BLBS102-c08 BLBS102-Simpson March 21, 2012 12:8 Trim: 276mm X 219mm Printer Name: Yet to Come
176 Part 2: Biotechnology and Enzymologyuseful in judging if there is a change in the rate-limiting step
of a sequential reaction when the line of the plot reveals
bending of different slopes at certain temperatures (Stauffer
1989).
Taken together, the reaction should be performed under a
constantly stable circumstance, with both temperature and pH
precisely controlled from the start to the finish of the assay,
for the enzyme to exhibit highly specific activity at appropriate
acidbase conditions and buffer constitutions. Whenever possi-
ble, the reactant molecules should be equilibrated at the required
assay condition following addition of the required components
and efficient mixing to provide a homogenous reaction mixture.
Because the enzyme to be added is usually stored at low
temperature, the reaction temperature will not be significantly
influenced when the enzyme volume added is at as low as 1–5%
of the total volume of the reaction mixture. A lag phase will be
noticed in the rate measurement as a function of time when the
temperature of the added enzyme stock eventually influences
the reaction. Significant temperature changes in the components
stored in different environments and atmospheres should be
avoided when the reaction mixture is mixed and the assay has
started.METHODS USED IN ENZYME ASSAYS
General ConsiderationsA suitable assay method is not only a prerequisite for detecting
the presence of enzyme activity in the extract or the purified ma-
terial: 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 assaying 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 latter is preferred because
a significant increase of signal 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 requirements, and
buffer constitutions, such as pH and ionic strength, to ensure
the consistency of the reaction. 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 enzyme activity,
one control experiment should be performed 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 re-
lated to the substrate concentration. Highly sufficient 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 of Assay MethodsOn the basis of measuring the decrease of the substrate or the
increase of the product, one common characteristic property
that is useful for distinguishing the substrate and the product is
their absorbance spectra, which can be determined using one of
thespectrophotometric methods. Observation of the change in
absorbance in the visible or near UV spectrum 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 disappearance of the substrate or the
appearance of the product. This is called acontinuous assay,
and it is also adirect assaybecause the catalyzed reaction it-
self 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 in-
formation during the measurement. However, methods based on
fluorescence, thespectrofluorometric methods, are more sensi-
tive 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 techniques 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 measurable signal when one
or several enzymatic reactions are coupled to the desired assay
reaction. These arecoupled assays, one of the particularindirect
assayswhose coupled reaction is not enzymatic. 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 reaching 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, theVmaxof 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 previously). To prevent this, control experiments
can be performed to check if theVmaxof the coupling reactions
is actually much higher than that of the preferred reaction. For
other indirect assays in which the desired catalyzed reaction
itself does not produce a measurable signal, a suitable reagent
that has no effect on enzyme activity can be added to the reaction
mixture to react with one of the products to form a measurable
signal.
Nevertheless, sometimes no easily readable differences be-
tween the spectrum of absorbance of the substrate and that of
the product can be measured. In such cases, it may be possi-
ble to measure the appearance of the colored product, by the