Preparation of the Heme Protein P-450 from the Adrenal Cortex and Some of Its Properties

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Abstract Details of a method for the preparation and purification of the heme protein P-450 from bovine adrenal glands are described. This method consists of the isolation of mitochondria, their ultrasonic disruption, followed by lyophilization, and extractions with acetone and 1-butanol in several steps, which remove 60 to 70% of the neutral and phospholipids. The heme protein P-450 is isolated by a fractional extraction with solutions of the neutral detergent Triton N-101, followed by ammonium sulfate precipitation and dialysis. The resulting optically clear preparation can be stored in the frozen state for several months without deterioration. After reconstitution with the specific FAD-containing reductase and the specific iron sulfur protein, both isolated and partially purified from the adrenal cortex, this P-450 preparation is highly active as steroid 11β-hydroxylase (EC 1.14.1.6) with TPNH as electron donor (pH optimum 6.3 to 6.5, turnover number 5 to 10 min-1 at 25°). The heme protein is also active in steroid 11β-hydroxylation with artificial electron donors in the presence of the specific iron sulfur protein. In addition, the reconstituted enzyme system catalyzes the side chain cleavage of cholesterol. The optical absorption spectra of the preparation exhibit Soret maxima at 416, 419, and 448 nm for the Fe3+, the Fe2+ form, and the CO-compound, respectively. The EPR spectrum shows the characteristic features of the low spin (S = ½ form of P-450 (Fe3+), with principal components 1.91, 2.24, and 2.42 of the g-tensor. P-450 accounts for 98% or more, and a P-420 type material for the remaining part of the measured protoheme IX content; these are the only heme proteins detectable. Specific concentration of P-450 in the preparation lies between 1.5 and 2.0 nmoles per mg of protein; the preparation contains also neutral and phospholipids (of the order of 10 to 15%), but negligible amounts of cholesterol (l0.1%). This enzymatically active material is suitable for optical and other spectroscopic studies as well as detailed investigations of the physical characteristics and the chemical nature of the heme protein P-450. A brief comparison is made with the properties of other particulate or solubilized P-450 preparations, obtained by several investigators from liver microsomes and other mammalian sources, and with the soluble P-450cam from Pseudomonas putida.