Mini-A-Crystallin Prevents Oxidation of Ascorbic Acid by Cu2+

Abstract

Purpose: : 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.

Methods: : 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.

Results: : 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.

Conclusions: : 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.

Keywords: crystallins • protein structure/function • oxidation/oxidative or free radical damage