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This article clarifies the difference between continuous assays and end point assays used for reaction velocity measurement.
Once an appropriate linear time period has been established, the researcher has two options for obtaining a velocity measurement.
First, the signal might be monitored at discrete intervals over the entire linear time period, or some convenient portion thereof.
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This strategy, referred to as a continuous assay, provides the safest means of accurately determining reaction velocity from the slope of a plot of signal versus time.
It is not always convenient to assay samples continuously, however, especially when one is using separation techniques, such as high performance liquid chromatography (HPLC) or electrophoresis. In such cases a second strategy, called end point or discontinuous assay, is often employed.
Having established a linear time period for an assay, one measure the signal at a single specific time point within the linear time period (most preferably, a time point near the middle of the linear phase). The reaction velocity is then determined from the difference in signal at that time point and at the initiation of the reaction, divided by the time
v = ∆I/∆t = It – I0/treading
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where the intensity of the signal being measured at time t and time zero is given by It and l0 respectively, and treading is the time interval between initiation of the reaction and measurement of the signal. In many instances it is much easier to take a single reading than to make multiple measurements during a reaction.
Inherent in the use of end point readings, however, is the danger of assuming that the signal will track linearly with time over the period chosen, under the conditions of the measurement. Changes in temperature, pH, substrate, and enzyme concentrations, as well as the presence of certain types of inhibitor can dramatically change the linearity of the signal over a fixed time window.
Hence, end point readings can be misleading. Whenever feasible, then, one should use continuous assays to monitor substrate loss or product formation. When this is impractical, end point readings can be used, but cautiously, with careful controls.
