IDENTIFICATION OF THE LOCI OF THE COLLAGEN-ASSOCIATED EHRLICH CHROMOGEN IN TYPE-I COLLAGEN CONFIRMS ITS ROLE AS A TRIVALENT CROSS-LINK

Collagenous peptides containing the Ehrlich chromogen (EC) were selectively isolated from a tryptic digest of bovine tendon by coupling to a diazotized polyacrylamide support. The isolated p-phenol-azo-EC peptides were purified and characterized by amino acid and sequence analyses. EC occurred in stoichiometric amounts in trimeric cross-linked chains originating from the known cross-link regions of type-I collagen. The major locus of the EC was alpha-2(I)Hyl-933 x alpha-1(I)Lys(Hyl)-9N x alpha-2(I)Lys(Hyl)-5N but it was also shown to occur at the loci alpha-1(I)Hyl-87 x alpha-1(I)Lys(Hyl)-16C x alpha-1(I)Lys(Hyl)-16C and alpha-1(I)Hyl-930 x alpha-1(I)Lys(Hyl)-9N x alpha-2(I)Lys(Hyl)-5N. After sequence analyses of the C-terminal helical cross-link region alpha-2(I)928-963, corrections are presented for residues 927, 930, 932 and 933 of the bovine alpha-2(I) chain. The collagen-associated EC is postulated to be a trisubstituted pyrrole formed by the reaction of the aldehyde form of a telopeptidyl lysine residue with a bifunctional keto amine cross-link. It is also proposed that when the telopeptidyl lysine residue is hydroxylated the above reaction will result in pyridinoline formation.