The role of zinc in pheomelanin synthesis. An EPR, LC/MS and chemical degradation assessment

Zinc levels in human hair are relatively high (up to 200 \uf06dg/g) suggesting a role in melanogenesis. We report herein a systematic investigation of the effects of zinc on the properties of synthetic pheomelanins prepared by oxidation of 5-S-cysteinyldopa (CD) with peroxidase/H2O2. For comparison a red hair pheomelanin obtained by a mild hydrolytic procedure was also investigated. The pigments were characterized by: a) EPR spectroscopy at X-band; b) alkaline hydrogen peroxide degradation followed by HPLC determination of benzothiazole-2-carboxylic acid (BTCA); and c) reverse phase HPLC coupled with UV or ESI(+)-MS detection. Pheomelanins prepared in the presence of Zn2+ (zinc pheomelanins) exhibited more distinct chromatographic patterns with some differences in product distribution depending on the amounts of oxidant used. The main components were identified by ESI-MS as benzothiazine dimers and trichochrome-related species. EPR spectra of pheomelanins showed a distinct three-line spectral feature characteristic for the pheomelanin semiquinonimine subunits. Saturation of pheomelanins with zinc ions at pH 5, or in situ irradiation in the resonant cavity with visible light, resulted in an enhancement of the intensity of the melanin EPR signals. Aerobic photoreactivity of the pheomelanins was also investigated by EPR-oximetry and EPR-spin trapping using DMPO as a spin trap. The highest photoinduced oxygen consumption was found for zinc pheomelanins, which was accompanied by the fastest photogeneration of superoxide anion. Our data indicate that zinc pheomelanin is significantly more photoreactive and when irradiated with visible light becomes substantially more reducing than pheomelanin synthesized in the absence of zinc. All these data will be comparatively presented and the role of zinc ions in directing CD oxidation and affecting the structure of the final pigment will be discussed