Continuous spectrofluorometric and spectrophotometric assays for NADPH-cytochrome P450 reductase activity using 5-cyano-2,3-ditolyl tetrazolium chloride.

Mammalian NADPH-cytochrome P450 reductase (CPR) transfers electrons from NADPH to cytochrome P450 enzymes and other several microsomal enzymes. It also catalyzes the one-electron reduction of many chemicals and drugs. Reduction of 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) by CPR was assessed as a method for monitoring CPR activity. The electrons released from NADPH by CPR were transferred to CTC in the reaction medium, and CTC reduction activity could be assessed spectrophotometrically and spectrofluorometrically. The reduction kinetics of CTC follows classical Michaelis-Menten kinetics (K(m) = 50 microM, k(cat) = 2,520 min(-1)). This method offers a continuous assay of the enzymatic activity of CPR.