Degree of polymerization of 5,6‐dihydroxyindole‐derived eumelanin from chemical degradation study

Eumelanin is a brown‐black pigment comprising 5,6‐dihydroxyindole (DHI) and its 2‐carboxy derivative (DHICA), but the detailed structure of eumelanin is unclear. Chemical degradation is a powerful tool for analyzing melanin. H₂O₂ oxidation degradation of eumelanin affords pyrrole‐2,3,5‐tricarboxylic acid (PTCA) and pyrrole‐2,3‐dicarboxylic acid (PDCA). The ratio of PDCA to PTCA provides information about the eumelanin structure. In this article, we propose simple equations on the basis of previous experimental results on dimer yields for evaluating the yields of PTCA and PDCA from any DHI oligomers. Assuming the chemical disorder model of DHI‐melanin, we solve an equation where a theoretical expression for the ratio of PDCA to PTCA is set to the corresponding experimental value to obtain a plausible Poisson distribution of DHI oligomers. The results demonstrate that the main contributors to DHI‐melanin are tetramers and pentamers as shown by the mass spectrometry.     

Composition of Mammalian Eumelanins: Analyses of DHICA-Derived Units in Pigments From Hair and Melanoma Cells

The proportions in which two eumelanin monomers, namely 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and 5,6-dihydroxyindole (DHI), compose the eumelanin polymer are believed to determine properties of the pigment including its color. These proportions are, however, not well elucidated for naturally occurring eumelanins, largely because of methodological difficulties. In this study we estimate the content of DHICA-derived units in mammalian eumelanins using a combination of two analytical techniques: 1) quantitation of DHICA-derived eumelanin by measuring the yield of pyrrole-2,3,5-tricarboxylic acid (PTCA index) and 2) spectrophotometrical quantitation of total (DHI + DHICA) eumelanin at 350 nm (A₃₅₀ index). The ratio of PTCA/A₃₅₀ measured for melanins synthesized from DHI and DHICA mixed in various molar proportions correlates well with the content of DHICA in synthetic polymers. Using this relationship as a standard curve we estimated the proportion of DHICA-derived units in mammalian eumelanins from hair and melanoma cells and found it to be much higher in rodent pigments (58.8%-98.3%; two species, mouse and hamster were examined) as compared to human eumelanins (19.2%-41.8%; one Caucasian and one Oriental individual were examined). No relationship between proportion of DHICA-derived units in eumelanin and hair color is found. The latter seems to be determined predominantly by the ratio of pheo- to eumelanin synthesis.     

Chemical Characterization of Melanins in Sheep Wool and Human Hair

The color of hair and wool in mammals and feathers in birds is mostly determined by the quantity and quality of melanins that are synthesized in follicular melanocytes and transferred to keratinocytes. There are two chemically distinct types of melanin pigments: the black to brown eumelanins and the yellow to reddish pheomelanins. Melanins in sheep wool and human hair of various colors were characterized by HPLC methods to estimate 5,6-dihydroxyindole-2-carboxylic acid (DHICA)-derived units in eumelanins and benzothiazine units in pheomelanins. Melanins were also characterized by spectrophotometric methods after differential solubilization in alkalies. It was demonstrated that 1) black wool in Asiatic sheep contains eumelanin with the DHICA content similar to black mouse melanin, while black to brown melanins from human hair contain much lower ratios of DHICA-derived units, comparable to the slaty mutation in mice, 2) dark brown to brown hair in human contains eumelanin whose chemical properties are indistinguishable from those of black hair, 3) dark red wool and red human hair contain pheomelanic pigments whose chemical properties are rather different from those of yellow pheomelanins in mice, and 4) light brown, blonde, and red hairs in human can be differentiated from each other with this methodology.     

