A major intermolecular cross-linking site in bovine dentine collagen involving the .alpha.2 chain and stabilizing the 4D overlap

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Abstract
Approximately 20% of the radioactivity incorporated into the dentine collagen of unerupted bovine molars after reduction with tritiated sodium borohydride was recovered in a cyanogen bromide peptide fraction of MW 61,000 following chromatography on agarose A5m. After rechromatography on agarose A1.5m, this fraction was resolved into 10 components by gel isoelectric focusing. Of these components, 9 (the most acidic) were tritiated and contained the reduced cross-links dihydroxylysinonorleucine and hydroxylysinonorleucine. The amino acid compositions were consistent with the identification of each of these components as .alpha.2CB3,5 linked to 1 or 2 small peptides. By limited Edman degradation, with and without prior digestion with pyroglutamate aminopeptidase (EC 3.4.11.8), these small peptides were identified as .alpha.1CB0,1 and .alpha.2CB1, occurring in a ratio of approximately 2:1. Specific cleavage with cathepsin D revealed that all the cross-link was associated with the C-terminal one-third of the .alpha.2 chain, thus fixing the displacement of the participating molecules at 4D. The content of the known reducible cross-links present in these peptides, calculated from the specific activity of the reductant, was sufficient to account for only 10-20% of the cross-linking actually found, suggesting that stabilization is mainly through nonreducible cross-links of as yet undetermined structure. By quantitative analysis of homoserine content and semiquantitative amino-terminal analyses, it was determined that virtually all of the .alpha.2 chain of bovine dentine collagen is cross-linked in this manner. One cross-link per molecule in this location could make a major contribution to the mechanical stability of the insoluble collagen fibrils in this tissue.