Photochemistry and Photobiology, 2003, 78(2): 138–145 Photophysical Properties of Xanthophylls in Carotenoproteins from Human Retina { Helena H. Billsten 1 , Prakash Bhosale 2 , Alexander Yemelyanov y2 , Paul S. Bernstein 2 and Toma ´s ˇ Polı ´vka* 1 1 Department of Chemical Physics, Lund University, Lund, Sweden and 2 Moran Eye Center, University of Utah School of Medicine, Salt Lake City, UT Received 17 December 2002; accepted 22 May 2003 ABSTRACT The macula of the human retina contains high amounts of the xanthophyll carotenoids lutein and zeaxanthin [a mixture of (3R,39R)-zeaxanthin and (3R,39S-meso)-zeaxanthin]. Recently, it was shown that the uptake and the stabilization of zeaxanthin and lutein into the retina are likely to be mediated by specific xanthophyll-binding proteins (XBP). Here, we have used femtosecond pump–probe spectroscopy to study the dynamics of the S 1 state of these xanthophylls in xanthophyll- enriched and native XBP. The results from the native XBP and the enriched XBP were then compared with those for carotenoids in organic solvents and in detergent micelles. Steady-state and transient absorption spectra show that the incorporation of xanthophylls into the protein causes a redshift of the spectra, which is stronger for lutein than for zeaxanthin. The transient absorption spectra further indicate that a part of the xanthophylls remains unbound in the xanthophyll- enriched XBP. The transient absorption spectra of the native XBP prove the presence of both xanthophylls in native XBP. Although the S 1 lifetime of lutein does not exhibit any changes when measured in solution, micelles or XBP, we have observed the influence of the environment on the S 1 lifetime of meso- zeaxanthin, which has a longer (12 ps) lifetime in XBP than in solution (9 ps). The most pronounced effect was found for vibrational relaxation in the S 1 state, which is significantly slower for xanthophylls in XBP compared with micelles and solution. This effect is more pronounced for meso-zeaxanthin, suggesting a specific site of binding of this carotenoid to XBP. INTRODUCTION Carotenoids are some of the most abundant pigments found in nature. They are present in most organisms, including humans, but they can be synthesized only by plants and microorganisms (1). Although they are perhaps best known for their bright colors, they have well-documented multiple functions in nature. They serve as light-harvesting pigments in photosynthetic organisms, covering a region of the visible spectrum not accessible by (bacterio)chloro- phylls (2), and they protect against excessive light by regulating energy flow via singlet and triplet states of (bacterio)chlorophylls (3). In addition, they are known to be efficient quenchers of dangerous singlet oxygen and various reactive radicals by interrupting the chain of oxidative reactions (4). There is accumulating evidence that this antioxidative function is a key mechanism of protection against various diseases, including cancer, atherosclerosis and age-related macular degeneration (AMD), in humans (5). The diversity of carotenoid function is unmatched by any other class of natural pigments and is directly related to the unique spectroscopic properties resulting from the structure of the carot- enoid molecule. The central pattern repeated in all carotenoids is a backbone consisting of alternating single and double carbon bonds that form a conjugated p-electron system responsible for most of the spectroscopic properties of carotenoids. The bright yellow-orange color of carotenoids is caused by a strong transition to the second excited state S 2 occurring in the blue part of the visible spectrum. The energy of this transition is given by the number of conjugated C‚C bonds; the shorter the carotenoid, the higher the energy of the S 0 –S 2 resonance (6). Because of symmetry (carotenoids belong to the C 2h symmetry group), the transition to the lowest-excited S 1 state is forbidden, thereby restricting observable absorption or fluorescence of this state (7). After being promoted to the S 2 state (or other higher allowed states), a carotenoid molecule will quickly (,300 fs) relax to the S 1 state (8,9), the lifetime of which is again determined by the conjugation length of the carotenoid. The S 1 lifetime can vary from 300 ps for short conjugated chains to ;1 ps for the longest (10). Large amounts of the two xanthophylls (oxygenated carote- noids), lutein and zeaxanthin, are accumulated in the yellow spot (macula lutea) of the human retina (11–13). About 50% of the total amount of the xanthophylls in the retina is concentrated in the macula (13), where zeaxanthin dominates over lutein by a ratio of 2:1 (13,14). At the center of the macula (the fovea), zeaxanthin is actually a 50:50 mixture of dietary (3R,39R)-zeaxanthin and nondietary (3R,39S-meso)-zeaxanthin, presumably a metabolite of dietary lutein or zeaxanthin (15,16). The concentration of xanthophylls increases progressively toward the center of the {Posted on the website on 5 June 2003 *To whom correspondence should be addressed at: Department of Chem- ical Physics, Lund University, P.O. Box 124, 221 00 Lund, Sweden. Fax: 46-46-2224119; e-mail: tomas.polivka@chemphys.lu.se  Current address: AMC Cancer Research Center, Denver, CO, USA. Abbreviations: AMD, age-related macular degeneration; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; ESA, ex- cited-state absorption; LHC, light-harvesting complexes; XBP, xantho- phyll-binding proteins. Ó 2003 American Society for Photobiology 0031-8655/03 $5.00 þ0.00 138