Natural Melanins Research Articles

X-ray characterization of melanins--I.

The intrinsic local structure characterization of natural sepia melanin and L-dopa and tyrosine synthetic melanin powder has been carried out by X-ray diffraction using synchrotron radiation. The derived structure factor, S(q), shows six significant diffuse peaks within the q-range from 0.3 A-1 to 16 A-1 in the reciprocal space (q = (4 pi sin theta)/lambda, 2 theta is the scattering angle). The Fourier transform of S(q), which yields the radial distribution function (RDF), gives us information in real space of a 1.42 A distance averaged over the C-C, C-O and C-N bond lengths as well as peaks at 2.40-2.41 A, 3.67-3.71 A and 4.67-4.70 A discrete neighbor distances. There is a great similarity in the scattering intensity profiles of the natural and synthetic melanins indicating that the synthetically prepared material may be essentially similar to "real" melanin in its local atomic arrangements. An evidence of a prepeak at q congruent to 0.45 A-1 has been confirmed which indicates a preferred length scale of approximately 13-20 A that corresponds to the initial particle size in colloidal melanin solutions.

Tunneling microscopy verification of an x-ray scattering-derived molecular model of tyrosine-based melanin

The molecular structure of melanins has been a focus of study for many years and only recently has a molecular model (MM) been proposed based upon x-ray scattering results. In order to test the validity of the model, scanning tunneling microscopy (STM) and spectroscopy have been performed on the same tyrosine-derived melanin, which has been characterized in the x-ray scattering studies and has been shown to exhibit characteristics of natural melanins. The STM measurements of the lateral and vertical dimensions provide a characteristic fundamental size which agree well with the x-ray scattering-derived MM. We find that the compact three-dimensional structure is best described as a planar morphology of finite lateral extent (≊2 nm) with a vertical extent (<1 nm) which is believed to consist of stacks of a basic two-dimensional amorphous polymer of 5,6 indolequinone units. Molecular mechanics and orbital calculations of representative amorphous models support the x-ray scattering and STM results.

A 13C solid-state NMR study of the structure and auto-oxidation process of natural and synthetic melanins

This paper presents a 13C CP/MAS NMR study of the melanin pigments obtained through natural synthetic origins: sepia-melanin from squid ink and three synthetic 5,6-dihydroxyindole-melanins prepared using different non-enzymatic oxidation pathways. The synthetic pigments can be distinguished from natural melanin by the absence of aliphatic carbons, thereby confirming the unreacted 3,4-dihydroxyphenylalanine and the proteinaceous origins of the aliphatic resonances in natural eumelanin. The spectra of selected non-protonated carbon resonances and those with only protonated carbon signals led to a quantitative analysis. An auto-oxidative experiment using a synthetic melanin, over a period of 130 h, has shown an usually slow disappearance of hydrogen peroxide formed in situ. The 13C-NMR spectrum of the insoluble oxidized synthetic melanin compared to that before auto-oxidation clearly demonstrates that the oxidation process is associated with chemical changes within the pigment; i.e., carbonyl functional group formation and an increase of the non-protonated carbons fraction.

Laser desorption ionization mass spectrometry in the study of natural and synthetic melanins. I—Tyrosine melanins

Nine synthetic, biosynthetic and natural tyrosine melanins, together with dopa and catechol melanins, have been studied by means of laser desorption ionization mass spectrometry. The results obtained have shown that all the melanins under study consist of homogeneous sets of oligomers and have led to definitive information on their molecular weight distribution (from 500 to 30,000 Da). A deeper analysis of the spectra, in terms of mass differences among the various oligomers, allowed the identification of some monomeric units, identical or analogous to those already proposed in the literature.

Characterization of melanins by pyrolysis/gas chromatography/mass spectrometry

AbstractVarious natural and synthetic melanins were studied by the pyrolysis/gas chromatography/mass spectrometry technique, using both resistive and Curie‐point heating. The results obtained by the two pyrolysis procedures are different and complementary. By means of Curie‐point pyrolysis, at long times and high temperatures low molecular weight gases are produced from various melanins; the yields of the common pyrolysis products are also dependent on the melanin under study. By resistive heating, higher molecular weight compounds are produced, mainly nitrogen and oxygen heterocyclic systems, and differences are still evident among the products from the different melanins.

