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B. J. Lassman, J. Griswold, D. Ghosh, F. Gonzalez-Fernandez; Purification and Potential Antioxidant Activity of Bovine IRBP. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5921.
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Interphotoreceptor retinoid-binding protein (IRBP) protects retinoids from isomeric and oxidative degradation (Crouch, et al 1992, Photochem Photobiol 56:251). IRBP is composed of "modules" each ~300 residues in length. A critical gap is that little is known about the structure of the full-length protein. Obtaining IRBP at the concentrations needed for X-ray crystallography has been problematic as the protein precipitates when concentrated above 3 mgs/ml.
Molar excesses of a thiol-based reducing agent, DTT were used to obtain bovine IRBP (bIRBP) in a soluble, stable, and active form, which yielded diffraction-quality crystals. Native bIRBP was purified by FPLC using a combination of ConA, ion exchange and size exclusion chromatography. Its ability to scavenge free radicals was compared to a known antioxidant, Trolox, a tocopherol analog. Antioxidant activity was assayed, using a peroxidase and H2O2 to produce a relatively stable, colored, free radical of ABTS (ABTS•). The addition of antioxidant produced a measurable decrease of ABTS• (absorbance at 750nm) proportional to the concentration of antioxidant (Miller, et al 1993, Biochem Soc Trans 21(2):95S).
bIRBP molecules aggregate, unfold, and denature irreversibly by cross-linking through free sulfahydryl groups, a process that was prevented by the addition of a thiol-based reducing agent, such as DTT. Non-thiol based reducing agents failed to provide similar protection. Also, IRBP demonstrated significant, concentration-dependent antioxidant activity corresponding to ~0.4 Trolox equivalents.
IRBP has a concentration-dependent free radical scavenging activity. bIRBP has free, exposed sulfhydryls capable of participating in cross-linking reactions. Recognition of this property allows concentration of IRBP without denaturing making possible ongoing crystallization trials, which have yielded large crystals and diffraction.
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