Fractionation of Peptides Obtained by Tryptic Hydrolysis of Baker's Yeast Cytochrome c

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Abstract
As the first step for the elucidation of the complete amino acid sequence of baker's yeast (Saccharomyces) cytochrome c, tryptic hydrolysis of the protein and the fractionation of the peptides in the hydrolysate were investigated. The hydrolysate (240 mg.) for 3.5 hours at 30°C and pH 8.0 were initially separated into nineteen fractions on a column of Amberlite CG-50 (2.0×100 cm.) by the gradient elution with pyridine-acetate buffers. The peptide fractions obtained by the column chromatography were subsequently purified or further fractionated by paper chrornatography, high-voltage electrophoresis or Sephadex G-25 column. Thus twenty-two peptides and more than three moles of free lysine could be isolated and their amino acid compositions were determined. Most of these peptides were obtained in enough amount for further structural studies, but not for some longer peptides containing aromatic amino acid residues owing to their remarkable losses during the fractionation and purification process. By the correlation of the isolated peptides with the spots on the peptide map, it was demonstrated that they were isolated, without overlooking.

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