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M. Raju, P. Santhoshkumar, K. Sharma; Mini-A-Crystallin Prevents Oxidation of Ascorbic Acid by Cu2+. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1599.
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Previous studies have shown that small heat shock protein α-crystallin binds Cu2+. The Cu2+ interaction with α-crystallin affects the chaperone activity. Earlier, we reported that αA70-88 peptide (also known as αA-mini-chaperone) can independently function as chaperone molecule. The present study was undertaken to determine whether mini-αA-crystallin posses Cu2+ binding property and inhibits copper induced oxidation of ascorbic acid.
Mini-αA [DFVIFLDVKHFSPEDLTVK] was supplied by GenScript Corporation. Cu2+ solution was prepared in buffer with glycine to avoid non-specific interaction. The recombinant αA-crystallin was expressed in E.coli cells and purified using chromatographic techniques. Deletion mutant αA-Δ70-77 was created by site directed mutagenesis method. Cu2+-mediated oxidation of ascorbic acid was measured as change in absorbance at 260 nm in a spectrophotometer. Secondary structure of mini-αA was analyzed by CD spectroscopy in presence or absence of Cu2+. Bis-ANS interaction with peptide was followed by fluorometry.
Four hundred µM of ascorbic acid was completely oxidized by 1µM of Cu2+ in 50mM phosphate buffer pH7.2 in 20 min. Addition of 33 nanomole of mini-αA-crystallin completely suppressed Cu2+-induced oxidation of ascorbic acid. The recombinant protein αA-Δ70-77 showed 80-90 percent reduced protection against Cu2+-mediated oxidation of ascorbic acid, whereas wild-type αA-crystallin completely prevented the oxidation of ascorbic acid during the experimental period. The binding of Cu2+ to mini-αA-crystallin increased the ellipticity of the peptide in far-UV region during CD spectroscopy. Prior binding of Cu2+ to the peptide resulted in diminished bis-ANS interaction with mini-αA-crystallin.
The present study demonstrates that the 70-77 region in αA-crystallin is a Cu2+ binding site. Mini-αA-crystallin prevents Cu2+-induced oxidation of ascorbic acid.
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