7 Enzyme Activities 167METHODS USED IN ENZYME
ASSAYS
GENERALCONSIDERATIONS
A suitable assay method is not only a prerequisite
for detecting the presence of enzyme activity in the
extract or the purified material: it is also an essential
vehicle for kinetic study and inhibitor evaluation.
Selection of an assay method that is appropriate to
the type of investigation and the purity of the assay-
ing material is of particular importance. It is known
that the concentration of the substrate will decrease
and that of product will increase when the enzyme is
incubated with its substrate. Therefore, an enzyme
assay is intended to measure either the decrease in
substrate concentration or the increase in product
concentration as a function of time. Usually the lat-
ter is preferred because a significant increase of sig-
nal is much easier to monitor. It is recommended
that a preliminary test be performed to determine the
optimal conditions for a reaction including substrate
concentration, reaction temperature, cofactor re-
quirements, and buffer constitutions, such as pH and
ionic strength, to ensure the consistency of the reac-
tion. Also an enzyme blank should be performed to
determine if the nonenzymatic reaction is negligible
or could be corrected. When a crude extract, rather
than purified enzyme, is used for determining en-
zyme activity, one control experiment should be per-
formed without adding substrate to determine if
there are any endogenous substrates present and to
prevent overcounting of the enzyme activity in the
extract.
Usually the initial reaction rate is measured, and it
is related to the substrate concentration. Highly suf-
ficient substrate concentration is required to prevent
a decrease in concentration during the assay period
of enzyme activity, thus allowing the concentration
of the substrate to be regarded as constant.
TYPES OFASSAYMETHODS
On the basis of measuring the decrease of the sub-
strate or the increase of the product, one common
characteristic property that is useful for distinguish-
ing the substrate and the product is their absorbance
spectra, which can be determined using one of the
spectrophotometric methods. Observation of the
change in absorbance in the visible or near UV spec-
trum is the method most commonly used in assaying
enzyme activity. NAD(P)is quite often a cofactor
of dehydrogenases, and NAD(P)H is the resulting
product. The absorbance spectrum is 340 nm for the
product, NAD(P)H, but not for the cofactor, NAD
(P). A continuous measurement at absorbance 340
nm is required to continuously monitor the disap-
pearance of the substrate or the appearance of the
product. This is called a continuous assay, and it is
also adirect assay because the catalyzed reaction
itself produces a measurable signal. The advantage
of continuous assays is that the progress curve is
immediately available, as is confirmation that the
initial rate is linear for the measuring period. They
are generally preferred because they give more
information during the measurement. However,
methods based on fluorescence, the spectrofluoro-
metric methods, are more sensitive than those
based on the changes in the absorbance spectrum.
The reaction is accomplished by the release or
uptake of protons and is the basis of performing the
assay. Detailed discussions on the assaying tech-
niques are available below.
Sometimes when a serial enzymatic reaction of a
metabolic pathway is being assayed, no significant
products, the substrates of the next reaction, can be
measured in the absorbance spectra due to rapid
changes in the reaction. Therefore, there may be no
suitable detection machinery available for this single
enzymatic study. Nevertheless, there is still a meas-
urable signal when one or several enzymatic reac-
tions are coupled to the desired assay reaction.
These are coupled assays, one of the particular
indirect assays whose coupled reaction is not enzy-
matic. This means that the coupled enzymes and the
substrates have to be present in excess to make sure
that the rate-limiting step is always the reaction of
the particular enzyme being assayed. Though a lag
in the appearance of the preferred product will be
noticed at the beginning of the assay, before reach-
ing a steady state, the phenomenon will only appear
for a short period of time if the concentration of the
coupling enzymes and substrates are kept in excess.
So the Vmaxof the coupling reactions will be greater
than that of the preferred reaction. Besides, not only
the substrate concentration but also other parameters
may affect the preferred reaction (see above). To
prevent this, control experiments can be performed
to check if the Vmaxof the coupling reactions is actu-
ally much higher than that of the preferred reaction.
For other indirect assays in which the desired