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Kinetic analysis was made of the dialysis of digests of ichthyocol gelatin prepared with collagenase. It was found that most, if not all, of the peptides in such digests are dialyzable, but at greatly different rates. First, there is a class of slowly dialyzable peptides that contain most of the tyrosine, ester groups, and carbohydrate moieties of the parent protein molecule. Second, there is a class of slowly dialyzable peptides, which are rich in the polar amino acids and appear to originate from the polar or “noncrystalline” regions of the protein molecule. Third, there is a class of rapidly dialyzable peptides that are rich in proline and hydroxyproline and appear to originate from the “crystalline” regions of the collagen molecule. After 48 hr., the most slowly dialyzable peptides remain in an aggregate fraction which, operationally, has been designated a “non-dialyzable” fraction. It has been established that the aggregate peptides have, relative to original gelatin, increased contents of glutamic acid, aspartic acid, and lysine, and contain major portions of the carbohydrate, tyrosine, and hydroxylamine-sensitive (“ester-like”) bonds of gelatin. However, they have relatively decreased contents of proline, hydroxyproline, arginine, and leucine. Their Contents of glycine and alanine are relatively unchanged. Dinitrophenylation studies indicated that the average peptide length of the “nondialyzable” peptides was 11–15 residues. On the basis of these and other studies, a representation is made of the polar and nonpolar regions of the collagen molecule.

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.