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 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 (Tm) of 70.2°C compared with untreated γS-crystallin (Tm=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.
Volume 48, 2023
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