Characterisation of a type-I collagen trimeric cross-linked peptide from calf aorta and its cross-linked structure. Detection of pyridinoline by time-of-flight secondary ion-mass spectroscopy and evidence for a new cross-link

Eur J Biochem. 1987 Jun 1;165(2):427-36. doi: 10.1111/j.1432-1033.1987.tb11456.x.

Abstract

A collagenous 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 [ColC(I)]2, is cross-linked to a tryptic peptide TN(I) from the N-terminal helical cross-link region of an adjacent type I molecule, forming the cross-linked peptide [ColC(I)]2 X TN(I). Amino acid sequence analysis of the peptide yielded a series of sequences corresponding to the cross-linking domains ColC(I) and TN(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 its ultraviolet absorption spectra, its fluorescence excitation and emission spectra and, for the first time, its time-of-flight secondary ion-mass spectrum. The high sensitivity of the latter method, exceeding that of fast-atom-bombardment 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.

MeSH terms

  • Amino Acid Sequence
  • Amino Acids / analysis
  • Amino Acids / isolation & purification*
  • Animals
  • Aorta / analysis*
  • Cattle
  • Chromatography, Gel
  • Chromatography, High Pressure Liquid
  • Chromatography, Ion Exchange
  • Collagen / isolation & purification*
  • Collagen Type I
  • Mass Spectrometry / methods
  • Peptides / isolation & purification*
  • Solubility
  • Spectrometry, Fluorescence
  • Spectrophotometry, Ultraviolet

Substances

  • Amino Acids
  • Collagen Type I
  • Peptides
  • collagen type I trimeric cross-linked peptide
  • pyridinoline
  • Collagen