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Effect of Oxidation of αA- and αB-Crystallins on their Structure, Oligomerization and Chaperone Function

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Abstract

The purpose of this study was to investigate the effect of metal-catalyzed oxidation by H2O2 on the structure, oligomerization, and chaperone function of αA- and αB-crystallins. Recombinant αA-and αB-crystallins were prepared by expressing them in E. coli and purifying by size-exclusion chromatography. They were incubated with 1.5 mM H2O2 and 0.1 mM FeCl3 at 37 ∘C for 24 hrs and the reaction was stopped by adding catalase. Structural changes due to oxidation were ascertained by circular dichroism (CD) measurements and chaperone activity was assayed with alcohol dehydrogenase (ADH) and insulin as target proteins. The oligomeric nature of the oxidized proteins was assessed by molecular sieve HPLC. The secondary structure of the oxidized αA- and αB-crystallins has been substantially altered due to significant increase in random coils, in addition to decrease in β-sheet or α-helix contents. The tertiary structure also showed significant changes indicative of different mode of folding of the secondary structural elements. Chaperone function was significantly compromised as supported by nearly 50% loss in chaperone activity. Oxidation also resulted in the formation of higher molecular weight (HMW) proteins as well as lower molecular weight (LMW) proteins. Thus, oxidation leads to disintegration of the oligomeric structure of αA- and αB-crystallins. Chaperone activity of the HMW fraction is normal whereas the LMW fraction lacks any chaperone activity. So, it appears that the formation of the LMW proteins is the primary cause of the chaperone activity loss due to oxidation.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, 4301 W. Markham St., Slot 516, 72205, Little Rock, Arkansas, USA

    Shanthi Rajan, Chad Horn & Edathara C. Abraham

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  1. Shanthi Rajan
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  2. Chad Horn
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  3. Edathara C. Abraham
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Correspondence to Edathara C. Abraham.

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Rajan, S., Horn, C. & Abraham, E.C. Effect of Oxidation of αA- and αB-Crystallins on their Structure, Oligomerization and Chaperone Function. Mol Cell Biochem 288, 125–134 (2006). https://doi.org/10.1007/s11010-006-9128-4

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  • DOI: https://doi.org/10.1007/s11010-006-9128-4

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