Extending the linear range for kinetic reactions by considering the relationship between enzyme activity of the reagent and measurement intervals.

We show that the rate-concentration curve is sigmoidal for enzyme-catalyzed procedures that are commonly applied to rapid automated analyzers. Linear data can be obtained by judicious selection of the reagent enzyme activity (Vm) and the measurement interval (t1 to t2). For determination of substrate concentrations much less than Km, conditions that balance linearity with sensitivity and accuracy are obtained when Vm/Km = [ln (t2/t1)]/(t2-t1). We also present theoretical and experimental data that show the linear range can be extended to concentrations exceeding the Km value. We illustrate the application of theoretically appropriate conditions by analysis of procedures reported to be optimized. Familiarity with these concepts can obviate laborious and potentially misleading experimentation.