Principles of Enzyme Assays

Abstract

Enzymology is a quantitative and exact science. Therefore it is important to understand how enzyme activity is measured and presented. A robust and reliable measure of the progress of an enzyme-catalyzed reaction is first and foremost requirement. Like with any other chemical reaction, progress of an enzyme-catalyzed reaction can be monitored either by the product formed (d[P]/dt) or by the substrate consumed (−d[A]/dt). The two rates are of course related by the reaction stoichiometry. It is desirable and often safe to follow the formation of product – a substance is better estimated when it is formed in a background where very little (or none) of it exists. On the other hand, to measure a decrease in the concentration of a reactant as it disappears – a small change in a large background – becomes daunting. In practice, a small decrease in substrate is relatively more difficult to observe than to follow a buildup of product from nothing. This is particularly relevant when we wish to record the initial rate (rate during very early time after the reaction is initiated, abbreviated as “v”), which is given by d[A]/dt when [P] ≈ 0. This is the rate at the beginning of the reaction or the instantaneous rate extrapolated to time zero.