Photodynamic cross-linking of polypeptides in intact rat lens

During aging and particularly during cataractogenesis, crystallin polypeptides in the human lens become cross-linked by non-disulfide covalent bonds. Recently it has been hypothesized that singlet oxygen generated photodynamically within the lens oxidized certain amino acid residues in lens crystallins leading to cross-link formation. That singlet oxygen can produce cross-links in lens protein solutions has been established. In the present work we demonstrate by electrophoresis in sodium dodecyl sulfate that photodynamic cross-linking of crystallins can occur within the intact rat lens in organ culture. The cross-linking was most pronounced in the urea insoluble fraction of the peripheral lens cortex. Urea insoluble protein was increased in the irradiated lenses relative to non-irradiated controls. The process can be supported by either the photosensitizing dye rose bengal or kynurenine, an oxidation product of tryptophan which is endogenous to human lens. Based on inhibition by scavengers we conclude that the cross-linking is mediated by singlet oxygen. Cross-linking within the intact lens appeared to have greater specificity with respect to the polypeptides involved than did cross-linking in protein solutions.