Dynamic ranges of detection-coupled assays and their effect on IC(50) measurements for inhibition of enzymatic reactions.

Most assays used to monitor enzymatic activities can be considered detection-coupled assays (i.e., indirect measurement of an analyte by using another chemical reaction for detection). The major reason for this is that the analyte that indicates the activity of the reaction cannot be directly measured or specifically directly measured by conventional means, such as optical density, fluorescence, and so on. By coupling another reaction to the enzymatic reaction of interest, the activity of the reaction can be monitored without modification to the analyte itself. However, due to the additional coupling reaction between the analyte and the detection reagents, the dynamic range of the assay could be greatly limited by the coupling reaction, and the apparent parameters of the enzymatic reaction could be much different from its uncoupled counterpart, causing significant deviations for IC(50) measurement for an inhibition reaction of the enzyme. By using exact solutions for both the enzymatic and the coupling reactions, the effects of the coupling reaction on the dynamic range and accuracy of the IC(50) measurement are reviewed and evaluated. In addition, real examples are provided to further illustrate the problem and validate the analysis.