Radiometric assay for cytochrome P-450-catalyzed progesterone 16α-hydroxylation and determination of an apparent isotope effect

Abstract

In the course of studies on the oxygenation of steroids by purified P-450 cytochromes, particularly rabbit liver microsomal cytochrome P-450 form 3b, a rapid and reliable radiometric assay has been devised for progesterone 16α-hydroxylation. In view of the lack of a commercially available, suitably tritiated substrate, [1,2,6,7,16,17- 3H]progesterone was treated with alkali to remove the label from potential hydroxylation sites other than the 16α position. The resulting [1,7,16- 3H]progesterone was added to a reconstituted enzyme system containing cytochrome P-450 form 3b, NADPH-cytochrome P-450 reductase, and NADPH, and the rate of 16α-hydroxylation was measured by the formation of 3H 2O. This reaction was shown to be linear with respect to time and to the cytochrome P-450 concentration. An apparent tritium isotope effect of 2.1 was observed by comparison of the rates of formation of tritium oxide and 16α-hydroxyprogesterone, and the magnitude of the isotope effect was confirmed by an isotope competition assay in which a mixture of [1,7,16- 3H]progesterone and [4- 14C]progesterone was employed.