Spectroscopic studies on the mixed disulfide formation of lens crystallin with glutathione.
Mixed disulfide was formed through thiol-disulfide exchange reaction of lens crystallin with oxidized glutathione (GSSG). The reaction was monitored by isoelectric focusing (IEF) and DTNB [5,5'-dithiobis(2-nitrobenzoic acid)] assay. The effects on protein conformation were studied by circular dichroism (CD) and fluorescence. The DTNB shows 22% and 49% decrease of SH groups after the exchange reaction in alpha-crystallin and gamma-crystallin, respectively. The exchange reaction was further shown by an acidic shifting in IEF pattern. The near ultraviolet CD shows a slight decrease in the GSSG-treated crystallins. The fluorescence measurements of the SH specific probe IANBD, 4-(N-iodoacetoxy)ethyl-N-methylamino-7-nitrobenz-2-oxa-1,3-diazole, indicate that the surface SH groups were oxidized in the GSSG-treated samples. The labeling with amine selectively reactive probe FITC, fluorescein-5-isothiocyanate, indicates an increase of amine reactivity with mixed disulfide formation. Polarization measurements show that bound FITC probes are in a less rigid structure in the mixed disulfide rich crystallin. All these results point out that the formation of mixed disulfide partially unfolds protein.
D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D003459:Crystallins / Q000032:analysis*; D004220:Disulfides / Q000032:analysis*; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005978:Glutathione / Q000494:pharmacology*; D007525:Isoelectric Focusing; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
D003459:Crystallins; D004220:Disulfides; D004228:Dithionitrobenzoic Acid; D005978:Glutathione