Oxidation of human lens recombinant alpha A-crystallin and cysteine-deficient mutants

Disulfide cross-linking, one of the results of oxidative stress, has been t hought to play an important role in cataractogenesis. High molecular mass ( HMM) protein aggregation also contributes to cataract development, and a pr evailing speculation is that disulfide cross-linking induces HMM aggregatio n. However, there is no direct evidence to support this speculation. Dimeri zation is an effect of disulfide cross-linking but cannot explain the size of HMM aggregates observed in the lens. alphaA-crystallin has two cysteine residues (Cys131 and Cys142) and we have prepared three Cys-deficient mutan ts, two single mutants (C131I and C142I) and one double mutant (C131I/C142I ). They were subjected to H2O2 oxidation in an ascorbate-FeCl3-EDTA-H2O2 sy stem. The effects of oxidation on the mutants, including changes in aggrega te size and conformation, were compared with those of the wild-type aA-crys tallin by FPLC gel filtration, absorption, fluorescence, and circular dichr oism measurements. The results indicated that other amino acid residues bes ides Cys, such as Trp and Tyr, were also oxidized by H2O2 Disulfide dimeriz ation alone seems to play a less important role in HMM ag,aggregation than does the secondary conformational change resulting from the combined effect of the oxidation of Trp and Tyr as well as Cys. (C) 2001 Academic Press.