title: Effect of oxidation of alphaA- and alphaB-crystallins on their structure, oligomerization and chaperone function.
abstract: The purpose of this study was to investigate the effect of metal-catalyzed oxidation by H(2)O(2) on the structure, oligomerization, and chaperone function of alphaA- and alphaB-crystallins. Recombinant alphaA-and alphaB-crystallins were prepared by expressing them in E. coli and purifying by size-exclusion chromatography. They were incubated with 1.5 mM H(2)O(2) and 0.1 mM FeCl(3) at 37 ( composite function)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 alphaA- and alphaB-crystallins has been substantially altered due to significant increase in random coils, in addition to decrease in beta-sheet or alpha-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 alphaA- and alphaB-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.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, 4301 W. Markham St., Slot 516, Little Rock, Arkansas 72205, USA.
authors.forename: Shanthi
authors.initials: S
authors.lastname: Rajan
authors.forename: Chad
authors.initials: C
authors.lastname: Horn
authors.forename: Edathara C
authors.initials: EC
authors.lastname: Abraham
mesh_terms: D000818:Animals; D002942:Circular Dichroism; D006861:Hydrogen Peroxide / Q000494:pharmacology; D010084:Oxidation-Reduction; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D038202:alpha-Crystallin A Chain / Q000737:chemistry* / Q000378:metabolism*; D038203:alpha-Crystallin B Chain / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D038202:alpha-Crystallin A Chain; D038203:alpha-Crystallin B Chain; D006861:Hydrogen Peroxide