Unexpected impact of esterification on the antioxidant activity and (photo)stability of a eumelanin from 5,6‐dihydroxyindole‐2‐carboxylic acid

To inquire into the role of the carboxyl group as determinant of the properties of 5,6‐dihydroxyindole melanins, melanins from aerial oxidation of 5,6‐dihydroxyindole‐2‐carboxylic acid (DHICA) and its DHICA methyl ester (MeDHICA) were comparatively tested for their antioxidant activity. MALDI MS spectrometry analysis of MeDHICA melanin provided evidence for a collection of intact oligomers. EPR analysis showed g‐values almost identical and signal amplitudes (ΔB) comparable to those of DHICA melanin, but spin density was one order of magnitude higher, with a different response to pH changes. Antioxidant assays were performed, and a model of lipid peroxidation was used to compare the protective effects of the melanins. In all cases, MeDHICA melanin performed better than DHICA melanin. This capacity was substantially maintained following exposure to air in aqueous buffer over 1 week or to solar simulator over 3 hr. Different from DHICA melanin, MeDHICA melanin was proved to be fairly soluble in different water‐miscible organic solvents, suggesting its use in dermocosmetic applications.     

Aggregation of eumelanin mitigates photogeneration of reactive oxygen species

Melanins protect tissue by absorption and rapid nonradiative, nonreactive dissipation of ultraviolet (UV) light. However, melanins also produce reactive oxygen species (ROS) upon UV illumination. A chemical understanding of this dichotomy of photoprotection and phototoxicity has not been established. Herein this issue is examined by studying the UV-B induced oxidation and reduction of cytochrome c by ROS generated by different aggregation states of eumelanin. The quantum yield for superoxide anion by unaggregated oligomers is 7.4 x 10(-3), an order of magnitude greater than that characteristic of the bulk pigment. The quantum efficiency of hydrogen peroxide production by oligomers is 5.7 x 10(-3), and its production is attributed to reaction between superoxide anion and hydroquinone groups on eumelanin oligomers. Aggregation of oligomers results in a reduction of these quantum yields, having a significantly greater effect on the efficiency of hydrogen peroxide production. This effect is attributed to the decrease in surface concentration of hydroquinone sites upon aggregation. The effect of aggregation on the photogeneration of ROS serves to provide a foundation for the understanding of the dichotomy of photoprotective and phototoxic properties of melanin.     

The DHICA Oxidase Activity of the Melanosomal Tyrosinases LEMT and HEMT

Although melanins can be formed in vitro by the unique action of tyrosinase on L-tyrosine, it is now well accepted that other enzymes termed tyrosinase-related proteins are involved in mammalian melanogenesis. However, some aspects of their roles in the regulation of the pathway are still unknown. The action of dopachrome tautomerase on L-dopachrome yields DHICA, a stable dihydroxyindole with a low rate of spontaneous oxidation. However, DHICA is efficiently incorporated to the pigment, as judged by the high content of carboxylated indole units in natural melanins. Therefore, the fate of this melanogenic intermediate and the mechanisms of its incorporation to the melanin polymer are major issues in the study of melanogenesis. We have recently shown that mouse melanosomes contain two electrophoretically distinguishable tyrosinase isoenzymes, LEMT and HEMT, that can be purified and completely resolved (Jiménez-Cervantes et al., 1993a). Herein, we have compared the ability of these tyrosinases to catalyze DHICA oxidation. Although highly purified LEMT shows a very low specific activity for dopa oxidation in comparison to HEMT, it is able to catalyze DHICA oxidation. However, the DHICA oxidase activity of HEMT was very low, if significant. The ability of purified LEMT to catalyze DHICA oxidation was abolished by heat, trypsin, or phenylthiourea treatments. LEMT acting on DHICA caused the formation of a brownish soluble color similar to DHICA-melanin. Immunoprecipitation of the DHICA oxidase activity of LEMT by specific antibodies suggests that this activity corresponds to TRP1. These results indicate that LEMT, most probably identical to the product of the b locus, is a tyrosinase having a specific DHICA oxidase activity. Opposite to HEMT, the true tyrosinase encoded by the albino locus, its role in melanogenesis would be related to the incorporation of DHICA into eumelanin rather than to the first steps of the pathway.     

