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.     

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 Å, 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 ～15 Å to ～5 Å) 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, Π–Π‐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 ～14 Å to ～6 Å). 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, Π–Π 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.     

Sugars in hydrolysates of fungal melanins and soil humic acids

Humic acids from four Brazilian topsoils of different origin and four fungal melanins, synthesized under two cultural conditions were subjected to a two step hydrolysis procedure and the released monosaccharides qualitatively and quantitatively determined by gas-liquid chromatography. The neutral sugars, glucose, galactose, mannose, arabinose, xylose, fucose, rhamnose and the alcohol sugar inositol, were detected in most of the soil humic acid samples. The fungal melanins showed the presence of glucose, galactose, mannose and arabinose. Ribose was present in two out of the eight samples tested. Some quantitative differences in the two types of humic polymers were noted and expected considering their origins. However, similarities were more apparent than differences and give further indication that melanic fungi may play a significant role in the formation of soil humic acids.     

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.     

Study of photochemical and surface-active melanins of some representatives of black fungi

The absorption spectra of melanins isolated from some black ascomycetes, as well as of synthetic melanin and natural melanin from Sepia officinalis, were recorded in the long-wavelength ultraviolet region A (320 nm < lambda < 400 nm) and in the blue-violet region of the electromagnetic spectrum at illumination intensities varying from 0.02 to 1 mW/cm2. The photochemical properties of fungal melanins were found to be dependent on both the producing strain and the conditions of its cultivation. The fungal melanins are more susceptible to photomodification and more biologically active than the synthetic melanin, indicating that these properties may be related. The data obtained suggest that the fungal melanins susceptible to photomodification possess higher biological activity than commercial melanins.     

Amino acid distribution in some fungal melanins and of soil humic acids from Brazil

Summary Humic acids from four Brazilian topsoils of different origins and four soil fungal melanins, synthesized under two cultural conditions, were subjected to 6N HCl hydrolysis and their amino acid distribution patterns qualitatively and quantitatively determined. Both soil and fungal polymers showed similar patterns with aspartic acid, glutamic acid, glycine and alanine as the dominant amino acids. Some variations noted were more quantitative than qualitative, the similarities were more pronounced than differences, indicating that the fungal melanins may play a significant role in the formation of soil humic acid polymers. The humic acids of Brazilian soils had amino acid distribution patterns similar to those reported for humic acids of other tropical and temperate soils.     

The inhibition of peroxyradical-induced chemiluminescence by melanins

Model melanins synthesized from adrenochrome, DOPA and pyrocatechol quench the peroxyradical-coupled chemiluminescence initiated by electrolysis, photolysis and redox reactions in several selected systems. Calculations of the selfadsorption (inner filter effect) of melanins that decreases chemiluminescence intensity as well as changes in absorption spectra of photolabile substrate-probes such as cytochrome c and rhodamine, lead to the conclusion that melanins scavenge peroxyradicals and inhibit chemiluminescence. The inhibition effect (beta-value) is much higher than that of most powerful antioxidants in all chemiluminescence systems excluding the luminol-hydrogen peroxide one. Donor-acceptor properties of melanins, pertinent to their antioxidizing activity, are discussed.     

Reversible and irreversible changes in hydrogen ion titration curves of melanins.

Hydrogen ion titration curves obtained on melanins prepared and modified differently show recognizable differences. Melanin prepared with NaOH exhibits irreversible loss of functional groups in the base range once titrated acidic. Melanin prepared with NH4OH shows two main branches selected by incubation at an extreme pH for 24 h. Longer exposure reveals other curves but they disappear once a 24-h schedule of titration is resumed. The existence of two major pH branches appears consistent with the oxidation-reduction properties of melanins. These results demonstrate the ability to obtain a reproducible feature in the hydrogen ion titration curves. Thus it is finally possible to quantitate CO2 adsorbed to melanin in solution at basic pH.     

