The Effects of Hyperbaric Oxygen on the Crystallins of Cultured Rabbit Lenses: a Possible Catalytic Role for Copper

Abstract

Oxidative effects on lens proteins have been linked with the formation of human age-related cataract, particularly nuclear cataract. This study investigated the effects of hyperbaric oxygen (HBO)-induced oxidative stress on nuclear and cortical α-, β- and γ-crystallins of cultured rabbit lenses, using high performance liquid chromatography (HPLC). The lenses were treated with 50atm of either 100% N2(control) or 100% O2(experimental) for 3, 6, 16 and 48hr. The levels of reduced glutathione (GSH) and water-soluble (WS) protein decreased more rapidly in the nucleus of the O2-treated lens than in the cortex. The first significant loss of WS protein in each of the two regions occurred when levels of GSH had decreased by at least 90% in either the nucleus (at 6hr) or the cortex (at 16hr). HPLC analysis of the nuclear WS proteins indicated that β-crystallins were the first proteins affected by the oxidative stress. Soon after HBO-treatment was initiated (at 6hr) and prior to insolubilization of protein, nuclear β- and γ-crystallins moved to the higher molecular weight α-crystallin fraction; 2-D gel electrophoresis and Western blotting indicated the presence of disulfide-crosslinked and non-crosslinked β- and γ-crystallins in this fraction. Significantly different HBO-induced effects were observed on lens cortical crystallins compared to those for the nucleus. For example, γ-crystallins in the cortex shifted very soon after HBO-treatment (at 3hr) to slightly higher molecular weights, possibly the result of protein/glutathione mixed disulfide formation; however, this phenomenon was not observed in the nucleus. Cortical β- and γ-crystallins remained in solution longer than nuclear proteins following HBO-treatment of the lenses, presumably the result of protection from the four-fold higher level of GSH (22 vs 6m M) present in the lens periphery. Surprisingly, there was no movement of β- and γ-crystallins to αH- and α-crystallin fractions in the cortex of the O2-treated lens, in contrast to that observed for the nucleus. Cortical crystallins appeared to go directly from being soluble to being insoluble with no high molecular weight intermediate stage. The data suggested a possible chaperone-like function for α-crystallin in the nucleus of the stressed lenses, but not in the cortex. HBO-induced effects on lens nuclear supernatants, which mimicked those observed for intact lenses, could be nearly completely prevented by the copper-chelator bathocuproine, but not by the iron-chelator deferoxamine. Overall, the results provide additional evidence demonstrating an increased susceptibility of the lens nucleus to oxidative stress; the greater protective ability of the cortex may be linked to a higher capacity for β- and γ-crystallin/glutathione mixed disulfide formation, inhibiting disulfide-crosslinked insolubilization. The data also implicate copper as a catalyst for the autoxidation of -SH groups in the lens, and suggest that α-crystallin chaperone-like activity may play a greater role in the lens nucleus than in the cortex in preventing oxidative insolubilization of crystallins.