A spectrophotometric method to monitor the catalytic activity of microsomal cytochrome P-450 IIB1/2: comparison with fluorometric assay

A simple spectrophotometric method to monitor the catalytic activity of microsomal cytochrome P-450 IIB1/2 has been developed. The method employs measurement of utilization of NADPH, consumption of the substrate, pentoxyresorufin (PRF) and formation of the product, resorufin (RF) in the same reaction mixture containing hepatic microsomes from phenobarbital treated rats. The velocity of NADPH utilization (16.36 nmole/min/nmole P-450), PRF consumption (1.58 nmole/min/nmole P-450) and RF formation (1.57 nmole/min/nmole P-450) suggested a stoichiometry of 1:1 between the substrate and the product alongwith utilization of 10 molecules of NADPH. However, the Km for the enzyme activity (nmole RF formed/min/nmole P-450) using varying concentrations of PRF and NADPH as substrates were found to be 11.6 and 20.2 microM, respectively. The spectrophotometric method was compared with fluorometric method in terms of linearity with time, P-450 content and Vmax, Km values observed for the reaction. Inhibition studies with metyrapone and SKF 525A in the utilization of NADPH, consumption of PRF and formation of RF suggested that the method could be useful in monitoring the effect of various inhibitors on the P-450 IIB1/2 reaction.