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P. Bhosale, P.S. Bernstein; Induced and Altered Circular Dichroism of Carotenoids Bound to Mammalian Xanthophyll Binding Proteins . Invest. Ophthalmol. Vis. Sci. 2003;44(13):404.
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Purpose: The human retina contains high amounts of the xanthophyll carotenoids lutein [(3R,3&rquot;R, 6&rquot; R)-ß,ε-carotene-3,3&rquot; diol] and zeaxanthin [a mixture of (3R,3&rquot;R)-ß,ß-carotene-3,3&rquot; diol and (3R,3&rquot;S-meso)-ß,ß-carotene-3,3 &rquot; diol]. They are likely to play an important role in protecting the eye against light-induced oxidative damage implicated in the pathogenesis of age-related macular degeneration. Their uptake and stabilization in the retina is thought to be mediated by specific xanthophyll binding proteins (XBP). We have examined the interactions of human retinal XBP and other mammalian xanthophyll carriers and transporters with their carotenoid ligands by circular dichroism (CD) spectroscopy. Methods: Xanthophyll-protein interactions were studied for their chiral characteristics using CD on detergent-solubilized preparations of XBP from human peripheral retinas in comparison with tubulin, high-density lipoprotein (HDL), low-density lipoprotein (LDL), albumin, and ß-lactoglobulin. Exogenous xanthophyll carotenoid ligands were added to the proteins in tetrahydrofuran (THF) and incubated overnight at 4°C. Unbound ligands were extracted into hexane. All CD experiments were performed at 4°C using suitable controls. Results: (3R, 3&rquot;S-meso)-zeaxanthin, an optically inactive nondietary xanthophyll carotenoid, exhibited a strong induced CD spectrum in association with XBP. Dietary (3R,3'R)-zeaxanthin and dietary (3R,3'R,6'R)-lutein displayed substantial alterations in their CD spectra in association with XBP. The other mammalian proteins studied also induced or altered CD spectra of bound carotenoids to varying extents, but always in a distinctly different manner relative to XBP. Conclusions: These results indicate that CD spectroscopy is an excellent method to probe specific interactions of mammalian xanthophyll binding proteins with their carotenoid ligands. Human retinal XBP exhibits a unique ensemble of CD spectra with its physiological ligands, yielding a CD spectral signature of this important ocular protein that can be compared with CD spectra of candidate proteins for human XBP identified from mass spectral sequencing.
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