Isolation and biophysical studies of natural eumelanins: applications of imaging technologies and ultrafast spectroscopy

The major pigments found in the skin, hair, and eyes of humans and other animals are melanins. Despite significant research efforts, the current understanding of the molecular structure of melanins, the assembly of the pigment within its organelle, and the structural consequences of the association of melanins with protein and metal cations is limited. Likewise, a detailed understanding of the photochemical and photophysical properties of melanins has remained elusive. Many types of melanins have been studied to date, including natural and synthetic model pigments. Such studies are often contradictory and to some extent the diversity of systems studied may have detracted from the development of a basic understanding of the structure and function of the natural pigment. Advances in the understanding of the structure and function of melanins require careful characterization of the pigments examined so as to assure the data obtained may be relevant to the properties of the pigment in vivo. To address this issue, herein the influence of isolation procedures on the resulting structure of the pigment is examined. Sections describing the applications of new technologies to the study of melanins follow this. Advanced imaging technologies such as scanning probe microscopies are providing new insights into the morphology of the pigment assembly. Recent photochemical studies on photoreduction of cytochrome c by different mass fraction of sonicated natural melanins reveal that the photogeneration of reactive oxygen species (ROS) depends upon aggregation of melanin. Specifically, aggregation mitigates ROS photoproduction by UV-excitation, suggesting the integrity of melanosomes in tissue may play an important role in the balance between the photoprotective and photodamaging behaviors attributed to melanins. Ultrafast laser spectroscopy studies of melanins are providing insights into the time scales and mechanisms by which melanin dissipates absorbed light energy.     

Effect of melanins from black yeast fungi on cultured human cells. II. Differentiation of keratinocytes in vitro

Data on the influence of the black yeast melanin (3 samples) on the in vitro differentiation of human keratinocytes are presented. The effect of melanins was estimated by the morphological state of keratinocytes using electron microscopy. The obtained differences in the state of the formed multilayer keratinocyte sheets depended on the melanin sample.     

Structural modifications in biosynthetic melanins induced by metal ions

A number of transition metal ions with a wide distribution in biological systems, e.g., Cu2+, Co2+ and Zn2+, 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.     

Melanin Standard Method: Empirical Formula 2

Natural melanins are composed of two distinct portions; a protein fraction and a chromophoric backbone. There is no unequivocal evidence for covalent bonding between these two fractions, and standard protocols used in protein purification have failed to separate the protein fraction from the chromophoric fraction. In order to study the chromophoric backbone, many workers have resorted to harsh isolation and purification protocols that are now known to degrade and damage the chromophoric portion. These artifactual melanin preparations are poor models for valid chemical, physical, and biological studies. We have developed a mild isolation and purification protocol for melanins that takes into consideration both the particulate nature of natural melanins and the stability characteristics of the chromophoric fraction. Mathematical factoring of the quantitative amino acid data into the elemental analysis was used to obtain the empirical formula of the chromophoric backbone of melanins. The analyses have shown that melanins from various sources have significantly different amino acid compositions and contents, molar C/N ratios, and empirical formulae. This method successfully differentiates melanins from a variety of sources, namely, human hair, Sepia officinalis, Sigma Chemical Company (cat. no. M8631), autoxidation of dopa, and from the feathers of Rhode Island Red chickens. Analytical results from these studies are presented and discussed.     

In vitro scavenger activity of some flavonoids and melanins against O2-(.).

The scavenger activity against O2-. of some flavonoids and melanins (synthetic melanins and melanins isolated from animal tissues, vegetable seeds, and mushroom spores) has been studied by ESR spectrometry. All these substances, except flavon and flavanone, diminish the signal of O2-. generated in vitro by a system containing H2O2 and acetone in an alkaline medium. It is shown that the presence of hydroxyl groups in the B ring of flavonoids is essential for their scavenger activity. Moreover, the presence of a hydroxyl at C-3 enhances the scavenger ability of flavonoids. Generally, aglycons are more active than their glycosides. It seems plausible that the antioxidant property of these substances comes from their scavenger activity against O2-(.). It is also pointed out that the scavenger activity shown by melanins, is strictly correlated with their nature of stable free radical.     

The Photochemical and Surface-Active Properties of Melanins Isolated from Some Black Fungi

The absorption spectra of melanins isolated from some black ascomycetes, as well as of synthetic melanin and natural melanin from Sepia officinalis, were recorded in the long-wavelength ultraviolet region A (320 nm < < 400 nm) and in the blue–violet region of the electromagnetic spectrum at illumination intensities varying from 0.02 to 1 mW/cm². The photochemical properties of fungal melanins were found to be dependent on both the producing strain and the conditions of its cultivation. The fungal melanins are more susceptible to photomodification and more biologically active than the synthetic melanin, indicating that these properties may be related. The data obtained suggest that the fungal melanins susceptible to photomodification possess higher biological activity than commercial melanins.     

