23] Dual-wavelength stopped-flow spectrophotometric measurement of NADPH-cytochrome P-450 reductase

Abstract

This chapter describes the stopped-flow spectrophotometric method for the determination of the activity of NADPH-cytochrome P-450 reductase in hepatic microsomes. The techniques applicable for the measurement of the rate of reduction of cytochrome P-450 in microsomal vesicles can be readily applied to other particulate systems. The involvement of hepatic microsomal NADPH-cytochrome P-450 reductase in the transfer of electrons to cytochrome P-450 has been known for a number of years. A major effort is directed to the measurement of the relative activity of this enzyme under different experimental conditions because of the possible role of this enzyme in the control of the monooxygenase reactions of cytochrome P-450. Recent results have shown that a number of the simple techniques that are used to measure the cytochrome P-450 reductase activity of this enzyme on the microsomal membrane have underestimated the rate of the reaction. The results of this experiment also illustrate the reproducibility of this analytical procedure. The experiments described in the chapter are repeated extensively, and the results obtained are reproducible within ±5%.