Dispersive Raman spectroscopy allows the identification and quantification of melanin types

Melanins are the most prevalent pigments in animals and are involved in visual communication by producing colored traits that often evolve as intraspecific signals of quality. Identifying and quantifying melanins are therefore essential to understand the function and evolution of melanin‐based signals. However, the analysis of melanins is difficult due to their insolubility and the lack of simple methods that allow the identification of their chemical forms. We recently proposed the use of Raman spectroscopy as a simple, noninvasive technique that can be used to identify and quantify melanins in feathers and hairs. Contrarily, other authors later stated that melanins are characterized by a lack of defined Raman signals. Here, we use confocal Raman microscopy to confirm previous analyses showing that the two main chemical forms of melanins (eumelanin and pheomelanin) exhibit distinct Raman signal and compare different excitation wavelengths to analyze synthetic pheomelanin and natural melanins in feathers of different species of birds. Our analyses indicate that only laser excitation wavelengths below 1064 nm are useful for the analysis of melanins by Raman spectroscopy, and only 780‐nm laser in the case of melanins in feathers. These findings show that the capacity of Raman spectroscopy to distinguish different chemical forms of melanins depends on laser power and integration time. As a consequence, Raman spectroscopy should be applied after preliminar analyses using a range of these parameters, especially in fragile biological tissues such as feathers.     

Insect cuticular melanins are distinctly different from those of mammalian epidermal melanins

Melanin from several insect samples was isolated and subjected to chemical degradation and HPLC analysis for melanin markers. Quantification of different melanin markers reveals that insect melanins are significantly different from that of the mammalian epidermal melanins. The eumelanin produced in mammals is derived from the oxidative polymerization of both 5,6‐dihydroxyindole and 5,6‐dihydroxyindole‐2‐carboxylic acids. The pheomelanin is formed by the oxidative polymerization of cysteinyldopa. Thus, dopa is the major precursor for both eumelanin and pheomelanin in mammals. But insect eumelanin appears to be mostly made from 5,6‐dihydroxyindole and originates from dopamine. More importantly, our study points out the wide spread occurrence of pheomelanin in many insect species. In addition, cysteinyldopamine and not cysteinyldopa is the major precursor for insect pheomelanin. Thus, both eumelanin and pheomelanin in insects differ from higher animals using dopamine and not dopa as the major precursor.     

Relationship of melanin degradation products to actual melanin content: application to human hair

Methods not only for characterizing but also for quantitating melanin subtypes from the two types of melanin found in hair--eumelanin and pheomelanin--have been established. In relation to testing for drugs of abuse in hair, these methods will allow for correction of drug binding to specific melanin subtypes and will serve to improve drug measurement in hair. 5,6-Dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) make up the majority of the eumelanin polymer while benzothiazene units derived from 2-cysteinyl-S-Dopa (2-CysDopa) and 5-cysteinyl-S-Dopa (5-CysDopa) compose the majority of the pheomelanin polymer. Our results show that: (1) pyrrole-2,3-dicarboxylic acid (PDCA) and pyrrole-2,3,5-tricarboxylic acid (PTCA), markers for DHI and DHICA units, respectively, are produced in 0.37 and 4.8% yields, respectively, when melanins are subjected to alkaline hydrogen peroxide degradation, (2) 3-aminotyrosine (3AT) and 4-amino-3-hydroxyphenylalanine (AHP), markers for 2-CysDopa and 5-CysDopa, respectively, are produced in 16 and 23% yield, respectively, when subjected to hydriodic acid hydrolysis, and (3) that black human hair contains approximately 99% eumelanin and 1% pheomelanin, brown and blond hair contain 95% eumelanin and 5% pheomelanin; and red hair contains 67% eumelanin and 33% pheomelanin. These data will allow deeper investigation into the relationship between melanin composition and drug incorporation into hair.     

Establishing structure-function relationships for eumelanin

The aggregation-dependent optical properties of eumelanin from human hair are examined. When aggregation is increased, the absorption spectrum extends to lower energy. The absorption spectra of oligomers isolated from black human hair and Sepia officinalis are comparable and are in quantitative agreement with the reported action spectra for photoinduced oxygen consumption and free-radical generation by eumelanin. The agreement between the optical properties of human hair and squid eumelanins suggests the fundamental molecular constituents of the pigments are similar and aggregation-dependent photophysical behavior is a general feature of all eumelanins.     

