The nonenzymatic glycosylation of collagen.

Calf skin acid-soluble collagen, containing about 34 residues of lysine plus hydroxylysine per 100,000 dalton polypeptide chain, was treated with [14C]glucose in the presence or absence of NaCNBH3. In 144 h, under the conditions employed, the presence of NaCNBH3 increased the extent of glycosylation from 8 to 15% of the total residues of lysine plus hydroxylysine. The extent of glycosylation was estimated, using acid hydrolysates of the protein, by isolation and determination of reduced adducts (1-lysinohexitols) employing a system of paper chromatography followed by chromatography on an amino acid analyzer. By those means the difficulties of using specific color reactions such as that with thiobarbituric acid were obviated. Identification of the reduced adducts as forms of 1-lysinohexitol was made by comparison of that substance prepared by treatment of polylysine with [14C]glucose in the presence of NaCNBH3. Of interest is the fact that treatment of the polymer with glucose for 144 h under conditions similar to those used for the collagen, resulted in an increase of extent of glycosylation from 3 to about 50% of the total lysine residues when NaCNBH3 was present in the incubation medium. The greater degree of glycosylation of lysine residues in polylysine as compared with collagen (15 versus about 50%) may be ascribed to the different orders of macromolecular structure in the protein that could sequester certain of the residues from reaction with glucose. 1-Lysinohexitol was also identified in hydrolysates of neutral salt-soluble guinea pig skin collagen that had been reacted with glucose and then treated with NaB3H4. The glycosylated collagens were fragmented by treatment with CNBr, and modified lysine residues were found to occur along the entire length of the collagen chains. The use of NaCNBH3 in the manner described above permits measurement of both aldimine and ketoamine forms of the adducts made with glucose. The possible physiological significance of the reversibility of the ketoamine form of adduct is discussed briefly.