The assay can be used to assay ATP and enzymes that utilise ATP by means of coupled
reactions. The use of excess reagents would ensure that each reaction went to
completion.Immunochemical methods
Monoclonal antibodies raised to a particular enzyme can be used as a basis for a
highly specific ELISA-based assay for the enzyme. Such assays can distinguish
between isoenzyme forms, which make the assay attractive for diagnostic purposes.
An important clinical example is creatine kinase. It is a dimer based on two different
subunits, M and B. The MB isoenzyme is important in the diagnosis of myocardial
infarction (heart attack) and an immunological assay is important in its assay.15.3.3 Analytical methods for pre-steady-state studies
The experimental techniques discussed in the previous section are not suitable for the
study of the progress of enzyme reactions in the short period of time (commonly
milliseconds) before steady-state conditions, with respect to the formation of enzyme-
substrate complex, are established. Figure 15.1 shows the progress curves for this
pre-steady-state initial stage of an enzymic reaction. The induction timetis related to
the rate constants for the formation and dissociation of the ES complex. Two main
types of method are available for the study of this pre-steady-state.Rapid mixing methods
In thecontinuous flow method, separate solutions of the enzyme and substrate are
introduced from syringes, each of 10 cm^3 maximum volume, into a mixing chamber
typically of 100 mm^3 capacity. The mixture is then pumped at a preselected speed
through a narrow tube that is illuminated by a light source and monitored by a
photomultiplier detector. Flow through the tube is fast, typically 10 m s^1 , so that it
is turbulent thus ensuring that the solution is homogeneous. The precise flow time
from the time of mixing to the observation point can be calculated from the known
flow rate. By varying the flow rate the reaction time at the observation point can be
varied, allowing the extent of reaction to be studied as a function of time. From these
data the various rate constants can be calculated. The technique uses relatively small
amounts of reactants and is limited only by the time required to mix the two reactants.
Thestopped-flow methodis a variant of the continuous flow method in that
shortly after the reactants emerge from the mixing chamber the flow is stopped and
the detector triggered to continuously monitor the change in the experimental
parameter such as absorbance or fluorescence (Fig. 15.12). Special flow cells are used
together with a detector that allows readings to be taken 180to the light source for
absorbance, transmittance or circular dichroism measurements, or at 90to the source
for fluorescence, fluorescence anisotropy or light scattering measurements.
A variant of the stopped-flow method is thequenching method. In this technique the
reactants from the mixing chamber are treated with a quenching agent from a third
syringe. The quenching agent, such as trichloroacetic acid, stops the reaction that is
then monitored by an appropriate analytical method for the build-up of intermediates.605 15.3 Analytical methods for the study of enzyme reactions