Spectroscopic studies of chemically modified synthetic melanins

The infrared and electron spin resonance spectra of synthetic 3,4-dihydroxyphenylalanine (DOPA) and tyrosine melanins and chemically modified melanin samples were determined, and it was shown that unmodified and reduced DOPA melanins exhibited similar ir spectra. Oxidized DOPA melanins showed a higher number of carboxy groups in the sample. A significant increase of free radical content in reduced DOPA melanin and a decrease of free radical content in oxidized DOPA melanin in comparison to unmodified samples were demonstrated by the use of ESR methodology. Methylation of tyrosine melanin with an excess of diazomethane gave very rich ir spectra as compared to melanins methylated with methanol saturated by gaseous HCl. In tyrosine melanin samples the esterification of carboxy groups with methanol caused a decrease in the free radical content. When diazomethane was used, the methylated melanin samples had free radical levels reduced to only about 4% of the total observed for unmodified tyrosine melanin.     

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.     

Melanin standard method: particle description.

Melanin isolated from the ink sac of Sepia officinalis (Sepia melanin) has been proposed as a standard for natural eumelanin. There are no standard methods for the isolation, purification, and storage of melanins. Mild methods designed to preserve the native composition and structure of melanin are needed. The specific aim of the present work, using Sepia melanin, was to develop a mild and generally applicable protocol for the isolation and purification of melanins. It is well established that melanin polymers contain a large number of free carboxylic acid residues. These anionic residues are responsible for the cation exchange properties observed for melanins. Heating melanins with hydrochloric acid at reflux has been demonstrated to lead to extensive decarboxylation. Indeed, heat alone has been shown to cause decarboxylation, and care must be exercised to avoid such conditions. By analogy with cation exchange resins, melanins should be isolated and named according to the associated counterion (e.g., Sepia melanin--K+ form). The method reported here avoided extremes in pH and temperature, and was designed to yield melanin in the K+ form. Physical disaggregation of particulate melanin using a wet milling step was also found to facilitate removal of significant quantities of adsorbed protein. The following physical parameters were used to monitor the purification and to characterize the resultant melanin: pH, conductance, particle size, and diffuse reflectance spectroscopy.     

Physical Studies on Melanins: II. X-Ray Diffraction

A number of purified natural and synthetic melanins have been examined by X-ray diffraction. A consistent finding with all samples was the lack of structure in the diffraction pattern corresponding to any significant crystallinity in these melanin preparations. A diffuse ring, centered at a Bragg spacing of 3.4 A was consistently found in samples of melanin from animal sources, and a similar ring at 4.2 A in all melanins obtained from plants. Models for these two polymer types, based upon the current concept that they primarily involve indole and catechol monomeric units respectively, were then evaluated by a Monte Carlo method. From the comparison of the observed spacings with the calculated ones it was concluded that the 4.2 A spacing in the catechol melanins is probably related to the average interaction between adjacent monomeric units, with mutually random orientations. The 3.4 A spacing observed in indole melanins appears to derive from the tendency of indole monomers (probably of adjacent chains) tending to aggregate in near parallel stacks. Some randomness in the form of translations and rotations parallel to the planar groups is consistent with the diffraction patterns. An interesting finding was that the diffraction pattern of synthetic melanin prepared by the alkaline auto-oxidation of catechol gave the 3.4 A spacing found in the indole melanins of natural origin.     

Effect of melanins on lipid peroxidation

Effects of melanins obtained from cultured Cladosporium cladosporidae fungi and Alpha grape on Fe(2+)-induced, Fe(2+)-ascorbate-induced, and NADPH-induced lipid peroxidation in rat liver, brain, and eye were studied. Melanins were shown to inhibit the accumulation of lipid peroxidation products in vitro. The inhibitory effects of melanins were not due to direct interactions of these pigments with superoxide anion (O2). However, melanins may interact with other free radicals. Melanins were demonstrated to have the ability to oxidize NADPH, which is probably one of the mechanisms of their antioxidant effects.