title: Expression, Purification and Properties of Redox-Sensitive Eye Lens Zeta-Crystallin of Arabian Camel.
abstract: The high protein concentration, unique composition and complex geometry of the lens makes it transparent. α-, &#946;-, and γ-crystallins are present in all the lenses. In addition, taxon-specific crystallins are present in lenses in bulk quantity. Zeta (ζ)-crystallin is an NADPH-dependent quinone oxidoreductase, which constitutes nearly 10 % of the total eye lens protein in the evolutionary divergent animals (Camel, guinea pig and Japanese frog eye lenses) living in different ecological conditions. ζ -Crystallin is also present in human and other animal lenses but at catalytic amount. The physiological role of γ-crystallin in the eye lens is not well understood, however, truncated ζ-crystallin causes congenital cataract in guinea pig. In earlier study, redox regulated reversible activity of ζ-crystallin was reported. In this study, recombinant camel ζ-crystallin was overexpressed in E.coli and purified to homogeneity. Effect of different concentrations of reducing agent, dithiothretol (DTT) on the quinone oxidoreductase activity of recombinant ζ-crystallin was studied by enzymatic assay. To evaluate the effect of the reducing agent on the ζ-crystallin conformation, we have used far-UV and near-UV CD, intrinsic fluorescence, ANS binding assay and size exclusion chromatography. Our results showed that nearly 50% of the of ζ-crystallin activity was lost at 50 µM DTT. However, no detectable changes in secondary structure were observed. No changes in the tertiary structure and surface hydrophobicity of ζ-crystallin were detected; however, marginal changes were seen at saturating concentration of DTT (1 mM).
authors.affiliation: Department of Biochemistry, Protein Research Chair, College of Sciences, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia. amalik@ksu.edu.sa.
authors.forename: Ajamaluddin
authors.initials: A
authors.lastname: Malik
authors.forename: Mohammed
authors.initials: M
authors.lastname: Rabbani
authors.forename: Nayyar
authors.initials: N
authors.lastname: Rabbani
authors.forename: Abdulrahman M
authors.initials: AM
authors.lastname: Al-Senaidy
authors.forename: Mohammad A
authors.initials: MA
authors.lastname: Alsenaidy
mesh_terms: D000818:Animals; D002162:Camelus; D004229:Dithiothreitol / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions / Q000187:drug effects; D007908:Lens, Crystalline / Q000378:metabolism*; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics; D038226:zeta-Crystallins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D038226:zeta-Crystallins; D004229:Dithiothreitol