Thiol-dependent antioxidant activity of interphotoreceptor retinoid-binding protein

Exp Eye Res. 2014 Mar;120:167-74. doi: 10.1016/j.exer.2014.01.002. Epub 2014 Jan 12.

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.

Keywords: X-ray structure; crystallography; interphotoreceptor matrix; interphotoreceptor-retinoid binding protein; photoreceptors; retina; retinoid binding protein; visual cycle.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antioxidants / chemistry*
  • Antioxidants / isolation & purification
  • Benzothiazoles / metabolism
  • Cattle
  • Chromatography, Affinity
  • Chromatography, Gel
  • Chromatography, Ion Exchange
  • Crystallization
  • Crystallography, X-Ray
  • Eye Proteins / chemistry*
  • Eye Proteins / isolation & purification
  • Free Radical Scavengers
  • Metmyoglobin / metabolism
  • Oxidation-Reduction
  • Retina / chemistry
  • Retinol-Binding Proteins / chemistry*
  • Retinol-Binding Proteins / isolation & purification
  • Spectrometry, Fluorescence
  • Sulfhydryl Compounds / chemistry*
  • Sulfonic Acids / metabolism
  • Tandem Mass Spectrometry

Substances

  • Antioxidants
  • Benzothiazoles
  • Eye Proteins
  • Free Radical Scavengers
  • Retinol-Binding Proteins
  • Sulfhydryl Compounds
  • Sulfonic Acids
  • interstitial retinol-binding protein
  • Metmyoglobin
  • 2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid