GLUTAMYL LINKAGES IN COLLAGEN

In the preceding paper (Bensusan, 1969) it was shown that the sequential digestion of collagen with collagenase, papain, and prolidase plus leucine aminopeptidase resulted in the release of essentially all of the glutamic acid residues as free glutamic acid. None of these enzymes is known to hydrolyze γ-glutamyl linkages. Since the presence of a relatively large number of such linkages has been reported previously (Franzblau et al., 1963), a resolution of the apparent discrepancy was deemed necessary. Preliminary experiments with the enzymes showed them incapable of hydrolyzing the γ-glutamyl peptide linkage in γ-glutamylalanine, γ-glutamylglutamic acid, or glutathione. The free carboxyl groups of ichthyocol gelatin were modified by coupling them with L-alanine methyl ester. When this derivative was subjected to the enzymic hydrolysis, it was found that only 2.6 residues out of a possible 50 residues of glutamic acid appeared in the digest. This demonstrated not only that the γ-carboxyl groups are free in ichthyocol but that the enzymes do not hydrolyze γ-glutamyl linkage. The method used by Franzblau et al. (1963) to demonstrate the presence of γ-glutamyl linkages in ichthyocol was then applied to ichthyocol, oxidized ribonuclease, oxidized insulin A, and oxidized insulin B. The results with ribonuclease and insulin B chains indicated the presence of γ-glutamyl linkages. Insulin A chains gave no reaction. A similar method based on the report by Ho are et al. (1968) was investigated using ichthyocol and salt-soluble collagen. The results showed that all reacting free carboxyl groups of glutamyl residues were the γ-carboxyl groups. It was concluded that essentially all the glutamyl residues in collagen are involved in the normal α-glutamyl linkages. © 1969, American Chemical Society. All rights reserved.