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Purification of the Components of Human Macular Carotenoid-Binding Complex, ,,, Rev A


Introduction
The macular region of the human retina is responsible for almost all color vision and most of the high resolution visual acuity. This part of the retina, known as the macula lutea, or yellow spot, accumulates approximately 200 ng of two closely-related compounds, lutein and zeaxanthin. These compounds are both dihydroxy-a-carotenes which differ in the position of one double bond in the -ionone ring. Neither of these compounds accumulates in any other human tissues and the macular is virtually devoid of the other major carotenoid, -carotene.

Isolation and purification of the proteins involved in the active transport and storage of carotenoids has been difficult. Since only primate eyes have maculae, source tissue was extremely limited in quantity and availability. The primate macula is only 56 mm in diameter and only about 0.4 mm thick, containing 25 mg of total protein. Protease activity is very high in maculae, owing to the high metabolic rate of this tissue, which hampers isolation of intact protein. The half life of the carotenoid-protein complex is about 12 hours and its intense yellow color allows it to be followed during the course of purification.


Purification
Four to six maculae were lysed by sonication in the cold in the presence of detergents and protease inhibitors, followed by centrifugation at 100,000 x g for 10 minutes. One ml of supernatant was applied to an UNO Q-1 column (Bio-Rad catalog # 720-0001) which was monitored at 280 and 436 nm; the latter wavelength being specific for carotenoid absorption (Figure 1). A yellow peak emerged at ab
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