Abstract
The high protein concentration, unique composition and complex geometry of the lens makes it transparent. alpha-, beta-, and gamma-crystallins are present in all the lenses. In addition, taxon-specific crystallins are present in lenses in bulk quantity. Zeta (zeta)-crystallin is an NADPH-dependent quinone oxidoreductase, which constitutes nearly 10% of the total eye lens protein in the evolutionary divergent animals (Camel, guinea pig and Japanese frog eye lenses) living in different ecological conditions zeta-Crystallin is also present in human and other animal lenses but at catalytic amount. The physiological role of zeta-crystallin in the eye lens is not well understood, however, truncated zeta-crystallin causes congenital cataract in guinea pig. In earlier study, redox regulated reversible activity of zeta-crystallin was reported. In this study, recombinant camel zeta-crystallin was overexpressed in E.coli and purified to homogeneity. Effect of different concentrations of reducing agent, dithiothretol (DTT) on the quinone oxidoreductase activity of recombinant zeta-crystallin was studied by enzymatic assay. To evaluate the effect of the reducing agent on the zeta-crystallin conformation, we have used far -UV and near -UV CD, intrinsic fluorescence, ANS binding assay and size exclusion chromatography. Our results showed that nearly 500/0 of the of zeta-crystallin activity was lost at 50 mu M DTT. However, no detectable changes in secondary structure were observed. No changes in the tertiary structure and surface hydrophobicity of -crystallin were detected; however, marginal changes were seen at saturating concentration of DTT (1 mM).