Fluorescent-labeled cross-links in collagen: pyrenesulfonylhydrazine

Aldol condensation products of two lysyl-derived aldehyde (allysine) residues are involved in cross-linking of collagen. However, the distribution of these cross-links and their age-related changes remain largely unanswered. We have found that the unsaturated aldehydes of aldol condensation cross-links can be fluorescent labeled. When labeled with pyrenesulfonylhydrazine, pyrene dimers and excimers fluoresce at 383 and 485, nm, respectively. (The pyrene dimer is stable in benzene, whereas in polar solvents it exhibits an exponential decay to monomer fluorescing at 378 nm.) Dimers bound to collagen also decay to monomers, but at a more complicated, nonexponential rate. This dissociation in collagen is also associated with gradual decrease in the excimer fluorescence. While dissociated monomers appear to be reassociated by redialysis, the excimer is not regenerated. During fibril formation in vitro of the labeled collagen, two fluorescence changes take place: a very rapid decrease of the excimer fluorescence and a gradual increase of the monomer fluorescence. These changes indicate very early and early conformational changes at the nonhelical terminal telopeptides. The excimer fluorescence also decreases upon thermal and guanidine denaturation. Two different environments for excimer formation are suggested by the latter. It is concluded that pyrenesulfonylhydrazine offers a unique and sensitive probe for the proximity of aldehyde groups as well as for the mobility and conformation changes of the telopeptides in collagen.