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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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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.
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.
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.
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.
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