Abstract
A trypsin-solubilized form of cytochrome b5 has been coupled with Sepharose 6 B, 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-Sepharose 6 B 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-4503B1, 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-450B1.
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 413nm. The reduced, carbon monoxide-bound form had absorption peaks at 551 and 449nm. 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 0.3M 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-4503, for cytochrome b5 indicate that the purified P-450B1, is a new form of cytochrome P-450, distinct from those previously reported.
