Purification and Characterization of Cytochrome P-450 with High Affinity for Cytochrome b51

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Abstract
A trypsin-solubilized form of cytochrome b5 has been coupled with Sepharose 6B, and the immobilized cytochrome b5 was used as an affinity adsorbent for the purification of cytochrome P-450. Chromatography of the partially purified cytochrome P-450 on cytochrome b5-Se-pharose 6B and CM-Sephadex resulted in purification of cytochrome P-450 to a gel-electro-phoretically homogeneous state with an overall yield of 5% from microsomes of untreated rabbit livers. The purified cytochrome, designated as P-45OB1, had a specific content of 15 nmol per mg of protein. The minimum molecular weight of the cytochrome was estimated to be 52,000 by sodium dodecyl sulfate-polyacrylamide gel-electrophoresis, and neither the phenobarbital-inducible nor the 3-methylcholanthrene-inducible form of cytochrome P-450 co-migrated with this purified P-45OB1. Oxidized P-450B1 exhibited absorption maxima at 567, 533, and 417 nm, characteristic of a hemoprotein of low-spin type; the dithionite-reduced form showed absorption maxima at 545 and 413 nm. The reduced, carbon monoxide-bound form had absorption peaks at 551 and 449 nm. The electron paramagnetic resonance spectrum of the oxidized state of P-450B1, was that of a low-spin ferric hemoprotein with g values of 1.91, 2.24, and 2.40. The absolute spectrum of the reduced P-450B1-phenyl isocyanide complex exhibited absorption maxima at 549, 454, and 429 nm, and the absorbance ratio of A454 to A429 in the difference spectrum was 6.5 in 0.3 m potassium phosphate, pH 7.4. The purified P-450B1 was active in the metabolism of benzphetamine, aminopyrine, and aniline with turnover numbers of 5.7, 6.4, and 2.7 nmol of product formed per min per nmol of cytochrome P-450, respectively. The electrophoretic, spectral, and catalytic properties together with the uniquely high affinity of P-450B1 for cytochrome bt indicate that the purified P-450B1, is a new form of cytochrome P-450, distinct from those previously reported.