Enzyme Kinetics via Open Circuit Potentiometry

Abstract

We demonstrate the application of open circuit potentiometry (OCP) to measure enzyme turnover kinetics, k_turn. The electrode surface will become poised by the addition of a well-behaved redox pair, such as ferrocenemethanol/ferrocenium methanol (FcMeOH/FcMeOH⁺), which acts as the cosubstrate for the enzymatic process. A measurable change in potential results when an enzyme consumes the one-electron transfer mediator. Glucose oxidase was studied as a test-case, but the method is generalizable across oxidoreductase enzymes that rely on electron transfer mediators. In the presence of glucose and FcMeOH⁺, glucose oxidase delivers electrons to FcMeOH⁺, and the potential changes with respect to the Nernst equation. A theoretical model incorporating enzymatic rate expressions into the Nernst equation was derived to explain the observed potential transients, and experimental data fit theory well. A similar experiment was performed using amperometry on ultramicroelectrodes (UMEs). Here, the same enzymatic rate expression may be incorporated into the equation for steady-state flux to an UME to obtain k_turn. While similar kinetic information was obtained from the potentiometric and amperometric responses, potentiometry is independent of electrode size and mass transfer effects. Finally, we show how k_turn changes as a function of one-electron mediator. Our results may eventually find applications to biosensors, where electrode fouling plagues long-term sensor performance.