Determination of C-terminal Arginine and Asparagine of Proteins by Catalytic Hydrazinolysis*

作者：Yasuhiro Kawanishi, Koichi Iwai, Toshio Ando · 发表于：The Journal of Biochemistry · 年份：1964 · DOI：10.1093/oxfordjournals.jbchem.a127995 · 被引用次数：30 · 研究领域：Amino Acid Enzymes and Metabolism、Biopolymer Synthesis and Applications、Mass Spectrometry Techniques and Applications

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.