Chemical analysis of constitutive pigmentation of human epidermis reveals constant eumelanin to pheomelanin ratio

The skin constitutive pigmentation is given by the amount of melanin pigment, its relative composition (eu/pheomelanin) and distribution within the epidermis, and is largely responsible for the sensitivity to UV exposure. Nevertheless, a precise knowledge of melanins in human skin is lacking. We characterized the melanin content of human breast skin samples with variable pigmentations rigorously classified through the Individual Typology Angle (ITA) by image analysis, spectrophotometry after solubilization with Soluene‐350 and high‐performance liquid chromatography (HPLC) after chemical degradation. ITA and total melanin content were found correlated, ITA and PTCA (degradation product of DHICA melanin), and TTCA (degradation product of benzothiazole‐type pheomelanin) as well but not 4‐AHP (degradation product of benzothiazine‐type pheomelanin). Results revealed that human epidermis comprises approximately 74% of eumelanin and 26% pheomelanin, regardless of the degree of pigmentation. They also confirm the low content of photoprotective eumelanin among lighter skins thereby explaining the higher sensitivity toward UV exposure.     

Synthesis and Characterization of Melanins From Dihydroxyindole-2-Carboxylic Acid and Dihydroxyindole

Several studies have confirmed that a melanocyte-specific enzyme, dopachrome tautomerase (EC 5.3.2.3), catalyzes the isomerization of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA) (Pawelek, 1991). Here we report that DHICA, produced either enzymatically with dopachrome tautomerase or through chemical synthesis, spontaneously polymerized to form brown melanin that was soluble in aqueous solutions above pH 5. Under the same reaction conditions, solutions of either DOPA, DOPAchrome, or 5,6-dihydroxyindole (DHI) formed black, insoluble melanin precipitates. When DHICA and DHI were mixed together, with DHICA in molar excess, little or no precipitation of DHI-melanin occurred and the rate and extent of soluble melanin formation was markedly enhanced over that achieved with DHICA alone, suggesting co-polymerization of DHICA and DHI. With or without DHI, DHICA-melanins absorbed throughout the ultraviolet and visible spectra (200-600 nm). The DHICA-melanins precipitated below pH 5, at least in part because of protonation of the carboxyl groups. DHICA-melanins could be passed through 0.22 μm filters but could not be dialyzed through semi-permeable membranes with exclusion limits of 12,000-14,000 daltons. HPLC/molecular sieve analyses revealed apparent molecular weights ranging from 20,000 to 200,000 daltons, corresponding to 100-1,000 DHICA monomers per molecule of melanin. DHICA-melanins were stable to boiling, lyophilization, freezing and thawing, and incubation at room temperature for more than 1 year. The natural occurrence of oligomers of DHICA was first reported by Ito and Nichol (1974) in their studies of the brown tapetal pigment in the eye of the sea catfish (Arius felis L.). In experiments reported here, brown, but not black, melanins from mouse hairs, human melanoma cells, and peacock feathers were soluble in aqueous buffers. Since DHICA-melanins are both soluble and brown, the results raise the possibility that they are determinants of brown colors in the animal kingdom.     

Isolation and characterization of mammalian eumelanins from hair and irides

A new enzymatic procedure was developed for isolation of eumelanin from black human hair which might provide a substantially intact pigment for structural characterization. Sequential digestion with protease, proteinase K and papaine in the presence of dithiothreitol afforded a pigment with a 6% w/w protein content. HPLC analysis of pyrrole acids resulting from alkaline H(2)O(2) degradation, carboxyl content determination, and ferricyanide titration showed that the isolated pigment is made up of 5,6-dihydroxyindole (DHI)- and 5, 6-dihydroxyindole-2-carboxylic acid (DHICA)-derived units at a 6:1 ratio, exhibiting a significant degree of oxidative degradation. For comparison, a different eumelanin isolated from black bovine irides by a similar enzymatic procedure was analyzed. Matrix-assisted laser desorption ionization (MALDI) mass spectrometry of the final pigment provided evidence for homologous series of DHICA oligomers, while chemical analysis allowed an estimate of 2:1 DHICA/DHI-derived units in the polymer, with a substantial proportion of intact o-diphenolic functions. Iris melanin proved able to promote the Fenton oxidation of deoxyribose while hair melanin was ineffective. Overall, these results provide, for the first time, unambiguous evidence for marked structural differences of mammalian eumelanins which may be directly related to the diversity of the sites of biosynthesis and storage, as well as to functional role of these pigments.     

