The isolation and purification of cytochrome c1 from bovine heart.
A large-scale isolation method for cytochrome c1 from beef heart is presented, based in principle on the procedure of Yu et al. (Yu, C.A., Yu, L. and King, T.E. (1972) J. Biol. Chem. 247, 1012--1019). Optimal solubilization of cytochrome c1 from succinate-cytochrome c oxidoreductase was achieved with 15% beta-mercaptoethanol, 1.5% cholate, 0.5% deoxycholate in 8% saturated ammoniun sulphate. The protein is purfied to a higher degree by chromatography on DEAE-cellulose and Ultrogel AcA 44. The method is reproducible and gives highly purified cytochrome c1 with a yield from succinate-cytochrome c oxidoreductase of 40%. The purified cytochrome c1 contains 32 nmol of heme/mg protein and has a spectral heme-to-protein ratio (Ared417nm/Ax276nm) of 2.7. Reduced cytochrome c1 is oxidized very rapidly by ferricytochrome c (k = 3 . 10(7) M-1 . S-1 at 10 degrees C, 100 mM potassium phosphate (pH 7.0) and 1% Tween 20). Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate shows that the isolated protein consists of one peptide, with a molecular weight of 31 000, carrying the chromophore. In the presence of 1% sodium cholate or 1% Tween 80, cytochrome c1 is in the monomeric state, whereas at lower concentrations of detergent the protein aggregates. The aggregation of cytochrome c1 is found to be reversible.
D000645:Ammonium Sulfate; D000818:Animals; D002417:Cattle; D002793:Cholic Acids; D002848:Chromatography, DEAE-Cellulose; D003574:Cytochrome c Group / Q000031:analogs & derivatives*; D003575:Cytochromes c1 / Q000032:analysis / Q000302:isolation & purification*; D003840:Deoxycholic Acid; D006418:Heme / Q000032:analysis; D008623:Mercaptoethanol; D008970:Molecular Weight; D009206:Myocardium / Q000032:analysis*; D010084:Oxidation-Reduction; D013057:Spectrum Analysis; D013384:Succinate Cytochrome c Oxidoreductase / Q000032:analysis
D002793:Cholic Acids; D003574:Cytochrome c Group; D003840:Deoxycholic Acid; D006418:Heme; D008623:Mercaptoethanol; D003575:Cytochromes c1; D013384:Succinate Cytochrome c Oxidoreductase; D000645:Ammonium Sulfate