Purification of rat liver microsomal cytochrome P‐450b without the use of nonionic detergent

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Abstract
Sodium cholate, Emulgen 911, and (3‐[(‐cholamidopropyl)‐dimethyl‐ ammonio]‐1‐propanesulfonate) (CHAPS) were selected to examine the effects of ionic, nonionic, and zwitterionic detergents on testosterone hydroxylation catalyzed by four purified isozymes of rat liver microsomal cytochrome P‐450, namely P‐450a, P‐450b, P‐450c, and P‐450h, in reconstituted systems containing optimal amounts of dilauroylphosphatidylcholine and saturating amounts of NADPH‐ cytochrome P‐450 reductase (reductase). The major phenobarbital‐inducible form of rat liver microsomal cytochrome P‐450, designated P‐450b, was extremely sensitive to the inhibitory effects of Emulgen 911, which is used in several procedures to purify this and other forms of cytochrome P‐450. In contrast, sodium cholate and CHAPS had little effect on the catalytic activity of cytochrome P‐450b, even at ten times the concentration of Emulgen 911 effecting 50% inhibition (IC‐50). By substituting the zwitterionic detergent CHAPS for Emulgen 911, we purified cytochrome P‐450b without the use of nonionic detergent. The protein is designated cytochrome P‐450b* to distinguish it from cytochrome P‐450b purified with the use of Emulgen 911. NADPH‐cytochrome P‐450 reductase was also purified both with and without the use of nonionic detergent.The absolute spectra of cytochrome P‐450b and P‐450b* were indistinguishable, as were the carbon monoxide (CO)‐ and metyrapone‐difference spectra of the dithionite‐reduced hemoproteins. When reconstituted with NADPH‐cytochrome P‐450 reductase and dilauroylphosphatidylcholine, cytochromes P‐450b and P‐450b* catalyzed the N‐demethylation of benzphetamine and aminopyrine, the 4‐hydroxylation of aniline, the O‐dealkylation of 7‐ethoxycoumarin, the 3‐hydroxylation of hexobarbital, and the 6‐hydroxylation of zoxazolamine. Both hemo‐proteins catalyzed the 16α‐ and 16β‐hydroxylation of testosterone, as well as the 17‐oxidation of testosterone to androstenedione. Both hemoproteins were poor catalysts of erythromycin demethylation and benzo[a]pyrene 3‐/9‐hydroxylation. The rate of biotransformation catalyzed by cytochrome P‐450b* was up to 50% greater than the rate catalyzed by cytochrome P‐450b when reconstituted with either reductase or reductase*. The activity of cytochrome P‐450b and P‐450b* increased up to 50% when reconstituted with reductase* instead of reductase. In addition to establishing the feasibility of purifying an isozyme of rat liver microsomal cytochrome P‐450 without the use of nonionic detergent, these results indicate that the catalytic activity of cytochrome P‐450 is not unduly compromised by residual contamination with the nonionic detergent Emulgen 911.