A NEW METHOD FOR SPECIFIC DETERMINATION OF GLUTATHIONE-PROTEIN MIXED DISULFIDES IN OCULAR LENS

Present studies were designed to develop a simple, specific and sensit ive method to determine protein-glutathione mixed disulfides (PS-SG) i n lens through which an accurate status of the constitutive PS-SG in l ens proteins could be assessed. The method described in this report is a modification of the previously described method for specific determ ination of glutathione (GSH) in which GSH is conjugated to 1-chloro-2, 4-dinitrobenzene through the reaction catalyzed by glutathione S-trans ferase and the conjugate (dinitrophenyl-S-glutathione, Dnp-SG) is meas ured by HPLC. In the present method, GSH is quantitatively removed fro m PS-SG by treating with 300 mM beta-mercaptoethanol, and enzymaticall y derivatized to Dnp-SG for its quantitation, by HPLC. Using this meth od, we demonstrate almost quantitative (96%) recovery of [H-3] GSH fro m [H-3] GSH-alpha-crystallin mixed disulfide containing known amounts of [H-3] GSH bound to a-crystallin. Our results also demonstrate that the PS-SG determined by this method represents mainly the constitutive PS-SG in lens and only a minimal (<5%) incorporation of [H-3] GSH occ urs in lens proteins during the processing of lens samples for analyse s. The amount of GSH present as PS-SG in fresh bovine as determined by this method was found to be 0.11 +/- 0.01 mu mol GSH/mg protein. In b ovine lenses stored at -20 degrees for one week, the PS-SG content inc reased by about two fold while in the lenses stored for 72 weeks this value increased over twenty fold. This may explain the wide range of v ariations reported for the PS-SG content in ocular lens. Quantitative recovery of protein bound GSH by this sensitive, specific and relative ly simple method in which there is minimal binding of free GSH to prot eins during the processing of samples indicates that an accurate deter mination of the constitutive PS-SG of lens and possibly of other tissu es can be achieved by this method.