April 2009
Volume 50, Issue 13
ARVO Annual Meeting Abstract  |   April 2009
Bovine IRBP Functions in vitro as a Thiol-Based Antioxidant
Author Affiliations & Notes
  • B. J. Lassman
    Ross Eye Institute, Ophthalmology, SUNY, Buffalo, New York
    V.A. Medical Center, Buffalo, New York
  • J. Griswold
    Hauptman-Woodward Institute, Buffalo, New York
  • F. Gonzalez-Fernandez
    Ross Eye Institute, Ophthalmology, SUNY, Buffalo, New York
    V.A. Medical Center, Buffalo, New York
  • D. Ghosh
    Hauptman-Woodward Institute, Buffalo, New York
    Roswell Park Cancer Institute, Buffalo, New York
  • Footnotes
    Commercial Relationships  B.J. Lassman, None; J. Griswold, None; F. Gonzalez-Fernandez, None; D. Ghosh, None.
  • Footnotes
    Support  NIH Grant EY009412, RPB Challenge Grant, VA Merit Review
Investigative Ophthalmology & Visual Science April 2009, Vol.50, 4504. doi:
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      B. J. Lassman, J. Griswold, F. Gonzalez-Fernandez, D. Ghosh; Bovine IRBP Functions in vitro as a Thiol-Based Antioxidant. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4504.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : Interphotoreceptor retinoid-binding protein (IRBP) shuttles retinoids in the visual cycle and has been implicated in preserving the isomeric and oxidative state of retinol (Crouch, et al 1992, Photochem Photobiol 56:251). The mechanism of this protective oxidative function is unknown. We have shown that molar excesses of a thiol-based reducing agent dithiothreitol (DTT) are required to purify bovine IRBP (bIRBP) in a soluble, stable and active form (Lassman, et al 2008, Invest. Ophthalmol. Vis. Sci. 49: E-Abstract 5921). Protein thiols are known to be catalytic centers for antioxidant activities. Free thiols in IRBP could serve to protect all-trans retinol from oxidation by reactive oxidative species. Here, we test the hypothesis that bIRBP has free thiol-based antioxidative property.

Methods: : Native bIRBP is purified to homogeneity in the presence of DTT. The antioxidant activity is assayed spectrophotometrically using the absorbance of the free radical ABTS°+ of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). The ABTS°+ absorbance at 750nm decreases with increasing concentration of bIRBP. bIRBP’s ability to scavenge free radicals is compared to thioredoxin, a thiol-based antioxidant, in the presence and absence of N-ethylamalimide (NEM). NEM covalently modifies free and accessible thiols. Mass spectroscopy is used to identify the thiols that are covalently modified.

Results: : bIRBP demonstrates a concentration-dependent antioxidant activity. The activity is greater than that of thioredoxin. When incubated with the thiol-alkylating agent NEM, bIRBP is 10% less active in reducing ABTS°+ compared to the unmodified bIRBP (p <0.007). Covalent modification by NEM is confirmed by mass spectroscopy. Thiols modified by NEM are: Cys304, Cys603, and Cys1051.

Conclusions: : Current study reveals that the antioxidant activity of bIRBP is at least in part thiol-dependent. The activity is impaired by the thiol-alkylating agent NEM that modifies 3 Cys residues possibly involved in the reduction process. Elucidation of relative locations of these residues with respect to the retinol-binding site will be crucial in ascertaining the biochemical and physiological relevance of the antioxidative roles of IRBPs.

Keywords: antioxidants • retina 

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