A simple and reliable method for determination of optimum pH in coupled enzyme assays.

Determination of the optimum pH in a coupled enzyme assay poses significant challenges because altering the pH of the reaction mixture can affect the performance of both enzymes. Here, we demonstrate a simple and reliable method to determine the pH optimum for pyruvate kinase using the pyruvate kinase/lactate dehydrogenase coupled enzyme assay. This simple and reliable method can be broadly adapted to determine the pH optimum for various enzymes that are assayed using a coupled enzyme assay. METHOD SUMMARY This article describes a simple method to determine the optimum pH for the activity of one enzyme in a coupled enzyme assay. Decoupling of the two-step reaction is achieved by heat denaturing the enzyme catalyzing the first reaction at different pHs followed by the addition of the second enzyme at a constant pH. The velocity of the second enzymatic reaction is determined by the concentration of the product produced in the first reaction, therefore, allowing the measurement of the optimum pH of the first enzyme by monitoring the rate of the second reaction.