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.     

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.     

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.     

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.     

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.     

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.     

Inhibition of human immunodeficiency virus type 1 replication and cytopathicity by synthetic soluble catecholamine melanins in vitro

Synthetic soluble melanins were synthesized by spontaneous oxidation of L-dopamine, norepinephrine or 5-hydroxytryptamine (serotonin) in weak alkaline solution. These three melanins inhibited infection of human CD4+ lymphoblastoid cells (MT-2) by cell-free human immunodeficiency virus type 1 (HIV-1), without cell toxicity, at concentrations of 0.15-10 micrograms/ml. Also, syncytium formation and resulting cytopathic effects when uninfected cells were mixed with chronic HIV-1-infected cells were blocked by these melanins. Antisyncytial activity was greater when infected cells were preincubated with melanin than when uninfected cells were preincubated with melanin, thus suggesting that interaction of melanin with viral proteins is an important aspect of the antiviral mechanism. These results make synthetic soluble melanins interesting candidates for further study as possible anti-HIV-1 therapeutics.     

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.     

Sensitization of surfactants on the chemiluminescence reaction of fluorescein isothiocyanate labeled proteins

The effect of surfactants on fluorescein isothiocyanate (FITC)-bovine serum albumin (BSA)-hypochlorite (ClO(-)), FITC-human serum albumin (HSA)-ClO(-), FITC-ovoconalbumin (OVA)-ClO(-), FITC-hemoglobin (Hb)-ClO(-) systems were investigated with chemiluminescence method by the reversed phase flow injection. It was found that the chemiluminescence (CL) intensity of each system was increased greatly in the presence of cationic surfactants. Cethyltrimethylammonium bromide (CTAB) is the optimal surfactant of these systems. The optimal conditions of the CL reaction and the optimal concentration of CTAB were examined and the function of cationic surfactant CTAB on the CL reaction was also discussed.     

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.     

Peroxynitrite mediated linoleic acid oxidation and tyrosine nitration in the presence of synthetic neuromelanins

Peroxynitrite-mediated linoleic acid oxidation and tyrosine nitration were analysed in the presence of synthetic model neuromelanins: dopamine (DA) -melanin, cysteinyldopamine (CysDA) -melanin and various DA/CysDA copolymers. The presence of melanin significantly decreased the amount of 3-nitrotyrosine formed. This inhibitory effect depended on the type and concentration of melanin polymer. It was found that incorporation of CysDA-derived units into melanin attenuated its protective effect on tyrosine nitration induced by peroxynitrite. In the presence of bicarbonate, the melanins also inhibited 3-nitrotyrosine formation in a concentration dependent manner, although the extent of inhibition was lower than in the absence of bicarbonate. The tested melanins inhibited peroxynitrite-induced formation of linoleic acid hydroperoxides, both in the absence and in the presence of bicarbonate. In the presence of bicarbonate, among the oxidation products appeared 4-hydroxynonenal (HNE). CysDA-melanin inhibited the formation of HNE, while DA-melanin did not affect the aldehyde level. The results of the presented study suggest that neuromelanin can act as a natural scavenger of peroxynitrite.     

MCLA为化学发光探针测定氧化低密度脂蛋白中维生素C诱导的单线态氧产生

以一种海萤荧光素类似物MCLA〔2 methyl 6 (p methoxyphenyl) 3,7 dihydroimidazo [1,2 a]pyrazin 3 one〕作为高灵敏且有选择性的化学发光探针 ,用化学发光的方法直接观测到了少量Cu2 氧化的低密度脂蛋白 (Ox LDL)中维生素C诱导的单线态氧 (1O2 )的产生。实验中通过叠氮化钠 (NaN3 )对MCLA介导的化学发光的猝灭作用进一步证实了上述体系中1O2的形成。根据实验观察的结果 ,分析了这一体系中1O2 形成的可能途径 ,认为首先是维生素C将Cu2 转变为还原态 ,而自身失去一个电子转变为维生素C自由基 ,从而刺激了过氧自由基和烷氧自由基的形成 ,过氧自由基的双分子反应很可能就是体系内1O2 产生的反应机制     

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 (T1) and the phase memory times (Tm) were obtained for the synthetic melanin system from 3-hydroxytyrosine (dopa) by means of electron spin echo spectroscopy at 77 degrees 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 microseconds, 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 Fe3+ of g = 4.3 and Mn2+ 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.     

Ability of melanins to protect against the radiolysis of thymine and thymidine

Individuals with black skin rarely get skin cancer, and melanomas, tumors arising from pigmented cells, are generally resistant to radiation therapy. The role of melanin in these two phenomena has not been defined, but oxygen-radical species have been implicated in both effects. These studies were undertaken to determine the ability of various melanins to compete for ionizing radiation-produced radicals which destroy nucleic acid bases. The ability of Sigma eumelanin (S-eumelanin) to protect against the radiolysis of thymidine in buffered solutions was compared to the protective ability of seven amino acids, including melanin precursors; bovine serum albumin, as a model protein; ficoll, as a model polysaccharide; and DNA. Both proteins and polysaccharides are known to scavenge hydroxyl radicals in cells. The concentration of thymidine after exposure to gamma radiation was determined by High Performance Liquid Chromatography (HPLC) analysis after removal of insoluble melanin by acid precipitation. S-eumelanin was more effective at competing with thymidine for free radicals than bovine serum albumin, Ficoll, or DNA, but less effective than certain of the small molecules. Several of the above compounds were also examined for ability to protect against thymine radiolysis. In addition, melanins from other sources were compared to S-eumelanin. Of these, enzymatically synthesized phaeomelanin was the most effective. The results indicate that melanins can compete for base- and nucleoside-damaging free radicals more effectively than other cellular macromolecules. Of the small molecules, the phenolic compounds had the greatest scavenging ability. In vivo, melanins are found in melanosomes bound to protein. Therefore, the relevance of these findings to the photo- and radiobiology of melanins in vivo has yet to be determined.     

Lipid peroxidation and function of some antioxidant enzyme systems in corn and oats in acute injury by hydrogen fluoride

Experimental research showed the HF main harmful effect at the maximal exposure time (12 h). Intensity of chemiluminescence processes was enhanced almost in 1.5-fold. In the maize leaves the reduced chemiluminescence intensity was observed under the maximal toxicant concentration (1 mg/m3) effect, whereas the further growth of chemiluminescence intensity was observed in the oat plants. The total amount of the lipid peroxidation products was increased dependently on the toxicant concentration in all experimental variants. Changes in the studied enzymatic system activities were multidirected. Analysis of all activities dynamics for the SOD, GGTP, GST, GR and GP showed that GGTP and sometimes GP were involved more rapidly then SOD into the processes of detoxication of lipid peroxidation products and metabolites formed under the HF effect. However, the correlation between changes both in the SOD and GP activities failed to be found. The obtained data give a basis to conclude that during period between 8 and 12 h of HF exposure the essential changes in the activities of SOD and glutathione enzymatic systems took place as an adaptive response of plant cell to the investigated stress-factor impact.     

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.