Quantitation of lens protein mixed disulfides by ion-exchange chromatography

Abstract

A sensitive and high resolution ion-exchange chromatographic method was employed to analyse lens protein mixed disulfides. The mixed disulfides were analysed and quantified as glutathione sulfonic acid and cysteic acid after the lens proteins were subjected to performic acid oxidation. The oxidized product was applied on a 0·9×15 cm column packed with BioRad Aminex A-28 resin (9 μm beads). The column was maintained at 55°C and eluted with 0·3 m sodium acetate buffer, pH 5·1 at 60 ml hr −1 for 40 min before switching to 0·5 m of the same buffer. The eluate was subsequently reacted with ninhydrin and monitored for absorbance at 570 nm. The glutathione sulfonic acid emerged at 69 min, cysteic acid at 35 min, and both were clearly resolved from neighboring peaks and could be detected at < nmol level. In the lens, both glutathione and cysteine protein mixed disulfides were only found in the TCA-insoluble protein fraction. Normal rat lens contained 0·35 nmol per lens of protein-bound glutathione but 10-fold this amount of protein-bound cysteine. The hyperglycemic condition had little effect on the protein thiol mixed disulfide level except that at a more advanced diabetic condition, equal depletion of cysteine and glutathione mixed disulfides were found. On the other hand, oxidative conditions induced a marked elevation of glutathione mixed disulfide but not of the cysteine mixed disulfide.