Cyanogen Bromide Peptides from Insoluble Skin and Dentin Bovine Collagens

Purified insoluble bovine corium (IC) and dentin (DC) collagens were degraded with CNBr. Neither collagen was totally soluble in the acidic digestion mixture, even after prolonged or repeated digestion. The soluble peptides were resolved by a combination of ion-exchange and gel filtration chromatographic procedures and compared with the peptides previously obtained from the isolated α1 and α2 chains from acid-soluble (SC) bovine corium or with β₁₂ from the same source. The majority of peptides from IC and DC could be matched with the SC CNBr peptides. However, three distinct new classes of peptides were detected in the IC and DC digests. Both contained an acid-stable intermolecular crosslink component, α1-CB6′[α1-CB6 + α1-CB(0,1)]. Both also contained two additional peptides attributed to the presence of an α1(III) chain, α1(III)-CB3, and α1(III)-CB(4,5). While the α1(I) peptides of IC and DC were quite similar except for degree of hydroxylation, the al(III) peptides showed more pronounced tissue specific differences. Evidence was also obtained indicating the presence of a peptide, α2-CB4′, consisting of α2-CB4 and a noncollagenous polypeptide attachment. The α2-CB4’ was present in both IC and DC but was not detected in SC. Each of the CNBr peptides common to both soluble and insoluble collagens was analyzed for hexose. In IC, α1(I)-CB5, α2-CB4, and α2-CB5 contained disaccharide units and α1(I)-CB6 contained a monosaccharide. In DC, only α1(I)-CB5 contained disaccharide, while α1(I)-CB3 and α1(I)-CB6 contained monosaccharide units. The DC α2-CB4 and α2-CB5 contained mixtures of mono- and disaccharide attachments. In addition to the hexose, DC α1(I)-CB6, α2-CB4, and α2-CB5 all contained covalently bound phosphate groups. The acid-insoluble CBNr digestion residues contained portions of collagen with uncleaved methionyl residues in close association, particularly in DC, with a highly acidic polypeptide. In DC, this polypeptide was rich in phosphoserine groups. These data indicate that the insoluble collagens do differ in several ways from acid-soluble collagen and it is suggested that these differences relate to the tissue-specific fibril organization as well as to the presence of intermolecular cross-linkages.