Ligand-binding characterization of xanthophyll carotenoids to solubilized membrane proteins derived from human retina

The macula of the human retina contains extraordinarily high concentrations of lutein and zeaxanthin, xanthophyll carotenoids that appear to play an i mportant role in protecting against age-related macular degeneration, the l eading cause of blindness among the elderly. It is likely that the uptake a nd stabilization of these carotenoids is mediated by specific xanthophyll-b inding proteins. In order to purify and characterize such a binding protein . a carotenoid-rich membrane fraction derived from human macula or peripher al retina was prepared by homogenization, differential centrifugation, and detergent solubilization. Further purification was carried out using ion-ex change chromatography and gelfiltration chromatography coupled with continu ous photodiode-array monitoring for endogenously associated xanthophyll car otenoids. The most highly purified preparations contained two major protein bands at 25 and 55 kDa that consistently co-eluted with endogenous lutein and zeaxanthin. The visible absorbance spectrum of the binding protein prep aration closely matches the spectral absorbance of the human macular pigmen t, and it is bathochromically shifted about 10 nm from the spectrum of lute in and zeaxanthin dissolved in organic solvents. Binding of exogenously add ed lutein and zeaxanthin is saturable and specific with an apparent K-d of approximately 1 muM. Canthaxanthin and p-carotene exhibit no significant bi nding activity to solubilized retinal membrane proteins when assayed under identical conditions. Other potential mammalian xanthophyll-binding protein s such as albumin, tubulin, lactoglobulin and serum lipoproteins possess on ly weak non-specific binding affinity for carotenoids when assayed under th e same stringent binding conditions. This investigation provides the first direct evidence for the existence of specific xanthophyll-binding protein(s ) in the vertebrate retina and macula. The possible roles of xanthophyll-bi nding proteins in normal macular function and in the pathogenesis of age-re lated macular degeneration remain to be elucidated. (C) 2001 Academic Press .