The identity of a cyanogen bromide fragment of bovine dentin collagen containing the site of an intermolecular cross-link

Abstract

A peptide fraction isolated from a cyanogen bromide digest of bovine dentin collagen had a molecular weight of 46000. Its size and amino acid composition indicated that it could not consist of peptides derived from the cleavage of a single alpha chain. On reduction with tritiated sodium borohydride, radioactivity was incorporated primarily into 5, 5'-dihydroxylysinonorleucine without degradation at the peptide backbone. Periodate cleavage of the reduced or nonreduced peptide fraction generated one fragment of molecular weight 28000 and one of 18000 completely accounting for the size of the parent peptide. On amino acid analysis the constituent single-chain peptides were determined to be alpha2CB4 and alpha1CB6. Both peptides isolated after periodate oxidation of the tritiated borohydride reduced cross-link peptide were found to contain (3H)hydroxynorvaline. These data show that some hydroxylysine of alpha2CB4, a helical region peptide, was present in aldehyde form and could act as the aldehyde donor icross-link, Schiff's base formation. The only cross-linkage of this alpha2CB4 acting as an aldehyde donor peptide to alpha1CB6 would be a helical region to helical region bond, perhaps accounting for the unusual stability and low solubility of dentin collagen.

Isolation and hydroxylysine glycoside content of some cyanogen bromide-cleaved fragments of collagen from bovine corneal stroma

“…Thus it is possible that in bovine corneal collagen too, this peptide is cross-linked and would therefore be eluted in a different position on the CM-cellulose column. Similarly fragment a2-CB4 is joined to peptide al-CB6 in dentin collagen (Scott et al, 1976), giving a cross-link peptide that appears on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with a relative mobility identical with that of the component with a mobility of 0.33 shown in Fig. 1.…”

The Chemistry and Biology of Collagen

“…At present, it would appear that aldehyde formation is limited to the terminal regions of the molecule in bone and tendon collagen. However, Scott et al (1976) have recently identified a cross-linked fragment from bovine dentin collagen which involves «1-CB6 and «2-CB4. In this fragment, a substantial fraction of the cross-link appears to be derived from the condensation of hydroxyallysine in ct2-CB4, located in the collagen helix.…”

Studies on the structure and chemistry of dentin collagen-phosphophoryn covalent complexes

“…Aldimines would result from allysine or hydroxyallysine reaction with amino groups of lysyl or hydroxylysyl residues [28]. Since 3H-dihydroxylysinonorleucine and 3H-hydroxylysinonorleucine are the major and minor tritiated amino acids in acid hydrolysates of reduced bone and dentin collagens, the major and minor reducible cross-links are dehydrohydroxylysino hydroxynorleucine and dehydrohydroxylysinonorleucine, respectively [16,[30][31][32][33]. Since 3H-dihydroxylysinonorleucine and 3H-hydroxylysinonorleucine are the major and minor tritiated amino acids in acid hydrolysates of reduced bone and dentin collagens, the major and minor reducible cross-links are dehydrohydroxylysino hydroxynorleucine and dehydrohydroxylysinonorleucine, respectively [16,[30][31][32][33].…”

“…After reduction with aH-borohydride, acid hydrolysis, and chromatography of the acid hydrolysate, every collagenous tissue exhibits a characteristic profile of :~H-containing reduced cross-link amino acids [29,30]. These cross-link components are not destroyed by CNBr digestion in 70% formic acid and have been recovered in their expected concentrations as :~H-dihydroxylysinonorleucine and :~H-hydroxylysinonorleucine among the soluble peptides even when CNBr digestion preceded borohydride reduction [16,30,33]. These cross-link components are not destroyed by CNBr digestion in 70% formic acid and have been recovered in their expected concentrations as :~H-dihydroxylysinonorleucine and :~H-hydroxylysinonorleucine among the soluble peptides even when CNBr digestion preceded borohydride reduction [16,30,33].…”

“…These cross-link components are not destroyed by CNBr digestion in 70% formic acid and have been recovered in their expected concentrations as :~H-dihydroxylysinonorleucine and :~H-hydroxylysinonorleucine among the soluble peptides even when CNBr digestion preceded borohydride reduction [16,30,33]. Since nearly all of the radioactivity eluted rapidly, far in advance of the known reduced cross-links and their precursors, and since this unidentified radioactive peak is typical in 3H-NaBH4reduced collagens [31,33], it is likely that this radioactive peak represents nonspecific reduction. When the collagen-bound phosphophoryn from bovine dentin was analyzed for reducible crosslinks, the results shown in Figure 8 were obtained.…”