[27] Assays for cytochrome P-450 peroxygenase activity

This chapter discusses assays for cytochrome P-450 peroxygenase activity. Toluene, which yields benzyl alcohol in the cytochrome P-450-catalyzed peroxygenase reaction, is a useful substrate in mechanistic studies. To determine benzyl alcohol formation at fixed time intervals, a gas chromatographic assay is employed. Cytochrome P-450 is versatile enzyme that catalyzes numerous types of chemical reactions with a variety of substrates, including the hydroxylation of xenobiotics, such as drugs, pesticides, dyes, organic solvents, anesthetics, and carcinogens, as well as naturally occurring lipids, including steroids, fatty acids, and prostaglandins. Molecular oxygen serves as the natural donor when electrons are supplied to cytochrome P-450 by nicotinamide adenine dinucleotide phosphate (NADPH) via proteins that serve as electron carriers. In the case of liver microsomes or the reconstituted enzyme system, NADPH-cytochrome-P-450 reductase serves as the carrier and in the overall reaction equimolar amounts of substrate, O2, and NADPH are consumed and equimolar amounts of hydroxylated substrate, H2O, and NADP are formed. Molecular oxygen can be replaced by a variety of hydroperoxides and related artificial donors. Following the discovery of the ability of cytochrome P-450-containing microsomal suspensions to promote the oxygenation of an organic substrate at the expense of an alkyl hydroperoxide, other oxidants, such as peroxy acids, periodate, iodosobenzene, iodobenzene diacetate, and N-oxides, are found to function in a similar capacity.