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E Gonzalez-Fernandez, Y-J Shiu, S-H Lin, F Gonzalez-Fernandez; Xanthophylls as Possible IRBP Ligands . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3606.
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Purpose: The sequestration of Xanthophylls in the neural retina from the choroidal circulation requires movement of the Xanthophyll across the interphotoreceptor matrix before the Xanthophyll can finally bind to targets in the retina. It is plausible that a protein in the matrix participates in the movement of Xanthophylls from the RPE/choroid. Interphotoreceptor retinoid-binding protein (IRBP) is a candidate for such a protein because it is found in the matrix and is able to bind hydrophobic molecules particularly visual-cycle retinoids and fatty acids. As a first step to evaluate the possible role of IRBP in Xanthophyll delivery, we here begin to characterize the binding of Xanthophylls to IRBP in vitro. Methods: Absorption spectroscopy was used to look for evidence of a direct interaction between lutein and native bovine IRBP or recombinant Xenopus IRBP. We compared the absorption spectrum (230 to 600 nm) of the following three solutions in phosphate buffered saline: lutein alone, IRBP alone, and IRBP in the presence of lutein (lutein/IRBP). Purified lutein and IRBPs were used at concentrations ranging from 1 to 4 micromolar. Results: The absorbance spectrum of leutin/IRBP showed a major peak at 280 nm, which is mainly due to the absorbance of the protein, and a smaller peak at 390 nm, which corresponds to the absorbance of leutin in PBS. The increase in the absorbance at 280 nm of leutin/IRBP compared to that of IRBP alone is simply the sum of the absorbance of leutin alone plus that of IRBP alone. However, at 390 nm the absorbance of leutin/IRBP was markedly greater than the sum of IRBP alone and of lutein alone. The IRBP/lutein complex appeared to be stable in that we saw no time dependent change in absorbance. Conclusion: The fact that the absorbance of IRBP/lutein at 390 nm was more than the sum of the absorbance of either lutein or IRBP alone indicates that IRBP is able to enhance the absorbance of lutein. This observation suggests that IRBP is able to bind lutein in a hydrophobic site. Ongoing studies are extending these experiments to photometric and fluorometric titrations, which will determine whether the enhancement is saturable and if so the stoichiometry and Kd of the interaction. E. Gonzalez-Fernandez, None.
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