Theoretical models of eumelanin protomolecules and their optical properties

The molecular structure of melanin, one of the most ubiquitous natural pigments in living organisms, is not known and its multifaceted biological role is still debated. We examine structural models for eumelanin protomolecules, based on tetramers consisting of four monomer units (hydroquinone, indolequinone, and its two tautomers), in arrangements that contain an interior porphyrin ring. These models reproduce convincingly many aspects of eumelanin's experimentally observed behavior. In particular, we present a plausible synthetic pathway of the tetramers and their further complexation through interlayer stacking, or through formation of helical superstructures, into eumelanin macromolecules. The unsaturated nature of C-C bonds in indolequinone units and the finite size of protomolecules introduce covalent bond formation between stacked layers. We employ time-dependent density functional theory to calculate the optical absorption spectrum of each molecule along the eumelanin synthesis pathway, which gradually develops into the characteristic broad-band adsorption of melanin pigment due to electron delocalization. These optical spectra may serve as signatures for identifying intermediate structures.     

Eumelanin buildup on the nanoscale: aggregate growth/assembly and visible absorption development in biomimetic 5,6-dihydroxyindole polymerization

Establishing structure-property relationships in the black insoluble eumelanins, the key determinants of human pigmentation and skin photoprotective system, is a considerable conceptual and experimental challenge in the current drive for elucidation of the biological roles of these biopolymers and their application as advanced materials for organoelectronics. Herein, we report a new breakthrough toward this goal by the first detailed investigation on the nanoscale level of the oxidative polymerization of 5,6-dihydroxyindole (DHI), a model process of eumelanin synthesis. On the basis of a combined use of spectrophotometry, dynamic light scattering (DLS), and small-angle neutron scattering (SANS) investigations, it was possible to unveil the dynamics of the aggregation process before precipitation, the key relationships with visible light absorption and the shape of fundamental aggregates. The results indicated a polymerization mechanism of the type: Polymer(n) + DHI(x) = Polymer(n+x), where DHI(x) indicates monomer, dimer, or low oligomers (x ≤ 5). During polymerization, visible absorption increases rapidly, reaching a plateau. Particle growth proceeds slowly, with formation of 2-D structures ～55 nm thick, until precipitation occurs, that is, when large aggregates with a maximum hydrodynamic radius (R(h)) of ～1200 nm are formed. Notably, markedly smaller R(h) values, up to ～110 nm, were determined in the presence of poly(vinyl alcohol) (PVA) that was shown to be an efficient aggregation-preventing agent for polymerizing DHI ensuring water solubilization. Finally, it is shown that DHI monomer can be efficiently and partially irreversibly depleted from aqueous solutions by the addition of eumelanin suspensions. This behavior is suggested to reflect oxidant-independent competing pathways of polymer synthesis and buildup via monomer conversion on the active aggregate surface contributing to particle growth. Besides filling crucial gaps in DHI polymerization, these results support the attractive hypothesis that eumelanins may behave as a peculiar example of living biopolymers. The potential of PVA as a powerful tool for solution chemistry-based investigations of eumelanin supramolecular organization and for technological manipulation purposes is underscored.     

Electron Transfer and Photoprotective Properties of Melanins in Solution

The polyquinoid nature of eumelanin(s) enables them to couple oxidation of electron donors with the reduction of electron acceptors. We have studied the ability of synthetic (Sigma) and “biological” (cuttlefish sepia) melanins to mediate electron transfer between hydroxybenzene donors (tyrosine, dopa, chemical depigmenters) and model acceptors (ferricyanide, tyrosinase). 1) Depending on the reductant, melanin either retards or accelerates ferricyanide reduction. Reaction kinetics are consistent with a mechanism involving non-interactive binding of both hydroxybenzene and ferricyanide to melanin prior to coupled electron transfer. 2) Melanins also act as an electron conduit in markedly accelerating the tyrosinase-catalyzed oxygenation of p-hydroxyanisole (MMEH). The active species appears to be a complex between melanin and MMEH. The magnitude of both effects depend on the type of melanin as well as its oxidation state. Sepia (eu)melanin appears to protect against UV-induced damage to acid-soluble collagen, as judged by irreversible loss of intrinsic collagen fluorescence. Photoprotection against this type of damage appears primarily to involve optical absorption/scattering by the pigment.     

