Stable, nonreducible cross-links of mature collagen.

During in vivo maturation, and also during in vitro incubation with physiological buffers, native collagen fibers display a progressive increase in tensile strength and insolubility. Paralleling these physiologically important changes is a progressive loss of the reducible cross-links which initially join the triple-chained subunits of collagen fibers. Although there is evidence suggesting that the reducible cross-links are gradually transformed into more stable, nonreducible cross-links during maturation, the nature of the transformation process and the structure of the stable "mature" cross-links has remained a mystery. In order to test the possibility that cross-link transformation involves addition of a nucleophilic amino acid residue to the reducible cross-links, histidine, arginine, glutamate, aspartate, lysine, and hydroxylysine residues were chemically modified, and the effect of each modification procedure on the in vitro transformation of reducible cross-links was ascertained. The results of these experiments indicated that destruction of histidine, arginine, glutamate, and aspartate residues has no measurable effect on the rate and extent of reducible cross-link transformation in hard tissue collagens. In contrast, modification of lysine and hydrocylysine residues with a wide variety of specific reagents completely blocks the transformation of reducible cross-links. Removal of the reversible blocking groups from lysine and hydroxlylysine residues then allows the transformation to proceed normally. These results indicate that collagen maturation involves nucleophilic addition of lysine and/or hydroxylysine residues to the electrophilic double bond of the reducible cross-links, yielding derivatives which are not only more stable but also capable of cross-linking more collagen molecules than their reducible precursors.