Purification of cytochrome P-450 from pig testis by aniline-sepharose 4B and isoelectric focusing

Testicular cytochrome P-450 was purified by a procedure including preparati ve isoelectrofocusing. The cytochrome P-450 was determined to have an isoel ectric point of 6.47 on analytical isoelectric focusing. The purified cytoc hrome P-450 was found to be homogeneous and its molecular weight was estima ted Lo be 52000 on polyacrylamide gel electrophoresis in the presence of so dium dodecyl sulfate. The carbon monoxide difference spectrum with a peak a t 448 nm exhibited absorption spectrum of a typical cytochrome P-450. 284-f old purification was achieved with an yield of 10.6 %. Following preparatio n of the microsomes,the purification is accomplished by a two-step procedur e utilizing Aniline-Sepharose 4B column chromatography and preparative isoe lectric focusing.