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 agerelated cataracts. The present study was designed to understand the effect of H₂O₂-induced oxidative stress on human γS-crystallin and its relationship to lens opacification and cataract. Human γS-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 γS-crystallin under H₂O₂ stress were studied using fluorescence and circular dichroism spectroscopy methods. H₂O₂ treatment did not show any significant effect on the γS-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°C compared with untreated γS-crystallin (T_m=71.4°C). The sample treated with H₂O₂ aggregated earlier in response to heating at 65°C. H₂O₂-induced oxidative stress alters the tryptophan microenvironment and the surface hydrophobicity of γS-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.