Structural differences in unbleached and mildly-bleached synthetic tyrosine-derived melanins identified by scanning probe microscopies

Using scanning tunneling microscopy (STM), we have imaged two types of mildly-bleached, synthetic tyrosine-derived melanins for comparison with the unbleached melanin from which they were prepared. These mildly-bleached melanins were generated by mild oxidation of the unbleached melanin, using either basic hydrogen peroxide or air/light. The unbleached melanin, and two mildly-bleached melanins, were independently deposited from very dilute tetrahydrofuran (THF) solutions onto highly oriented pyrolytic graphite (HOPG) substrate for STM imaging. Lateral dimensions (23 A, average of two directions) of structures from each of the three samples showed no differences. However, structures from both mildly-bleached melanins showed similar dramatic decreases (from approximately 15 A to approximately 5 A) in their STM-measured apparent heights, compared with structures from the unbleached melanin sample. These STM observations are compatible with structural models for unbleached and mildly-bleached melanins, incorporating a three-dimensional structure for unbleached melanin composed of multi-layered, pi-pi-stacked, carboxylic and amino variants of polyaromatic polymeric sheets. The STM-observed decrease in apparent heights after mild oxidation, which we associate with a change in stack height, has been confirmed by experiments using tapping mode atomic force microscopy (TM-AFM) for the unbleached and mildly-hydrogen-peroXide-bleached melanins (from approximately 14 A to approximately 6 A). In these TM-AFM experiments, the melanins were deposited directly onto magnesium cation-treated glass substrates in contact with methanolic solutions of each of the melanins. We interpret our mild-bleaching results as an oxidative conversion of the multi-layered, stacked sheets of mainly carboxylic and amino variants of polyquinhydrone-like moieties, to largely de-stacked, mildly-bleached melanin sheets. These oxidized and, hence, electron-deficient sheets should not readily form multi-layered, pi-pi interacting stacks, but instead appear to be either single-layer polyquinone sheets or, at most, double-layer polyquinhydrone sheets. The effects of such de-stacking on in vivo melanin photoprotection, and structural similarities between melanin derived from natural sources and the synthetic melanin samples used in this work are discussed.