Isolation and properties of somatic and testicular cytochromes c from rat tissues

Abstract

Somatic ( c s ) and a testis-specific ( c t I) cytochromes c were purified to homogeneity from rat tissues (heart, liver, kidney, and testis). The purification procedure involved (1) homogenization of tissues at pH 4.5, (2) treatment with methanol-chloroform solvents, (3) hydroxylapatite column chromatography, (4) carboxymethyl-cellulose column chromatography, and (5) Sephacryl S-200 gel filtration. The isolated cytochromes c were free from polymeric and other “modified” forms, and did not bind CO, azide, or cyanide. The absorption maxima and the molecular weights of both cytochromes c s and c t I were identical. The ratio of A 549.5 nm ( reduced) A 280 nm ( oxidized) for cytochromes c s averaged 1.28. The unique properties of cytochrome c t I, compared to somatic cytochrome c, were as follows: (1) different elution profiles from hydroxylapatite and carboxymethyl-cellulose column chromatography experiments, (2) less basic intrinsic molecular charge shown by the slow mobility in native polyacrylamide gel electrophoresis, (3) probable asymmetric molecular shape as evidenced from gel filtration experiments, (4) significantly higher millimolar extinction coefficient values (33.6 at 549.5 nm), (5) a low ratio (1.04) of A 549.5 nm ( reduced) A 280 nm ( oxidized) , and (6) difference of about 20 amino acid residues per mole.