Two-step purification of cytochrome P-450 from adult pig testis by isoelectric focusing

Adult testicular cytochrome P-450 was purified by a two-step procedure util izing preparative isoelectrofocusing. Purification was achieved 1132 times with a yield of 4.82%. 17 alpha-hydroxylase activity was shown to be 14.5 n mol of prod uct/min/nmol of P-450. The cytochrome P-450 was determined to h ave an isoelectric point of 6.45 on analytical isoelectric focusing. The pu rified cytochrome P-450 was found to be homogeneous and its molecular weigh t was estimated to be 52000 on polyacrylamide gel electrophoresis in the pr esence of sodium dodecyl sulfate. The carbon monoxide difference spectrum w ith a peak at 448 nm exhibited the absorption spectrum of a typical cytochr ome P-450.