Cytochrome P450 Reconstitution Systems

Human liver microsomes contain multiple forms of cytochrome P450 (CYP or P450) that catalyze oxidation of a number of xenobiotic and endobiotic chemicals. Individual P450 forms have unique, but overlapping, substrate specificities. It is necessary to determine which P450s play more important roles in the oxidation of these chemicals. A good way of studying the roles of P450s in the metabolism of these chemicals is to reconstitute the activities by mixing purified P450s and nicotinamide adenine dinucleotide phosphate-cytochrome P450 reductase in the membranes of phospholipid vesicles. However, our studies have suggested that the conditions for reconstitution of activities vary depending on the P450 enzymes used. For example, some reactions catalyzed by P450s require cytochrome-b ₅ and a particular phospholipid environment for exerting their full catalytic activities. In this chapter, we describe optimal conditions that have been determined in our laboratories for the reconstitution of drug oxidation activities catalyzed by purified human CYP1A2, 2C9, 2E1, and 3A4.