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P. P. Vachali, B. Li, P. S. Bernstein; Surface Plasmon Resonance (SPR) Studies of the Interactions of Carotenoids and Their Binding Proteins. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1291.
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SPR-based biosensors have drawn attention in recent years because of their ability to analyze protein-ligand interactions rapidly and sensitively. The main advantages of this technology are that assays can be performed directly and that the kinetics of analyte-target interaction can be easily determined. In this study, we explored the binding interactions of a recombinant CBP, a new member of the steroidogenic acute regulatory (StAR) protein family from silk worm (Bombyx mori) gland with significant homology to many human StAR proteins, and GSTP1 a xanthophyll-binding protein in human macula with different carotenoid ligands.
Purified rCBP and GSTP1 were immobilized on sensor chip (a planar mixture of hydroxyls and carboxyls in a 4:1 ratio of hydroxyl to carboxyl) using standard amine-coupling protocols to obtain a surface density of 1000-1200 RU. The carotenoids were tested in 2-fold serial dilutions. The running buffer contained 50 mM Tris pH 8 and 5% DMSO. All surface plasmon resonance measurements were recorded on a SensiQ SPR instrument (Icx Nomadics) at a controlled temperature of 25ºC.
The rCBP binding responses were analyzed using Qdat Software (Icx Nomadics). Lutein showed a high affinity towards rCBP with a KD of 130 nM. The rCBP-lutein binding result is in excellent agreement with the earlier reports using Protein A -sepharose-rCBP pull down assay. The GSTP1 binding responses were analyzed with a heterogenous binding model using GraphPad Prism software. Out of the five carotenoids tested with GSTP1, (3R, 3’R)-zeaxanthin showed the highest affinity toward GSTP1 with a KD of 52.9 nM, followed by (3R, 3’S)-meso-zeaxanthin and astaxanthin with KD of 55.2nM and146 nM respectively. Zeaxanthin, meso-zeaxanthin and astaxanthin showed a second low affinity site with a KD of 5.29 µM, 5.17 µM and 4.13 µM respectively. Beta-carotene and lutein did not show a significant affinity towards GSTP1.
The results demonstrate that biosensor technology can be employed to study carotenoid affinities with target proteins reliably and reproducibly. The GSTP1 results confirm our published findings that GSTP1 is the physiologically relevant binding protein for zeaxanthin in the human macula. We recently reported a Human Retinal Lutein binding protein (HR-LBP), which share many features similar to CBP. Biosensor-based assays should facilitate further study of the functional roles of xanthophyll-binding proteins in the human retina.
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