Photochemistry and Photobiology, 2003, 77(2): 115–120 Structural Studies of Bleached Melanin by Synchrotron Small-angle X-ray Scattering { Kenneth C. Littrell 1 , James M. Gallas* 2 , Gerry W. Zajac 3 and Pappannan Thiyagarajan 1 1 Intense Pulsed Neutron Source, Argonne National Laboratory, 9700 Cass Avenue, Argonne, IL; 2 Department of Physics and Astronomy, University of Texas at San Antonio, Rt 10 and 1604, San Antonio, Texas and 3 BP Research Center, P.O. Box 3011, Naperville, IL Received 20 June 2002; accepted 10 November 2002 ABSTRACT Small-angle X-ray scattering was used to measure the effects of chemical bleaching on the size and morphology of tyrosine- derived synthetic melanin dispersed in aqueous media. The average size as measured by the radius of gyration of the melanin particles in solution, at neutral to mildly basic pH, decreases from 16.5 to 12.5  with increased bleaching. The melanin particles exhibit scattering characteristic of sheet-like structures with a thickness of approximately 11  at all but the highest levels of bleaching. The scattering data are well described by the form factor for scattering from a pancake- like circular cylinder. These data are consistent with the hypothesis that unbleached melanin, at neutral to mildly basic pH, is a planar aggregate of 6- to 10-nm-sized melanin proto- molecules, hydrogen bonded through their quinone and phenolic perimeters. The observed decrease in melanin par- ticle size with increased bleaching is interpreted as evidence for deaggregation, most probably the result of oxidative dis- ruption of hydrogen bonds and an increase in the number of charged, carboxylic acid groups, whereby the melanin aggregates disassociate into units composed of decreasing numbers of protomolecules. INTRODUCTION Melanin is the pigment that imparts color to the hair, skin and eyes in humans and animals (1,2). It filters the wavelengths of light roughly in proportion to their ability to cause actinic damage to tissue (3,4) and is able to scavenge and neutralize a variety of free radicals (5), proving its role in the highly evolved, photobiological protection system. The colors of melanin range from black through brown and red to yellow and are dependent on the choice of precursors and the conditions of synthesis (6). Color changes can be induced in melanin by the addition of bleaching or oxidizing agents such as hydrogen peroxide (7). The general phenomenon of peroxide or oxidative bleaching of dyes and its chemical basis are of broad interest to many researchers. The more specific phenomenon of peroxide bleaching is of particular interest in melanin research because it influences color, solubility and antioxidant behavior. The phenomenon of bleaching in melanin has important consequences for its complex biofunctionality. For example, melanin’s ability to function as a redox polymer is reduced after bleaching, lessening its capacity to deactivate poten- tially harmful free radicals (8). In addition, the optical absorbance of bleached or yellow melanin is markedly lower in the red end of the spectrum, where such a change may affect its photoprotective function (9). Relating melanin’s biofunctionality to its structure is an active area of research (10), and the study of this relationship with regard to bleaching represents a research direction of great practical importance. Such studies may also prove important in resolving the key issues of the size and structure of functionally relevant melanin particles. Direct imaging of melanin particles on nanometer-size scales has been done previously using scanning tunneling microscopy (STM) in studies of synthetic tyrosine-derived melanin deposited onto highly oriented pyrolitic graphite crystals from well-dispersed tetrahydrofuran solutions (11). In this work, only the smallest uniformly sized particles were imaged. These particles have lateral dimensions that were nominally in the range of 15–20 A ˚ and heights of 10–15 A ˚ . These structures were subsequently related to an extensively analyzed structural model developed to explain the wide-angle X-ray scattering (WAXS) data from melanin powders (12,13). This model, illustrated in Fig. 1, describes a ‘‘local structure’’ consisting of five to seven monomers of 5-6 indol- equinone arranged in a plane to form a layered system with 4–5 layers stacked 3.4 A ˚ apart. This local, ‘‘homo-polymeric’’ structure corresponds roughly to the nanometer-sized images observed by STM in both size and shape, suggesting that this structure is the actual melanin ‘‘protomolecule’’ or fundamental unit. Miyake et al. (14) used small-angle X-ray scattering (SAXS) to determine the sizes of unbleached melanins derived from precursors of various origins, including 3,4-dihydroxyphenylala- nine (L-Dopa), dopamine and natural melanins (14). These sizes, reported as radii of gyration (R g ) values, ranged from 15 to 50 A ˚ . In these studies, no attempt was made to study the state of aggregation of the unbleached melanin or to determine the size and shape of completely deaggregated melanin. However, the R g value calculated for the melanin protomolecule described above is only 5 A ˚ , suggesting that the experimental R g values determined by {Posted on the website on 25 November 2002. *To whom correspondence should be addressed at: Department of Physics and Astronomy, University of Texas at San Antonio, Rt 10 and 1604, San Antonio, TX 78249, USA. Fax: 210-458-4919; e-mail: jgallas@utsa.edu Abbreviations: AFM, atomic force microscopy; L-Dopa, dihydroxypheny- lalanine; R g , radii of gyration; SAXS, small-angle X-ray scattering; STM, scanning tunneling microscopy; WAXS, wide-angle X-ray scattering. Ó 2003 American Society for Photobiology 0031-8655/01 $5.0010.00 115