The presence in collagen of γ‐glutamyl peptide linkages

A method is described for formation of hydroxamic acids by direct coupling, in aqueous medium, of hydroxylamine hydrochloride and the sodium salt of a carboxylic acid. The reaction is mediated by a water‐soluble carbodiimide, 1‐cyclohexyl‐3‐[2‐morpholinyl‐(4)‐ethyl]‐carbodiimide metho‐ p ‐toluene sulfonate. Using model compounds, the production of α‐, β‐ and γ‐acyl hydroxamic acids was studied. Hydroxamic acid derivatives of α‐polyglutamic and γ‐polyglutamic acids were also prepared. Dinitro‐phenylhydroxamate derivatives were prepared and subjected to Lossen rearrangement, and the reaction mixtures hydrolyzed. Analysis showed that α‐polyglutamic acid yielded as many molecules of α,γ‐diaminobutyric acid as there had been glutamic acid residues in the form of the dinitrophenylhydroxamate, and gave rise to no ammonia and succinic semialdehyde. In contrast, γ‐polyglutamic acid yielded exactly twice the molar quantity of ammonia as there had been glutamic acid residues in the form of dinitrophenylhydroxamate, and also gave rise to significant quantity of succinic semi‐aldehyde but no α,γ‐diaminobutyric acid. Since production of the latter is characteristic of α‐glutamyl hydroxamates and production of the former is associated with γ‐glutamyl hydroxamates, the results indicated that the side‐chain carboxyl groups of either polymer retained their identities during formation of the hydroxamic acid derivative, and no interchange had occurred between α‐ and γ‐carboxyl groups. These methods were then used to establish that gelatin of ichthyocol contains (per 1000 total residues) at least 20 residues of glutamic acid in γ‐peptide linkage. Due to the incomplete dinitrophenylation of the hydroxamic acid derivative of the protein, gelatin of calf skin collagen gave a lower figure of 10 such linkages per 1000 total amino acid residues–a value which must be considered a minimum value. These results show that γ‐glutamyl peptide bonds exist in collagen.