Application of a specific radio-immunoassay for the peptide alpha 1CB6 to quantitative studies on intermolecular cross-linking in bovine dentine collagen

Abstract

Antisera from rabbits immunized against bovine Type I collagen were used to develop a specific radio-immunoassay for the antigenic determinant located within the extra-helical carboxy-terminal sequence of the alpha 1 chain. This assay was applied to mixtures of cyanogen bromide peptides of bovine dentine collagen fractionated by (a) gel chromatography on agarose and (b) preparative gel electrophoresis in the presence of sodium dodecylsulfate. The data confirm previous estimates that only about 25% of the total alpha 1CB6 (the cyanogen bromide peptide containing the antigenic determinant) could be isolated in a free uncross-linked state. Antigenically active cross-linked alpha 1CB6 was recovered in three fractions from preparative gel electrophoresis. Two of these (apparent Mr 21,000 and 48,000) contain alpha 1CB6 linked through its carboxy-terminal extra-helical sequence and appear to result from the same, now well-established, 4 D intermolecular relationship within the collagen fibrils. Considered together, these fractions were recovered in amounts which reflect the occurrence of cross-links in these locations at a frequency of close to one for each collagen molecule. About 30% of the total cross-linked alpha 1CB6 was recovered in high molecular weight material barely penetrating the electrophoresis gel. This may be a mixture of products of the further reaction of the initially-formed double-chain cross-linked peptides involving alpha 1CB6 and perhaps also of incompletely cleaved sequences of alpha 1 or alpha 2 chain linked to alpha 1CB6. The absence of a fraction corresponding to a dimer of alpha 1CB6, as reported for bovine corneal and scleral collagens, suggests tissue specificity in the location of intermolecular crosslinks.