Potentiometric determination of coenzyme‐dependent oxidoreductase enzymes with recycling of the coenzyme

Abstract

SummaryBy enzymatically establishing a rapid (essentially equilibrium) coupling of a redox coenzyme such as NAD with the components of the ferrocyanide-fenicyanide halfcell (e.g., using excess diaphorase) the half-cell potential can be used to monitor another enzymatic reaction involving the same coenzyme. This approach provides a general, rapid potentiometric method of assaying coenzyme-dependent oxidoreductase enzymes. We show that these assay systems can be designed for multiple turnover of coenzyme (in our case NAD) during a single assay thereby amplifying the rate of electromotive force (emf) change with a concomitant increase in sensitivity of enzyme assay. This allows the use of small concentrations of coenzyme and extension of the range of enzyme concentrations that may be assayed.