DETERMINATION OF C-TERMINAL ARGININE AND ASPARAGINE OF PROTEINS BY CATALYTIC HYDRAZINOLYSIS.

Abstract

Some improvements made on the fractional extraction procedure (see Scheme 1) of dinitrophenylated hydrazinolyzate of peptide or protein, could cause a quantitative separation of DNP-compounds, especially diDNP-derivatives, with free carboxyl group derived from the C-terminal.Aspartic acid β-hydrazide and glutamic acid γ-hydrazide, both owing to the C-terminal residues, and aspartic or glutamic acid α-hydrazide owing to the intrachain residues were stabilized as the diDNP-derivatives, and successfully separated and identified on the two-dimensional paper chrornatogram.Catalytic hydrazinolysis according to Bradbury's method (heating at 60°C in hydrazine containing M hydrazine sulfate as catalyst) gave a high and reproducible yield of ornithine, aspartic acid β-hydrazide or glutamic acid γ-hydrazide as the diDNP-derivative from the C-terminal arginine or asparagine residue, or from glutathione.Thus, combining the catalytic hydrazinolysis with the modified extraction and the paper chromatography, it became possible to determine quantitatively the C-terminal arginine and asparagine (and glutamine) residues of proteins in amounts necessary for the paper-chromatographic identification. The method was confirmed to be effective on such proteins as protamines and insulins from several sources, egg-white lysozyme, and carboxypeptidase-A. Some improvements made on the fractional extraction procedure (see Scheme 1) of dinitrophenylated hydrazinolyzate of peptide or protein, could cause a quantitative separation of DNP-compounds, especially diDNP-derivatives, with free carboxyl group derived from the C-terminal. Aspartic acid β-hydrazide and glutamic acid γ-hydrazide, both owing to the C-terminal residues, and aspartic or glutamic acid α-hydrazide owing to the intrachain residues were stabilized as the diDNP-derivatives, and successfully separated and identified on the two-dimensional paper chrornatogram. Catalytic hydrazinolysis according to Bradbury's method (heating at 60°C in hydrazine containing M hydrazine sulfate as catalyst) gave a high and reproducible yield of ornithine, aspartic acid β-hydrazide or glutamic acid γ-hydrazide as the diDNP-derivative from the C-terminal arginine or asparagine residue, or from glutathione. Thus, combining the catalytic hydrazinolysis with the modified extraction and the paper chromatography, it became possible to determine quantitatively the C-terminal arginine and asparagine (and glutamine) residues of proteins in amounts necessary for the paper-chromatographic identification. The method was confirmed to be effective on such proteins as protamines and insulins from several sources, egg-white lysozyme, and carboxypeptidase-A.