Regulatory Mechanisms of Melanogenesis: Beyond the Tyrosinase Concept

The last decade has witnessed a quantum jump in our knowledge of the chemistry of melanogenesis and the emergence of some understanding as to how the chemical and enzymatic activities are integrated and regulated at cellular level. To an unusual extent, most of these advances stemmed from a molecular approach involving direct analysis of natural melanins coupled with studies of the chemical reactivity of the putative intermediates under biologically relevant conditions. Thus, bit by bit it has been possible to reconstruct in vitro the overall chemical activity of melanocytes, and to identify a number of regulatory factors other than tyrosinase. This and other approaches, especially the molecular biology of the pigment-related genes, have radically changed and expanded the traditional concept of melanogenesis far beyond the boundaries of the Raper-Mason pathway. Recently, I suggested an improved scheme of melanogenesis accounting for the formation of melanins and related products of melanocyte activity. The present contribution surveys some verifications of the predictions, and certain new break-throughs that have been made in the understanding of the regulatory steps after the dopaquinone stage. It also aims to provide an overview of what is presently known about the structure of the melanin polymers, and to draw attention to some problems pertaining to the definition and classification of the various types of pigments that can be found in epidermal tissues.

Renal excretion of plasma soluble melanins by healthy human adults.

The soluble melanins of blood plasma form in vivo and in vitro from dopa, catecholamines, catechol, hydroquinone, homogentisic acid, 3-hydroxykynurenine, 3-hydroxyanthranilic acid, p-aminophenol, p-phenylenediamine and other structurally related end(ex)ogenous compounds by oxidative polymerization. The mean quantity of natural melanins in normal plasma is 1.61 +/- 0.10 (standard deviation) mg/ml, (n = 20) and in uraemic plasma 2.72 +/- 0.38 mg/ml, (n = 16). The plasma melanins (approximately 3%), are associated with proteins (approximately 85%), mucoproteins (approximately 0.25%), lipids (approximately 0.4%), as soluble lipofuscins, and probably are associated with proteins without lipids as soluble melanoproteins. Fluorescence, UV-VIS and IR spectroscopies and the melanin isolation method show the presence of soluble melanins in the urine of healthy people. Soluble melanins can also be formed in vitro in the urine by oxidative polymerization of the precursors. In most of the urine samples we studied, melanins were present in larger amounts than the urinary proteins, indicating that the kidneys can selectively excrete the melanin components of the lipofuscins, and that the solubility of melanins does not depend upon combination with proteins. The quantities of purified melanins precipitated with 6 N HCl at 110 degrees C during 72 h from urine samples collected during 24 h periods ranged from 0.1460 g to 3.7627 g (mean 1.1303 +/- 1.1739 g, n = 8) and the plasma clearance rates ranged from 0.06 ml/min to 1.56 ml/min (mean 0.48 +/- 0.48 ml/min, n = 8). From the individual 24 h urine samples we obtained from 9 to 216 mg/dl of precipitated melanins while the individual plasma samples contained from 145 to 175 mg/dl.

Modulation by neuromelanin of the availability and reactivity of metal ions.

Presence of neuromelanin is likely to alter significantly the amount, distribution, reactivity, and consequences of reactivity of metal ions in those parts of the brain that contain neuromelanin. The effects are complex and can be predicted only with detailed knowledge of the system because (1) melanins are strong binders of metal ions and many organic molecules; (2) the effect of binding, depending on the circumstances, can increase or decrease reactivity of metal ions; and (3) melanins can generate and react with oxidation-reduction-active species such as hydrogen peroxide, hydroxyl radicals, superoxide anions, and singlet oxygen. Neuromelanin has some significant differences from most other natural melanins because of its mode of formation (by autooxidation rather than enzymatically) and its composition (it probably is a copolymer derived from dopamine and glutathione). It probably is not possible to understand fully the role of metal ions in oxidative damage in the brain without having an adequate understanding of the structure and reactivity of neuromelanin.

Selective antiviral activity of synthetic soluble l-tyrosine and l-dopa melanins against human immunodeficiency virus in vitro

Melanins are pigments found in hair, skin, irides of the eye, and brain. Their functions in mammals include protection from exposure to sunlight, camouflage from predators, sexual recognition within species, and possible electron transfer reactants. Most natural melanins exist in an insoluble form, which is one reason there is little information on the biological properties of soluble melanins. Here, synthetic soluble melanins were obtained by chemical oxidation of l-tyrosine or spontaneous oxidation of l-β-3,4-dihydroxyphenylalanine ( l-dopa). Replication of human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) was inhibited by soluble melanin in two human lymphoblastoid cell lines (MT-2 and H9) and in phytohemagglutinin-stimulated human T cells. Effective concentrations of 0.15-10 μg/ml had no cell toxicity. Melanin blocked infection by cell-free virus and interfered with HIV-induced syncytium formation and cytopathic effects when fusion-susceptible, uninfected cells, were mixed with chronically infected cells. Melanin also impeded the HIV-1 envelope surface glycoprotein, and T cell specific monoclonal antibody leu-3a (CD4), but not leu-5b (CD2), from binding to the surface of MT-2 cells. No effect on HIV-1 reverse transcriptase activity in viral lysates was observed. These results identify a unique biological property of melanin, and suggest that soluble melanins may represent a new class of pharmacologically active substances which should be further investigated for potential therapeutic utility in the treatment of Acquired immune Deficiency Syndrome (AIDS).

