Studies on the steroid hydroxylation system in adrenal cortex microsomes. Purification and characterization of cytochrome P-450 specific for steroid C-21 hydroxylation.

Cytochrome P-450 was purified to a specific content of 16 nmol/mg of protein from bovine adrenal cortex microsomes by hydrophobic chromatography using an w-amino-n-octyl Sepharose column in the presence of Emulgen 913, a nonionic detergent. The purified preparation exhibited a single polypeptide band (Mr = 47,000 f 1,000) when submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The cytochrome P-450 was immunochemically different from mitochondrial cytochrome P-450 catalyzing the sidechain cleavage of cholesterol and cytochrome P-450 catalyzing the 11s and 18 hydroxylation of ll-deoxycorticosterone. The purified cytochrome P-450 catalyzed the C-21 hydroxylation of l7a-hydroxyprogesterone and progesterone when mixed with NADPH-cytochrome P-450 reductase, which was purified separately from the microsomes. The absorption spectrum of the oxidized cytochrome P-450 had maxima at 419,535, and 569 nm, characteristic of a low spin form. Upon the addition of 17a-hydroxyprogesterone or progesterone, the cytochrome P-450 showed maxima at 396 and 651 nm, characteristic of a high spin form. The effects of steroid binding were studied optically for the cytochrome P-450 both in the low and the high spin forms. Some steroids caused spectral changes different from the type I spectral changes upon addition to the cytochrome P-450 in the low spin form. These steroids were found to cause the reverse type I spectral changes effectively upon addition to the cytochrome P-450 in the high spin form. A hypothesis to explain the mechanism of these spectral changes was proposed.