The location of sulphydryl groups in alpha-crystallin.
The microenvironments of the sulphydryl groups in the multimeric protein, alpha-crystallin, were studied by examining: the rate of the reaction of the groups with DTNB; the effect of increasing urea concentrations on their accessibilities; and the quenching of a fluorescent probe. In foetal bovine alpha-crystallin (1 SH/alpha A subunit) both kinetic and quenching studies indicated that over 90% of the sulphydryl groups fell into a single buried class; the remainder was exposed. In the human protein (2 SH/alpha A subunit), half of the groups were buried and the other half exposed. Accessible sulphydryl groups increased gradually as the urea concentration was increased, with complete exposure at about 4.0 M. Sedimentation velocity analyses revealed that no significant dissociation of the aggregates into subunits occurred below 3.5 M urea, at which point over 80% of the sulphydryl groups were exposed. An age-dependent increase (3-35%) was found in the proportion of exposed sulphydryl groups in bovine alpha-crystallin and a decrease in the urea concentration required to expose the remainder. It was concluded that the single cysteine is buried in the newly synthesized protein, but becomes solvent-exposed as a result of age-related conformational changes. Our observations are consistent with a quaternary structure in which all alpha A subunits occupy equivalent sites.
D000375:Aging / Q000378:metabolism; D000818:Animals; D002417:Cattle; D003459:Crystallins / Q000032:analysis*; D004228:Dithionitrobenzoic Acid; D005456:Fluorescent Dyes; D006801:Humans; D007700:Kinetics; D007908:Lens, Crystalline / Q000032:analysis* / Q000196:embryology; D009282:Naphthalenesulfonates; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000032:analysis*
D003459:Crystallins; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D013438:Sulfhydryl Compounds; C006421:1,5-I-AEDANS; D004228:Dithionitrobenzoic Acid