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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. 2008;49(13):4970.
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Surface plasmon resonance (SPR)-based biosensors have drawn attention in recent years because of their ability to analyze protein-ligand interactions rapidly and sensitively. In this study, binding interactions of GSTP1, a xanthophyll-binding protein in human macula, and human serum albumin (HSA) with five different carotenoid ligands were explored.
HSA and GSTP1 were immobilized on sensor chips (CM5 modified) using standard amine-coupling protocols to obtain a surface density of 5 - 10K RU. The carotenoids were tested in 2-fold serial dilutions. The running buffer contained 50mM Tris-CHAPS (8mM) pH 7.8. Each compound series was assayed 3 times, and assays were performed at 4 degrees. All binding studies were completed in 30 seconds.
Out of the five carotenoids tested, beta-carotene showed a very high affinity toward HSA with a Kd of 12.2 µM, followed by zeaxanthin and lutein with Kd of 22.6 and 60.1 µM, respectively. GSTP1 showed a high affinity toward zeaxanthin with a Kd of 26.9 µM followed by beta-carotene at 43.2 µM and lutein at 73 µM. GSTP1 and HSA showed a Kd of 163 µM and 151 µM, respectively for meso-zeaxanthin. Astaxanthin did not show a significant affinity towards either HSA or GSTP1.
The results demonstrate that biosensor technology can be employed to study carotenoid affinities with target proteins reliably and reproducibly. This rapid method could replace classical equilibrium binding experiments which require much more time and labor; however, differences in relative and absolute Kd values between these two methods still need to be explored, and other xanthophyll-binding proteins need to be examined. Biosensor-based assays should facilitate further study of the functional roles of xanthophyll-binding proteins in the human retina.
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