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.
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.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis / Q000302:isolation & purification*; D000818:Animals; D001011:Aorta / Q000032:analysis*; D002417:Cattle; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003094:Collagen / Q000302:isolation & purification*; D024042:Collagen Type I; D013058:Mass Spectrometry / Q000379:methods; D010455:Peptides / Q000302:isolation & purification*; D012995:Solubility; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D000596:Amino Acids; D024042:Collagen Type I; D010455:Peptides; C052929:collagen type I trimeric cross-linked peptide; C015484:pyridinoline; D003094:Collagen