The cyanate end group method is a means of obtaining a quantitative determination of the NH,-terminal residue (1) and it can give an unequivocal demonstration of a blocked NH, terminus. Thus it was demonstrated that there are no free NH,-terminal residues in cytochrome C (1)) which possesses an N-acetyl terminal residue, and in pyrrolidonyl peptides (2) in which the NH, termini are blocked by intramolecular acylation. The method was selected for use in the characterization of insulin derivatives formed by acylation at one or both of the two cr-NHz groups.
Procedure. The insulin derivative was allowed to react with cyanate and the carbamylated protein was maintained in GN-HCl at 100°C for 1 hr (or under stronger hydrolytic conditions in this study) to release the NH,-terminal residue as a hydantoin and a mixture of peptides and amino acids. The hydantoin, which has no NHz-group, was isolated from the mixture by passage through a column of Dowex 50 X 2 and was hydrolysed to the corresponding amino acid for quantitative identification.
Results and Discussion. When the method was applied to N-acylated derivatives of insulin, which were devoid of NH, groups, the absence of NH,-terminal residues was confirmed in N,N',N"-triacetylinsulin and N,N',N"-tribenzoylinsulin.
N,N',N"-Trisuccinylinsulin, however, gave 0.5 residue of gIycine and 0.3 residue of phenylalanine. The anomalous result might be explained by incomplete deacylation of N-succinylglycine and N-succinylphenylalanine in the cyclization step. The N-acyl amino acids would not be retained by the Dowex 50 column and would pass into the "hydantoin fraction." On hydrolysis the N-acyl amino acid would yield the corresponding amino acid.
Model experiments were carried out to determine the rates of deacylation of N-succinyl, N-glutaryl and N-acetyl amino acids. At 110°C in GN-HCl for 2 hr, succinylglycine and succinylphenylalanine underwent 62% and 74% deacylation (Table 1) ; under the same conditions, glutarylglycine and glutarylphenylalanine released more than 90% of glycine and phenylalanine.
At 100°C in GN-HCl, glycine and phenylalanine were released quantitatively from the N-acetyl amino acids in less than 1 hr.
The data suggested that the normal conditions of the cyclization step 311