Advanced chemical methods in melanin determination

Among the biopolymers, melanins are unique in many respects. The other essential biopolymers - proteins, nucleic acids, and carbohydrates - are chemically well characterized; their precursors (monomer units) and modes of connection between the monomer units are known, and sequences of their connection can be determined with well-established methodologies. In contrast, we still do not have a method to determine accurately the ratio of various units present in melanins. This is largely because of the chemical properties of melanins, such as their insolubility over a broad range of pH, the heterogeneity in their structural features, and also because of the lack of methods that can split melanin polymers into their monomer units (all other biopolymers can be hydrolysed to the corresponding monomer units). To overcome this difficulty, we developed a rapid and sensitive method for quantitatively analysing eumelanin and pheomelanin in biological samples by chemical degradation methods followed by HPLC determination. This HPLC microanalytical method for characterizing eumelanin and pheomelanin has become a useful tool for the study of melanogenesis. This review will summarize the usefulness and limitations of the various chemical and spectrophotometric methods used to analyse melanins at the biochemical, cellular, and tissue levels. Emphasis is given on the usefulness of 4-amino-3-hydroxyphenylalanine as a specific marker of pheomelanin.     

Quantitative scattering of melanin solutions

The optical scattering coefficient of a dilute, well-solubilized eumelanin solution has been accurately measured as a function of incident wavelength, and found to contribute <6% of the total optical attenuation between 210 and 325 nm. At longer wavelengths (325-800 nm), the scattering was less than the minimum sensitivity of our instrument. This indicates that ultraviolet and visible optical density spectra can be interpreted as true absorption with a high degree of confidence. The scattering coefficient versus wavelength was found to be consistent with Rayleigh theory for a particle radius of 38 +/- 1 nm. Our results shed important light on the role of melanins as photoprotectants.     

Current challenges in understanding melanogenesis: bridging chemistry,biological control,morphology, and function

Melanin is a natural pigment produced within organelles, melanosomes, located in melanocytes. Biological functions of melanosomes are often attributed to the unique chemical properties of the melanins they contain; however, the molecular structure of melanins, the mechanism by which the pigment is produced, and how the pigment is organized within the melanosome remains to be fully understood. In this review, we examine the current understanding of the initial chemical steps in the melanogenesis. Most natural melanins are mixtures of eumelanin and pheomelanin, and so after presenting the current understanding of the individual pigments, we focus on the mixed melanin systems, with a critical eye towards understanding how studies on individual melanin do and do not provide insight in the molecular aspects of their structures. We conclude the review with a discussion of important issues that must be addressed in future research efforts to more fully understand the relationship between molecular and functional properties of this important class of natural pigments.     

Raman spectroscopy as a non‐invasive technique for the quantification of melanins in feathers and hairs

The quantification of melanins is a complex task due to the chemical heterogeneity of the pigments and the difficulty of their isolation. The best accepted procedure currently consists in the chemical cleavage of melanins and the subsequent detection of degradation products by HPLC, which implies the destruction of samples. Here, we show that Raman spectroscopy is a non‐invasive technique that can be used to quantify melanins. We made parallel analyses of the characteristics of pheomelanin and eumelanin Raman spectra as measured by confocal Raman microscopy and of degradation products of pheomelanin (4‐amino‐3‐hydroxyphenylalanine, 4‐AHP) and eumelanin (pyrrole‐2,3,5‐tricarboxylic acid, PTCA) as measured by HPLC in feathers of red‐legged partridges and hairs of wild boars and humans. We found strong correlations between the spectral Raman characteristics and 4‐AHP and PTCA levels, which indicates that the Raman spectra of melanins can be used to determine their content.