title: Thiol-dependent antioxidant activity of interphotoreceptor retinoid-binding protein.
abstract: Interphotoreceptor retinoid-binding protein (IRBP), which is critical to photoreceptor survival and function, is comprised of homologous tandem modules each ∼300 amino acids, and contains 10 cysteines, possibly 8 as free thiols. Purification of IRBP has historically been difficult due to aggregation, denaturation and precipitation. Our observation that reducing agent 1,4-dithiothreitol dramatically prevents aggregation prompted investigation of possible functions for IRBP's free thiols. Bovine IRBP (bIRBP) was purified from retina saline washes by a combination of concanavalin A, ion exchange and size exclusion chromatography. Antioxidant activity of the purified protein was measured by its ability to inhibit oxidation of 2,2'-azinobis [3-ethylbenzothiazoline-6-sulfonate] by metmyoglobin. Homology modeling predicted the relationship of the retinoid binding sites to cysteine residues. As a free radical scavenger, bIRBP was more active than ovalbumin, thioredoxin, and vitamin E analog Trolox. Alkylation of free cysteines by N-ethylmaleimide inhibited bIRBP's antioxidant activity, but not its ability to bind all-trans retinol. Structural modeling predicted that Cys 1051 is at the mouth of the module 4 hydrophobic ligand-binding site. Its free radical scavenging activity points to a new function for IRBP in defining the redox environment in the subretinal space.
authors.affiliation: Medical Research Service, Veterans Affairs Medical Center, Buffalo, NY, USA; Departments of Ophthalmology (Ross Eye Institute) and Pathology & Anatomic Sciences, the State University of New York, Buffalo, NY, USA; SUNY Eye Institute, State University of New York, NY, USA. Electronic address: fg23@buffalo.edu.
authors.forename: Federico
authors.initials: F
authors.lastname: Gonzalez-Fernandez
authors.affiliation: Departments of Ophthalmology (Ross Eye Institute) and Pathology & Anatomic Sciences, the State University of New York, Buffalo, NY, USA.
authors.forename: Dongjin
authors.initials: D
authors.lastname: Sung
authors.affiliation: Department of Pharmacology, SUNY Upstate Medical University, Syracuse, NY, USA.
authors.forename: Karen M
authors.initials: KM
authors.lastname: Haswell
authors.affiliation: Department of Biology, The University of Texas at San Antonio, San Antonio, TX, USA.
authors.forename: Andrew
authors.initials: A
authors.lastname: Tsin
authors.affiliation: Department of Pharmacology, SUNY Upstate Medical University, Syracuse, NY, USA; Upstate Cancer Research Institute, SUNY Upstate Medical University, Syracuse, NY, USA. Electronic address: ghoshd@upstate.edu.
authors.forename: Debashis
authors.initials: D
authors.lastname: Ghosh
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000737:chemistry* / Q000302:isolation & purification; D052160:Benzothiazoles / Q000378:metabolism; D002417:Cattle; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003460:Crystallization; D018360:Crystallography, X-Ray; D005136:Eye Proteins / Q000737:chemistry* / Q000302:isolation & purification; D016166:Free Radical Scavengers; D008786:Metmyoglobin / Q000378:metabolism; D010084:Oxidation-Reduction; D012160:Retina / Q000737:chemistry; D012177:Retinol-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D013451:Sulfonic Acids / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D000099214:Retinol-Binding Proteins, Interstitial
chemical_list: D000975:Antioxidants; D052160:Benzothiazoles; D005136:Eye Proteins; D016166:Free Radical Scavengers; D008786:Metmyoglobin; D012177:Retinol-Binding Proteins; D013438:Sulfhydryl Compounds; D013451:Sulfonic Acids; D000099214:Retinol-Binding Proteins, Interstitial; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid
keywords: X-ray structure; crystallography; interphotoreceptor matrix; interphotoreceptor-retinoid binding protein; photoreceptors; retina; retinoid binding protein; visual cycle