYMEACTIVITYThe unit of enzyme activity is usually expressed as
either micromoles of substrate converted or product
formed per unit time, or unit of activity per milliliter
under a standardized set of conditions. Though any
unit of enzyme activity can be used, the Com-
mission on Enzymes of the International Union of
Biochemistry 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 which will catalyze the
conversion of one micromole of substrate per
minute, 1 U 1 mol/min, under defined condi-
tions. The conditions include substrate concentra-
tion, pH, temperature, buffer composition, and other
parameters that may affect the sensitivity and speci-
ficity of the reaction, and usually a continuous spec-
trophotometric method or a pH stat method is pre-
ferred. 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 speci-
fied enzyme from an extract, it is necessary to com-
pare 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 con-
centration. The fold purification is an index reflect-
ing only the increase in the specific activity with
respect to the extract, not the purity of the specified
enzyme.REFERENCES
Barry MJ. 1997. Emzymes and symmetrical molecules.
Trends Biochem Sci 22:228–230.
Bashford CL, Harris DA. 1987. Spectrophotometry and
Spectrofluorimetry: A Practical Approach. Washing-
ton, DC: IRL Press.
Bell JE, Bell ET. 1988. Proteins and Enzymes. New
Jersey: Prentice-Hall.