Nonreducible Cross-link in Dentin Collagen

Isolation and Characterization of Cross-linked Peptides Containing Pyridinoline

Abstract

Dentin collagen which comprises the main organic component of dentin is known to be extre-mely resistant to solubilization and swelling compared with that of other tissues. This property has been ascribed to the unique distribution of cross-links in this collagen fiber. The aim of this paper is to investigate the distribution of pyridinoline, one of the major cross-links in dentin col-lagen, by isolating and characterizing the cross-linked peptides.
NaBH₄-reduced bovine dentin collagen was completely solubilized by trypsin digestion and heating at 60°C. From these digests two cross-linked peptides, peptide A and peptide B, containing pyridinoline were isolated by sequential chromatographic procedures on Sephadex G50, phosphocel-lulose and DEAE cellulose columns. These peptides were abundant in dentin and fetal bone colla-gen but were scarce in adult bone collagen. Amino acid analyses showed that the two peptides were quite similar in amino acid composition, rich in serine, methionine, tyrosine and histidine, suggesting that the isolated peptides contained the telopeptide portion of the collagen molecule. The molecular weights of the peptides A and B were approximated by gel filtration and SDS-polyacrylamide gel electrophoresis to be 6000-7000 and 5000-6000, respectively.
The changes in the contents of pyridinoline and dihydroxylysinonorleucine, the other major cross-link in dentin collagen, during in vitro aging were examined. When the collagen was incubated in physiological buffer at 37°C, it was found that up to 6 weeks the content of dihy-droxylysinonorleucine decreased strikingly with the incubation period, while the content of pyridinoline increased. After 6 weeks, the pyridinoline content showed a slight decrease. Elution profiles of Sephadex G50 chromatography of tryptic peptides of in vitro-aged dentin collagen re-vealed that there were one main radioactive peak which contained dihydroxylysinonorleucine and two fluorescent peaks containing pyridinoline. The amount of radioactive peak decreased marked-ly and that of the first fluorescent peak varied during the incubation. However, it was found that the second fluorescent peak containing peptides A and B did not change at all.
The results supported the hypothesis of precursor-product relationship between dihydroxyly-sinonorleucine and pyridinoline, but the decrease in the amount of reducible cross-link with aging could not be fully explained by the increase of pyridinoline, and the other possible route to maturation in the dentin collagen cross-links was discussed.