May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Purification and Potential Antioxidant Activity of Bovine IRBP
Author Affiliations & Notes
  • B. J. Lassman
    Ophthalmology, SUNY at Buffalo, Buffalo, New York
    Veterans Affairs Medical Center, Buffalo, New York
  • J. Griswold
    Hauptman-Woodward Institute, Buffalo, New York
  • D. Ghosh
    Hauptman-Woodward Institute, Buffalo, New York
    Roswell Park Cancer Institute, Buffalo, New York
  • F. Gonzalez-Fernandez
    Ophthalmology, SUNY at Buffalo, Buffalo, New York
    Veterans Affairs Medical Center, Buffalo, New York
  • Footnotes
    Commercial Relationships  B.J. Lassman, None; J. Griswold, None; D. Ghosh, None; F. Gonzalez-Fernandez, None.
  • Footnotes
    Support  R01 EY09412; VA Merit Review
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 5921. doi:https://doi.org/
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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. doi: https://doi.org/.

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

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Abstract

Purpose: : 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.

Methods: : 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).

Results: : 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.

Conclusions: : 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.

Keywords: antioxidants • retina 
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