Characterisation of a type‐I collagen trimeric cross‐linked peptide from calf aorta and its cross‐linked structure

Abstract

A collageneous trimeric cross‐linked peptide has been isolated from the insoluble matrix of calf aorta, using trypsin solubilisation, and purified by gel filtration, cation‐exchange chromatography and reversed‐phase HPLC. Molecular mass and amino acid composition indicated that the C‐terminal, non‐helical region of type I collagen in its dimer form, designated as [Col c (I)] 2 , is cross‐linked to a tryptic peptide T N (I) from the N‐terminal helical cross‐link region of an adjacent type I molecule, forming the cross‐linked peptide [Col c (I)] 2 × T N (I). Amino acid sequence analysis of the peptide yielded a series of sequences corresponding to the cross‐linking domains Col C (I) and T N (I) and furnished the first direct chemical evidence for the 4D staggered arrangement of type I molecules within native fibers. The trifunctional cross‐linking amino acid pyridinoline was shown to occur in the peptide, confirming the peptides three‐chain structure. Pyridinoline was isolated from the cross‐linked peptide by preparative amino acid analysis and reversed‐phase HPLC and identified by (a) its ultraviolet absorption spectra, (b) its fluorescence excitation and emission spectra and, for the first time, (c) its time‐of‐flight secondary ion‐mass spectrum. The high sensitivity of the latter method, exceeding that of fast‐atom‐bombardement mass spectroscopy by three orders of magnitude, allowed detection of pyridinoline in the picomole range.The occurrence of pyridinoline in non‐stoichiometric amounts, the presence of hydroxylysine in hydrolysates of all cross‐linked peptides and the finding that hydrolysates also contained an unidentified component indicated that there is at least one cross‐link form that is different from pyridinoline and is hydrolysable.