Subcellular distribution of cytochrome c in rat liver. Methods for its extraction and purification.

Abstract

1. A method for the extraction and purification of cytochrome c from rat liver is described. The method depends on multiple chromatography on Amberlite IRC-50 with elution with ammonium phosphate buffers of differing ionic composition and pH, interspersed with gel filtration with Sephadex G-25. Conditions leading to denaturation are avoided and the product is chromatographically pure. 2. The method may be used for the quantitative analysis of cytochrome c either in unfractionated liver or in subcellular fractions. 3. Two pools of cytochrome c were detected, one extractable at pH4.0 with distilled water and the other extracted from the residues of the first extraction with 0.15m-sodium chloride. 4. For subcellular distribution studies the liver was homogenized in 0.3m-sucrose and a nuclear fraction (washed thoroughly to remove trapped mitochondria), a mitochondrial fraction, a heavy microsomal fraction, a standard microsomal fraction and the cell sap were isolated. The mitochondrial fraction was subfractionated further by density-gradient centrifugation. Each fraction was analysed for protein, RNA, DNA, succinate-neotetrazolium oxidoreductase and glucose 6-phosphatase. 5. A total of 123mug. of cytochrome c was obtained/g. wet wt. of rat liver. 6. Values for the percentage subcellular distribution of cytochrome c are: nuclear fraction, 24.4; mitochondrial fraction, 57.2; heavy microsomal fraction, 5.2; standard microsomal fraction, 10.6; cell sap, 2.7. 7. Three out of the eight mitochondrial subfractions separated by gradient centrifugation contained 76% of the cytochrome c and 85% of the succinate-neotetrazolium oxidoreductase present in the mitochondrial fraction. 8. In unfractionated liver 94% of the cytochrome c was extracted at pH4.0 with water whereas in most of the subcellular fractions the corresponding value was approx. 75-80%.