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M.D. Linetsky, R.D. LeGrand; Thiolation of human lens proteins by glutathione through the formation of thioether bond . Invest. Ophthalmol. Vis. Sci. 2004;45(13):1689.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Formation of dehydroalanine cross–links has been associated with human lens aging and cataractogenesis. In this study we determined if modification of lens proteins by glutathione could proceed through an alternative pathway, via formation of a non–reducible thioether bond between protein and GSH. Methods: Water–soluble (WS) and water–insoluble (WI) lens protein fractions from human brunescent cataractous, human aged and bovine lenses were exhaustively dialyzed, reduced with dithiothreitol under denaturing basic conditions and their SH–groups were blocked with N–ethyl maleimide. After purification of these proteins by gel–filtration and exhaustive dialysis they were tested for immunoreactivity toward protein–bound non–reducible glutathione (nGSP) with anti–glutathione monoclonal antibody by direct and competitive ELISA. N–terminal amino acid determination in the lens proteins was performed by derivatization of these preparations with dansyl chloride, followed by an exhaustive dialysis, acid hydrolysis and detection of dansylated amino acids by RP–HPLC with fluorescence detection at Eex/Eem= 340/540 nm. Results: Direct Elisa of the reduced WS and WI lens proteins from human brunescent cataractous, human aged and bovine lenses showed a concentration–dependent immunoreactivity toward nGSP in human lens proteins only. These data were confirmed by dot–blot analysis. The reduced WI cataractous lens proteins showed the highest immunoreactivity, while bovine lens protein exhibited no reaction. An age–dependent accumulation of non–reducible GSP was detected in both water–soluble and water–insoluble lens proteins. According to the competitive Elisa results the levels of nGSP were 11.2 and 2.3 fold higher in the WI cataractous lens proteins than the levels of nGSP in the WI proteins from young and old lenses respectively. The derivatization of the reduced lens proteins with dansyl chloride followed by acid hydrolysis and RP–HPLC separation showed a presence of N–terminal glutamic acid in the reduced human lens proteins. This evidence points out that N–terminal amino group glutathione in the reduced human lens proteins is not involved in any covalent bond formation. Since no disulfides were determined in the reduced human lens proteins, it is most likely that GSH is attached to lens proteins through thioether bonds. Conclusions:These results provide, for the first time, the evidence that glutathiolation of the human lens proteins could occur through the formation of non–reducible thioether bond. The levels of non–reducible glutathione in the human lens proteins were determined to be in the sub–nanomolar range.
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