A sensitive method for quantitation of steroid hydroxylase activities of individual P-450 in tissue homogenates

Assay methods which allow measurements of the level of individual P-450's in a homogenate of steroidogenic tissues have been developed. The assay is based simply on the determination of the specific products of steroid monooxygenase reactions under conditions in which sufficient amounts of the purified electron-donating components as well as lipids and detergents are supplemented so that the membrane-associated P-450 is able to exhibit its maximum activity. Under these conditions, an at least 10-fold increase in the NADPH-dependent monooxygenase activity of P-450 occurred as compared to that measured in the unenriched system. In addition, the present assay used ascorbate as an O2- -scavenger which effectively prevented possible inhibition caused by initiation of O2- -formation. Under the present assay conditions, nearly quantitative recovery of activities was accomplished of each purified P-450 that had been added to the homogenates as an internal standard. Furthermore, the high sensitivity of this assay allows the measurement of P-450 in small specimens (at least 100 mg of tissues), and could be used for measurements in autopsied cases or the glands of small animals.