A novel approach to study the activity and stoichiometry simultaneously for microsomal pentoxyresorufin-O-dealkylase...

A simple approach to study the activity and stoichiometry of cytochrome P-450 IIB1-catalyzed metabolism of pentoxyresorufin (PRF) has been investigated. It involves the continuous spectral analysis of reaction mixture containing PRF, microsomes from phenobarbital-induced rats and NADPH. The kinetics of NADPH consumption, PRF utilization, NADP and resorufin formation was monitored at lambda(max) of 338, 484, 260 and 572 nm, respectively. The stoichiometry of the enzyme reaction tabulated either by specific activity or by V(max) value showed that 10 molecules of NADPH were required for the conversion of one molecule of PRF to one molecule of resorufin along with 10 molecules of NADP. Further, it was observed that almost six molecules of NADPH are consumed in the incubation mixture devoid of PRF indicating the possibility of metabolism of endogenous substrates. Interestingly, the stoichiometry ratio of 1:1 for PRF and resorufin was established even in the presence of P-450 inhibitors with a lower rate of metabolism. However, the ratio of NADPH to PRF was altered in the presence of inhibitors, suggesting that the simultaneous monitoring of the substrate, electron donor and the products could be useful in understanding the modifications of stoichiometry of electron donor and substrate/product.