Thermogravimetry and differential scanning calorimetry of natural and synthetic melanins

The thermal stability of natural melanins from bovine eyes, black human hair and the hard core of banana peel, synthetic melanins obtained enzymatically or by autoxidation of various precursors, and chemically modified synthetic melanins was studied by DSC and TG analysis. It was shown that the resistance of melanins to thermal degradation depends on their origin. Synthetic melanins were found to be more stable to thermal decomposition than natural melanins. Methylation of melanins caused a significant increase in thermal stability. The DSC curves of melanins reveal typical relaxation phenomena in the temperature range 293–413 K.

Interactions of flavins with melanin

Natural melanins are photoprotective pigments that in mammals are principally found in the skin, hair, and eyes. Although the molecular mechanism of photoprotection of pigmented cells has not yet been established, several hypotheses have been proposed with melanin acting as a light filter, free radical scavenger, and quencher of electronically excited states of reactive intermediates. It can be expected that the detoxicating efficiency of melanin should be enhanced if the melanin and potentially cytotoxic species are brought close together. Such a situation may occur for a number of photosensitizing dyes that have the ability to bind to melanin. The interaction of melanin with flavins has been studied under strictly controlled experimental conditions. The equilibrium dialysis method has been employed to determine dissociation constants and the number of binding sites in melanin at pH 5-9. The data reveal that synthetic DOPA-melanin has two different classes of binding sites with dissociation constants of 10(-6) and 10(-5) M, respectively. The overall binding capacity of melanin, at pH 7, is 250 nmol RF/mg melanin. The amount of bound-to-melanin RF increases with pH. The absorption spectra of melanin complexes with RF and lumiflavin indicate that hydrophobic interaction may be involved in the binding of these flavins by melanin. No changes in flavin fluorescence have been detected after binding of flavin to melanin. It appears that, contrary to cationic photosensitizing dyes, the singlet excited state of flavin molecules is not quenched by melanin.

The formation of glyoxylate from glycine by melanin

Natural and synthetic melanins catalyze the conversion of glycine to glyoxylate and formic acid in vitro . The conversion depends upon the concentration of melanin.

Optimization of Conditions for Preparing Synthetic Pheomelanin

Synthetic melanins are often used as the standards for natural melanins. Conditions for preparing synthetic pheomelanin from L-dopa and L-cysteine were studied under different temperatures (25 and 37 degrees C), different amounts of cysteine (1.5 and 2.0 eq), and different amounts of tyrosinase (10 and 20 mg). Effects of addition of catalase and of replacement of oxygen with air were also examined. The best preparation of pheomelanin was obtained in a good yield, when 1 mmol of L-dopa was oxidized at 25 degrees C under oxygen current in 100 ml of a pH 6.8 buffer by 20 mg of mushroom tyrosinase inn the presence of 1.5 mmol of L-cysteine. The pheomelanin thus obtained had an elemental composition and an absorption spectra that were very similar to those from a pheomelanin prepared by tyrosinase oxidation of 5-S-cysteinyldopa. Hydrolysis of synthetic pheomelanins and pheomelanic tissues with hydriodic acid gave aminohydroxyphenylalanines (AHP) as a major product and hydroxybenzothiazolylalanines as a minor product. It is proposed that AHP can be used as a specific indicator of purity of pheomelanins.

Analysis of the structure of synthetic and natural melanins by solid-phase NMR.

The structures of one synthetic and two natural melanins are examined by solid-state NMR using cross polarization, magic angle sample spinning, and high-power proton decoupling. The structural features of synthetic dopa melanin are compared to those of melanin from malignant melanoma cells grown in culture and sepia melanin from squid ink. Natural abundance 13C and 15N spectra show resonances consistent with known pyrrolic and indolic structures within the heterogeneous biopolymer; 13C spectra indicate the presence of aliphatic residues in all three materials. These solid-phase experiments illustrate the promise of solid-phase NMR for elucidating structural information from insoluble biomaterials.

Analysis and Quantification of Pheomelanins by Radioimmunoassay

An antiserum against alpha-amino-(4-hydroxy-6-benzothiazolyl)propionic acid (AHBP), a major product obtained after hydriodic acid hydrolysis of pheomelanin, was raised in rabbits immunized with AHBP coupled to bovine serum albumin (BSA). The antiserum was used to develop a radioimmunoassay (RIA) to AHBP. The limit of detection of the RIA is 0.1 ng of AHBP. The antiserum does not crossreact with other major hydriodic acid degradation products, and the assay has been used to estimate the amounts of AHBP in synthetic and natural melanins.

