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Preejith P. Vachali, Mary A. Garlipp, Federico Gonzalez-Fernandez, Paul S. Bernstein; Carotenoids as Possible Interphotoreceptor Retinoid-binding Protein (IRBP) Ligands: A Surface Plasmon Resonance (SPR)-based Study. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3640.
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The transport of macular pigment carotenoids across the interphotoreceptor matrix may be mediated by a protein in the matrix, which could help as a carrier of these hydrophobic molecules from RPE/choroid to the retina. Interphotoreceptor retinoid-binding protein (IRBP) is a candidate because it is abundant in the matrix and binds hydrophobic molecules such as visual-cycle retinoids and fatty acids. SPR-based biosensors have drawn attention in recent years because of their ability to analyze protein-ligand interactions rapidly and sensitively. In this study, we explored the binding interactions of various carotenoids with IRBP.
Bovine IRBP was purified to homogeneity by a combination of Con-A affinity, ion exchange, and size-exclusion chromatography. The protein at 10 µg/mL in 10 mM sodium acetate, pH 4.5-5.0 was immobilized on the sensor chip surface using standard amine-coupling. Each of the five carotenoids was dissolved in sucrose monolaurate (SML) (Mitsubishi Chemicals, Japan) to achieve a high concentration, and 10 mM PBS (pH 7.4) with 0.01% Triton X-100 and 0.4 mM SML was used as the running buffer. The carotenoid concentration series was prepared as two-fold dilutions in running buffer. Typically, the carotenoid concentration series spanned 0.01-10 µM. All SPR measurements were recorded on a SensiQ SPR instrument (ICx Nomadics) at 25ºC.
The binding responses were analyzed using Qdat Software (ICx Nomadics). Out of the five carotenoids tested with bovine IRBP, lutein showed an affinity of 1.26 µM, while (3R, 3’R)-zeaxanthin and (3R, 3’S)-meso-zeaxanthin exhibited somewhat lower affinities with KD values of ~1.82 and 2.4 µM, respectively. Beta-carotene had a relatively high affinity for IRBP with a KD value of 0.873 µM. Astaxanthin showed the lowest affinity with a KD value of 2.77 µM.
The results demonstrate that IRBP is able to bind carotenoids, although with relatively low specificity. Biosensor technology is useful to study carotenoid affinities with target proteins with high reliability and reproducibility. Biosensor-based SPR assays are promising approaches for the study of functional roles of IRBP, and they can likely be extended to other physiological ligands such as retinoids and fatty acids.
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