( Introduction ...)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 about 12% B containing 78 species by SDS-PAGE (Figure 2, lane 3) with molecular weights ranging from 2575 kDa. These fractions were pooled, concentrated and applied to an UNO S-1 column (Bio-Rad catalog # 720-0021). The strongly colored peak eluted from this column at 40% B and had about 4 components by SDS-PAGE (Figure 2, lane 2 and Figure 3).

Final purification was performed by isoelectric focusing using the Mini Rotofor Cell (Bio-Rad catalog # 170-2988) and BioLytes covering the pH range of 310 (Bio-Rad catalog # 163-1112, 163-1172 and 163-1182). The experiment was scaled up to 28 maculae yielding 2 ml of highly purified, bright yellow carotenoids which were injected into the 18 ml Rotofor core. After focusing for 6 hours at 10 W constant power, 2 colored zones were isolated from fractions with pH values of about 7.0 and 9.0. Fractions were concentrated and analyzed by SDS-PAGE (Figure 4). HPLC analysis indicated that the fraction (Figure 4, lane 6) with a pH of 8.5 contained over 40% of the initial quantity of macular carotenoids and the SDS-PAGE indicated one major protein component.

The 70 kDa protein in this fraction appears analogous to a recently described -carotene binding protein which required a four step, more complex isolation procedure.1 The proteins in fraction 6 are currently undergoing sequence studies.

Acknowledgment
Data courtesy of Dr. N. Balashov, of the John A. Moran Eye Center and Department of Opthamology, University of Utah, Salt Lake City, UT 84112.

Reference
1. Rao, M. N., Ghosh, P., and Lakshman, M. R., J. Biol. Chem., 26, 2445524460 (1997).

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