Impact of hydrogen peroxide on structure, stability, and aggregational properties of human γS-crystallin

Cataract is the leading cause of blindness worldwide. Oxidative stress is one of the known risk factors for age-related cataracts. The present study was designed to understand the effect of H2O2-induced oxidative stress on human gamma S-crystallin and its relationship to lens opacification and cataract. Human gamma S-crystallin cDNA was cloned into the pET-20b vector, overexpressed in BL21 Star (DE3) cells, and was purified using ion-exchange and gel filtration chromatography. The structure, stability, and aggregational properties of human gamma S-crystallin under H2O2 stress were studied using fluorescence and circular dichroism spectroscopy methods. H2O2 treatment did not show any significant effect on the gamma S-crystallin secondary structure but showed an effect on its tertiary structure, resulting in N '-formylkynurenine formation. The H2O2-treated sample showed increased surface hydrophobicity, was less stable, and opened its Greek key motifs earlier with a midpoint of thermal unfolding curve (T-m) of 70.2 degrees C compared with untreated gamma S-crystallin (T-m=71.4 degrees C). The sample treated with H2O2 aggregated earlier in response to heating at 65 degrees C. H2O2-induced oxidative stress alters the tryptophan microenvironment and the surface hydrophobicity of gamma S-crystallin, and these changes decrease its thermal stability and increase its tendency to aggregate, consistent with its role as a risk factor in age-related cataract.