Does Binding To Lens Lipid Slow -Crystallin Oxidation?

Abstract

Abstract: : Purpose: Oxidative stress may be an initiating or early event in the development of cataracts. Elevated levels of hydrogen peroxide have been found in cataractous lenses. In this study we explored in vitro if the rate and degree of α-crystallin oxidation is influenced by lipid binding. Methods: Lens lipid was extracted from porcine lenses. α-Crystallin and chemical reagents were purchased from the Sigma Chemical Co, St Louis, MO. Light scattering of lens lipid vesicles and α-crystallin was measured using a fluorometer. Quenching of native tryptophan fluorescence was used to measure protein oxidation by hydrogen peroxide (H₂O₂) or t-butyl-hydroperoxide. Results: It was found that treatment for 20 hours with 1.0 mM of H₂O₂ induced a 10-fold increase of light scattering from α-crystallin and a 1.14-fold increase from pig lens lipid vesicles. H₂O₂ reduced the native tryptophan fluorescence of α-crystallin by 60%. However, this reduction was only 40% when α-crystallin was bound to lens lipid. The addition of t-butyl-hydroperoxide reduced tryptophan fluorescence of both α-crystallin and α-crystallin bound to lens lipid by comparable amounts (40%). Conclusions: The addition of H₂O₂ induced the aggregation of α-crystallin. The presence of lens lipid lowered the rate and degree of α-crystallin oxidation by H₂O₂. When using t-butyl hydroperoxide, the degree of oxidation of α-crystallin alone was comparable to that of the α-crystallin-lipid complex. These findings suggest that the hydrophobic nature of the lipid complex reduces the oxidation α-crystallin when H₂O₂, the more polar oxidant is used. Support: NIH grant EY07975, The Kentucky Lions Eye Foundation (Louisville, KY) and an unrestricted grant from Research to Prevent Blindness, Inc., New York.

Keywords: lipids • oxidation/oxidative or free radical damage • cataract