Cross-linking and fluorescence changes of collagen by glycation and oxidation

The non-enzymatic glucosylation of collagen in vivo and in vitro produces blue-fluorescent cross-links very slowly. The mechanism of their formation is unknown. We investigated the role of oxidation in glycation. When native fluorescent collagen from old-rat tail tendon and its CNBr peptides were oxidized by chemically generated singlet oxygen, cross-linking occurred immediately, and the cross-linked products showed an increased blue fluorescence. Further cross-linking and development of blue fluorescence also were accelerated by singlet oxygen when oxidizing in vitro glucosylated collagen CNBr peptides. It was noted that the blue fluorescence developed at the expense of a near-UV fluorescence. This near-UV fluorophore, which is also present in native collagen, was found to be produced by the in vitro glucosylation of collagen and during the cross-linking by glucosylation was slowly converted to the blue fluorophore. These changes indicate the autoxidation of near-UV fluorescent intermediates to blue fluorescent cross-links during glucosylation. Non-enzymatic fructosylation, which occurs in vivo in certain proteins, was more effective than glucosylation in forming fluorophores and cross-links with collagen in vitro. Fructosylated fluorophores were found different from glucosylated products in their oxidation reactivities with singlet oxygen.