Dark- or Light-Colored Melanins: Generating Pigments Using Fe2+ and H2O2

Abstract

We have studied the formation of melanin-like pigments from catechol or pyrogallol and a wide range of other phenolic compounds using Fe<sup>2+</sup> and H<sub>2</sub>O<sub>2</sub>. Combining UV_Vis spectroscopic measurements and size-exclusion chromatography analyses we evaluated the impact of the intensity of the oxidation reaction by varying the concentration of H<sub>2</sub>O<sub>2</sub> present in the reaction mixtures. All compounds tested, except tyrosine, reacted readily leading to mixtures that were black, brown or yellow-orange in color. For many compounds tested, the use of increasing concentrations of H<sub>2</sub>O<sub>2 </sub>resulted in either precipitation of the pigment or the formation of a soluble, lighter-colored pigment. With catechol or pyrogallol as model compounds, and using different concentrations of H<sub>2</sub>O<sub>2</sub>, several materials were synthesized, purified and dried. The physic-chemical properties of these materials were compared to the properties of melanin-like pigments synthesized from the same precursors using air-oxidation in an alkaline environment. For both precursors, a distinct chemical change, as judged from FT-IR spectroscopy, was introduced in the melanin structures when using H<sub>2</sub>O<sub>2</sub> as the oxidizing agent and the relative intensity of this distinct signal strengthened with increasing concentration of H<sub>2</sub>O<sub>2</sub> used in the reaction. In general, our results suggest that depending on the precursor molecule and the intensity of the oxidizing reaction conditions involved, light- or dark-colored melanin-like pigments can be generated. This may be an important factor when evaluating the visible outlook of histological or archeological specimens: the presence of a lighter color or the absence of a dark color may not necessarily mean the absence of melanin-like biomolecules.

Light- or Dark-Colored, L-DOPA-Based Melanins

“…Similar estimates of  for MN-like pigments generated from catechol or pyrogallol ranged between 1.25 and 6.35 (mg/mL) -1 depending on the reaction conditions used to generated the pigments. 15 The results presented in Figure 2, panel B, indicate that pigment B exhibited very little fluorescence emission. Pigment A exhibited fluorescence emission that appeared to increase with increasing concentration of pigment present.…”

“…Such retention times are in line with those reported for MN-like pigments generated from other phenolic or catecholic compounds. [14][15][16]23 For each of the purified pigment materials a stock solution at 1mg/mL was prepared in distilled water. Figure 1 presents a photograph of these stock solutions.…”

“…Such an absorbance shoulder is weakly present in the spectra of pigment B and absent in the spectra of pigment C. This distinct feature in the FT-IR spectra has been observed for other MN-like pigments generated through Fe 2+ /H 2 O 2 -mediated oxidation. [15][16] An absorbance at that wavenumber can be attributed to the presence of a carbonyl entity (C=O); either from a ketone or a carboxylic acid functionality. 26 This feature in the FT-IR spectra may be due to a chemical reaction whereby a phenolic functional group was converted into a ketone functional group or whereby an aromatic unit was opened and two carboxylic acids were generated into the structure.…”

“…This has been observed before for MNlike pigments generated from other precursors. 15 For the study of the interleukin release from immune cells, the three preparations of each type of pigment were pooled together and stock solutions were prepared in distilled water. Figure 4, panels A and B respectively, present the average (± standard deviation; n=3) IL-1 and IL-6 released from immune cells in the presence of increasing concentration of pigments A, B or C. The results presented in Figure 4 indicate that the various pigments generated from L-DOPA are capable of enhancing the IL-1 and IL-6 release from a mixture of immune cells.…”

“…13 In previous reports we have presented our observations regarding the synthesis of MN-like pigments from a multitude of precursors using Fe 2+ and H 2 O 2 as the oxidizing reagents and observed that light-or dark-colored materials could be generated depending on the intensity of the oxidizing reaction conditions. [14][15][16] We have expanded these earlier observations by evaluating the generation of MN-like pigments from L-DOPA using air-oxidation in an alkaline environment and Fe 2+ /H 2 O 2 -mediated oxidation. In addition, we have explored the possibility that the physic-chemically distinct materials generated from L-DOPA could have a different effect on the interleukin release from immune cells.…”