Structural modifications in biosynthetic melanins induced by metal ions

A number of transition metal ions with a wide distribution in biological systems, e.g., Cu 2+, Co 2+ and Zn 2+, are shown to affect markedly the chemical properties of melanins formed by the tyrosinase-catalysed oxidation of dopa. Acid decarboxylation and permanganate degradation provide evidence that melanins prepared in the presence of metal ions contain a high content of carboxyl groups arising from the incorporation of 5,6-dihydroxyindole-2-carboxylic acid (DICA) into the pigment polymer. Naturally occurring melanins from cephalopod ink and B16 mouse melanoma were found to be much more similar to melanins prepared in the presence of metal ions than to standard melanins prepared in the absence of metal ions. These results suggest that the presence of carboxylated indole units in natural melanins is probably due to the intervention in the biochemical pathway of metal ions which, as recently shown, catalyse the formation of DICA versus 5,6-dihydroxyindole in the rearrangement of dopachrome.

Incorporation of sulfhydryl compounds into melanins in vitro

Cysteine is known to be involved in pheomelanin synthesis. Available evidence, however, indicates that glutathione may also play an important role in melanogenesis. This in vitro study clarifies how cysteine and glutathione participate in melanogenesis. Melanins were prepared by tyrosinase oxidation of 3,4-dihydroxyphenylalanine (dopa) with various amountsof cysteine or glutathione, and were subjected to HCl hydrolysis. Synthetic melanins and the hydrolyzed melanins gave N/S molar ratios corresponding to those calculated from the ratio of dopa to cysteine or glutathione. On hydriodic acid hydrolysis, dopa plus cysteine-melanins and dopa plus glutathione-melanins gave aminohydroxyphenylalanine and cysteine, respectively, the yields of which were proportional to the sulfur content. The results indicate that cysteine is integrated into benzothiazine units (pheomelanins) while glutathione is connected to dihydroxyindole units (eumelanins)_ with the retention of glutathione moiety. Analysis of natural melanins, prepared by HCl hydrolysis of Sepia, B16, and Harding-Passey melanosomes, indicates that sulfur (0.4–1.4%) in these natural melanins may be derived artificially from the reaction of melanins with cysteine or cystine in the course of HCl hydrolysis.

Electrochemical properties of natural and synthetic melanins

Electrochemical properties of natural and synthetic melanins

Effects of Melanin-Induced Free Radicals on the Isolated Rat Peritoneal Mast Cells

Pheomelanin from human red hair (RHM) produces considerably more cellular damage in Ehrlich ascites carcinoma cells when subjected to radiations of wavelength 320-700 nm than eumelanin from black hair (BHM). Irradiation of RHM generated large amounts of superoxide while BHM did not produce detectable amounts of superoxide. The present investigations describe the effects of irradiation of mast cells in the presence of various natural and synthetic melanins. Irradiation of mast cells in the presence of RHM and red hair melanoprotein released large amounts of histamine while BHM and synthetic melanins prepared from dopa, cysteinyldopa, or a mixture of dopa and cysteinyldopa did not release histamine. The release of histamine at lower concentrations of RHM was not accompanied by the release of 51Cr from chromium-loaded cells, suggesting that this release was of noncytotoxic nature. On the other hand, the release of histamine at higher concentrations of RHM was due to cell lysis since both histamine and cytoplasmic marker 51Cr were released to the same extent. The release evoked by large concentration RHM was not inhibited by superoxide dismutase or catalase. This suggests that the cell lysis under these conditions was not due to H2O2 or O-2. The finding that mast cells release histamine when irradiated in the presence of RHM suggests that the immediate and late-phase reactions seen in sunburn may in part be due to the release of mediators from these cells.

Electron spin relaxation of synthetic melanin and melanin-containing human tissues as studied by electron spin echo and electron spin resonance

Electron spin lattice relaxation times (T 1) and the phase memory times ( T m) were obtained for the synthetic melanin system from 3-hydroxytyrosine (dopa) by means of electron spin echo spectroscopy at 77 °K. Saturation behavior of the ESR spectra of melanins in melanin-containing tissue and of the synthetic melanin was also determined at the same temperature. The spin lattice relaxation time and the spectral diffusion time of the synthetic melanin are very long (4.3 ms and 101 μs, respectively, in the solid state), and the ESR signal saturates readily at low microwave powers. On the other hand, ESR spectra of natural melanins from the tissues chosen for this study, as well as those of synthetic melanins which contain Fe 3+ of g = 4.3 and Mn 2+ of g = 2, are relatively difficult to saturate compared with samples without such metal ions. These results show clearly that a large part of those two metal ions in sites responsible for the ESR spectral components with these particular g values are coordinated to melanin in melanin-containing tissue, and modify the magnetic relaxation behavior of the melanin. Accumulations of these metal ions in melanins are different from system to system, and they increase in the order: hair (black), retina and choroid (brown), malignant melanoma of eye and skin, and lentigo and nevus of skin.