Three classes of sulfhydryl group in bovine alpha-crystallin according to reactivity to various reagents

alpha-Crystallin from calf eye lens is found to have 30 sulfhydryl groups per 800 000 daltons by modification with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and 4,4'-dithiopyridine in 6 M urea. These -SH groups can be divided into three different classes in native alpha-crystallin from their reactivity with DTNB, 4,4'-dithiopyridine, iodoacetamide and ethylenimine. Results obtained with these reagents point to the presence of 7 +/- 1 thiol groups (Class I) which are likely to be surface exposed, with a concomitant 40--45% quenching of tryptophan fluorescence in DTNB-modified alpha-crystallin. Another 10 +/- 1 thiol groups (Class II) must be in a hydrophobic environment since they react only with the hydrophobic reagents, causing a further decrease in fluorescence intensity. Class III, 13 +/- 2 thiol groups, is inaccesible to any of these reagents. Introduction of up to 15 negatively charged thionitrobenzoate groups or seven positively charged aminoethyl groups in the alpha-crystallin molecule at pH 7.5--8.0 did not change the state of aggregation as judged from the sedimentation coefficients.