Purification and amino acid analysis of cytochrome c fromUstilago violacea

Cytochrome c fromUstilago violacea was further analyzed in order to characterize the pink phenotype. NaOH-extracted cytochrome c was purified in three steps, which included ammonium sulfate precipitation, CM-Sephadex ion-exchange chromatography with 0.5 N NaCl elution, and CM-Sephadex ion-exchange chromatography with a 0.0–0.6 N NaCl gradient elution. Polyacrylamide gel electrophoresis of the purified protein yielded a single red band, which was the only band detected upon Coumassie brilliant blue staining. HCl-hydrolysates of the protein were examined for their amino acid composition, which indicated that the cytochrome c fromU. violacea contains approximately 104 residues, with high levels of alanine, histidine, serine, and low levels of phenylalanine and arginine.