Hydrazinolysis of Peptides and Proteins. II. Fundamental Studies on the Determination of the Carboxyl-ends of Proteins

Abstract

1. Fundamental experiments on the hydrazinolysis method, a method for characterization of C-terminal amino acids of proteins, were carried out. 2. Amino acids were found to decompose to various extents by heating with anhydrous hydrazine. 3. Amino acid hydrazides were hydrolysed about 1% or less by heating with anhydrous hydrazine (ca. 99%) for ten hours. Hydrolyses of them in neutral and slightly acidic solutions at room temperature are negligible, but in alkaline solution the extent of hydrolyses are somewhat greater though less than 0.2% in five hours in 1% bicarbonate solution. 4. Procedures for separating free amino acids from amino acid hydrazides as DNP-derivatives with or without treatment of iso-valeraldehyde prior to dinitrophenylation were reinvestigated thoroughly. 5. Some experiments on the hydrazino-lyses of peptides were done. 6. Fundamental investigations of the characterization of the mode of linkage of monoaminodicarboxylic acids in peptide chains by the hydrazinolysis method indicate its availability. 7. Standard methods for quantitative estimation of C-terminal amino acids or proteins are established based upon the results obtained by the present investigation. 8. Brief consideration on the mechanism of the hydrazinolysis was described and the scope of this method was discussed in conection with that of the carboxypeptidase method

The staphylococcal enterotoxins

“…Schantzl on rechecking the partial specific volume for enterotoxin B reports that the experimental value he obtained by pycnometry was 0.730 which is in agreement with the values calculated from the amino acid composition. The values, 0.732, obtained for enterotoxin C1 by pycnometry, is in good 1 Personal communication. This discrepancy is being rechecked by Schantz' and preliminary information indicates the molecular weight may be less than 30,000.…”

Functional characterization of constituent enzyme fractions of mycobacillin synthetase

“…This mixture was incubated for 20min, and the peptides from the incubation mixture were isolated by using the method described previously by Sengupta & Bose (1972). Each of the new peptides so isolated was then analysed to determine the amino acid composition and the Cterminal and N-terminal amino acids by the method of Akabori et al (1956) and Sanger (1945) as described by Sengupta & Bose (1972).…”

The Staphylococcal Enterotoxins

“…Schantzl on rechecking the partial specific volume for enterotoxin B reports that the experimental value he obtained by pycnometry was 0.730 which is in agreement with the values calculated from the amino acid composition. The values, 0.732, obtained for enterotoxin C1 by pycnometry, is in good 1 Personal communication. [43] from the amino acid composition.…”

“…These workers determined the C-terminal amino acid sequence to be lysine-lysine-tyrosineleucine by modification of the hydrozinolytic procedure of Akabori et al [1] and by treatment with carboxypeptidases A and B.…”