PURIFICATION OF CYTOCHROME B5 FROM PIG TESTIS MICROSOMES BY ISOELECTRIC-FOCUSING IN AN IMMOBILINE PH GRADIENT

Testicular cytochrome b5 was purified by a procedure including prepara tive isoelectrofocusing. The cytochrome b5 was determined to have an i soelectric point of 4.45 on analytical isoelectric focusing. The purif ied cytochrome b5 was found to be homogeneous and its molecular weight was estimated to be 16,000 on polyacrylamide gel electrophoresis in t he presence of sodium dodecyl sulfate. The oxidized and reduced forms of the purified preparation exhibited absorption spectra of a typical cytochrome b5. A 69-fold purification was achieved with an overall yie ld of 6.2%. Following preparation of the microsomes, the purification is accomplished by a two-step procedure utilizing column chromatograph y and preparative isoelectric focusing. (C) 1998 Academic Press.