X-ray diffraction and ESR studies on armorphous melanin

By using X-ray diffraction and ESR (electron spin resonance) techniques, some physical properties of synthetic melanin, such as intrinsic local atomic structure, structure of adsorbed water and stable free radical content and the influence of water on these free radicals, were studied in detail. Using the corrected X-ray intensities of wet and dry samples, the difference intensity (wet-dry), which refers to the structure of the adsorbed water, appears roughly similar to bulk liquid water for the part that can be desorbed at 100°C. The remaining, or more tightly bound water, which comes off at 150°C and higher, seems similar to amorphous ice. Both Cu and Ag radiations were used to obtain the intensity data of a 150°C dried melanin sample. A model study of the intrinsic local atomic structure of dry melanin suggests a 'best' or most possible structure model, which has its i(K), the interference function, fit most successfully to the measured i(K) over the entire range from K = 1.5 A-1 to K = 19 A-1 . This model fit consisted of an array of planar connected monomers of from 4-8 members per layer with three layers stacked at about the graphite spacing of 3.4 A and included some disorder of layer orientations, such as rotations and shifting with respect to each other, and also a (maximum) 0.02A random tilting of the oxygen and nitrogen side atoms. This supergroup or 1 paracrystalline1 array is then assumed to represent a well-correlated piece of the fully connected 3-D melanin polymer. The ESR study shows a spin density and ESR line-width which increase with heating and pumping, and decrease with readsorbing water in a somewhat reversible fashion. Heating at 150°C and higher which drives out the small amount of more tightly bound water, caused the major increase of the ESR signal. Readsorption of water can not quench the entire ESR signal, but rather causes the signal to decrease slowly and to approach its original equilibrium value. The reversible part of the ESR signal, the difference ESR intensity of (dry-wet), was found to be quenched by water in a manner resembling a 2nd order rate process. The temperature dependence of the ESR intensities of both dry and wet samples shows that the paramagnetism follows the Curie Law with the dry sample having a greater slope. Changes of relaxation time due to water were relatively small. Considering the information we have obtained here, it seems that melanin is a random polymer composed of stacked- layer groups, has a relative large granule size, with trapped free radicals and bound water. The bound water that diffuses (or penetrates) into part (but not all) of the melanin granule induces free radicals to 'pair off' within that region. This 'macroscopic' melanin model which describes the free radical properties and also the influence of water on these radicals is expected to be associated with the biological functions of melanin.