An enzyme assay measures the catalytic activity of an enzyme by following consumption of a substrate or appearance of a reaction product under specified conditions. The signal may be chemical concentration directly or a calibrated optical readout, such as absorbance or fluorescence.
For example, if product concentration increases from 0 to 6 micromolar in the first two minutes, a linear initial interval gives a rate of 3 micromolar per minute. A no-enzyme control tests whether the signal also changes without the catalyst. Comparing rates requires the same temperature, pH, substrate concentration and detection conditions; raw brightness alone is not an activity unit.
The initial interval matters because later measurements can be affected by substrate depletion, product accumulation or loss of enzyme activity. Assay-development guidance therefore recommends establishing the linear initial-rate region before comparing inhibitors or enzyme preparations.[ref]
Basics of Enzymatic Assays for HTS by Harold B. Brooks and others. Initial velocity, linear reaction range, substrates and controls
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