Characterization of chick bone collagen and compositional changes associated with maturation.

Collagen extracted from the diaphyseal region of the tibiae of normal and lathyritic chicks with 0.5 M acetic acid and 5 M guanidine hydrochloride at neutral pH has been studied with regard to chain organization and amino acid composition. Decalcification by dialysis against large quantities of 0.5 M acetic acid and subsequent exposure of the decalcified bones to fresh 0.5 M acetic acid followed by 5 M guanidine resulted in the solubilization of 85% of the total collagen in the bones from lathyritic animals. Collagen in the bones of control animals was almost entirely resistant to extraction with 0.5 M acetic acid, and less than 20 % of the total collagen could be extracted in 5 M guanidine. These observations suggest a high degree of intermolecular cross-linking. Chromatography of the collagen extracted by both solvents from the bones of lathyritic animals on columns of carboxymethyl cellulose indicated that the extracts were composed almost entirely of singleand double-chain components. Carboxymethyl cellulose chromatography of the collagen extracted with 5 M guanidine from the bones of normal chicks revealed that only 30% of the dissolved collagen (5 % of the total collagen) could be recovered as these components. Chromatography on an agarose molecular sieve column showed that the remainder could be accounted for in part by high molecular weight aggregates and in part by low molecular weight, degraded material. These results suggest that bone collagen fibrils are stabilized through the formation of intraand intermolecular cross-links and that lathyrogens exert their effects by inhibiting cross-link formation, as previously shown for soft tissue collagens. The extractability of normal bone collagen in guanidine appears to result from degradation in the presence of the denaturing agent to yield a highly heterogeneous mixture. Carboxymethyl cellulose chromatography of acid-extracted collagen from the bones of lathyritic animals indicated that the bone collagen molecule contains two chains (al) that are identical or similar and one chain ((~2) that is unlike the other two. Their compositions are analogous to those of other vertebrate collagens. Amino acid analyses of the chromatographically separated cy chains in successive extracts of collagen from the lathyritic bones indicated that during development there was a progressive decrease in hydroxylysine content, accompanied by an equivalent increase in lysine content, with no significant change in the content of any other amino acid. This observation was verified by amino acid analyses of the total bone collagen of normal chicks of various ages. The hydroxylysine content decreased by nearly one-half, from 11 to 6 residues per 1000, between the 1st and the 49th day after hatching. This result suggests that the hydroxylation of bone collagen lysyl residues is dependent in part upon factors extrinsic to the collagen molecule.