A comparison of glycopeptides derived from soluble and insoluble collagens.

Digestion of preparations of insoluble collagen of guinea fraction, “soluble collagen,” decreased with the age of the animal (2) have led to a considerable interest in the chemical basis of this “aging” phenomenon. The concept of the progressive formation, extracellularly, of covalent cross-links in collagen fibrils has recently received direct support from the reports of Bornstein, Kang, and Piez (3) and of Bornstein and Piez (4) and the isolation of peptides in which covalent bonds between collagen o( chains have been found to result from the oxidative deamination of lysine residues in the NHz-terminal region and a subsequent aldol type condensation between residues in adjacent chains. It is by no means certain, however, that this bond constitutes the only interchain cross-link in collagen. One of the earliest postulates concerning cross-linking mechanisms was that of Grassman and Kuhn (5), who pointed to the ubiquitous presence of carbohydrate in collagen and to the dramatic solubilizing effect of periodate upon insoluble collagen. Nishihara (6) has reported a similar increase in solubility following treatment of insoluble collagens with oc-amylase, and Oneson and Zacharias (7) have reported a direct correlation between the viscosity and the carbohydrate content of dispersions of tendon collagen. These and relat,ed studies have been incorporated by Hormann (8, 9) and by Gallop, Seifter, and Meilman (10) into a hypothetical cross-link in which a carbohydrate residue is involved in glycosidic linkage, ester linkage, or both to the collagen chains. More recently, Ulumenfeld et al. (11) have presented evidence which they interpreted as indicating that glucose and galactose are present only as monosaccharides linked glycosidically to the protein and that the only available site for ester linkage was C-6 of glucose. pig skin with collagenase and trypsin led to the production of two distinct glycopeptide fractions. The lower molecular weight fraction was similar in amount and in composition to the single predominant glycopeptide fraction obtained by similar treatment of soluble collagen. The major component in this fraction had the same chemical and physical characteristics as the glycohexapeptide, Gly-Met-Hyl(-Gal-Glc)Gly-His-Arg, which had been characterized previously in similar digests of soluble collagen. The second and higher molecular weight glycopeptide fraction (which contained no hydroxylysine) was found only in insoluble collagen. The major amino acids and carbohydrates present in this fraction were aspartic and glutamic acids, glycine, alanine, serine, proline, glucose, galactose, mannose, glucosamine, galactosamine, and sialic acid. The fraction appeared to be quite heterogeneous, and it has not yet been possible to establish whether interpeptide crosslinks were present. This fraction was, however, increased in samples of perchlorate-insoluble collagen identical with those in which Hormann reported evidence for covalent cross-links involving carbohydrate. Alkaline hydrolysis of soluble or insoluble collagen led to the isolation of closely similar quantities of both O-Hyl(-Gal-Glc) and 0-Hyl(-Gal), thus establishing the presence of limited numbers of monosaccharide side chains in both forms of collagen. These analyses, as well as studies of partial acid hydrolysates of purified hydroxylysine-containing glycopeptides, showed the presence in mammalian skin collagen of a disaccharide in which C-l of D-glucose is linked to D-galactose, and C-l of D-galactose to the B-hydroxyl group of hydroxylysine. No evidence was found for the participation of this carbohydrate prosthetic group in crosslinking related to the conversion of soluble collagen to insoluble collagen.