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.     

Melanosomes from liver and skin of Rana esculenta L. A comparative chemical study

1. Melanosomes from skin and liver of Rana esculenta L. have been isolated and some chemical properties of the relevant melanin and protein components were compared. 2. In both cases the pigments show spectroscopic (ESR) and chemical characteristics similar to those of eumelanins. The melanin content in skin melanosomes is higher than in the liver counterparts. 3. Amino acid patterns of the two protein components are different in their quantitative composition and both are characterized by high levels of glycine and proline. 4. The results as a whole indicate that skin and liver melanosomes from the same animal markedly differ in their chemical composition.     

Regulation of Melanogenesis in Melanocytes

Melanogenesis refers to the biosynthesis of melanin pigment in pigment cells called melanocytes. Melanins are mixed biopolymers formed during a series of oxidation/reduction reactions that are initiated by the enzymatic hydroxylation of L-tyrosine to L-dopa. In living cells, melanogenesis is limited to melanosomes, the membrane bounded microscopic secretory granules of melanocytes. Melanosomes may be secreted into the environment as, for example, from the squid's ink gland; or be transferred to neighboring cells, such as the keratinocytes in human skin and hair; or they may remain within the pigment cell and change only their subcellular localization, as in the rapidly color-changing dermis of lower vertebrates. Regulation of the melanocytic phenotype involves synthesis of the biosynthetically active subcellular apparatus of melanogenesis, premelanosomes and tyrosinase, and the utilization of the final product, melanized melanosomes, in the translocation and secretory processes mentioned above. Genetic information for this regulation is stored in the nuclear genome whose expression is controlled by the intra- and extracellular environment. As premelanosomes become biosynthetically active, they mature into melanosomes by fusing with vesicles derived from the trans-Golgi network and the plasmalemma, thereby internalizing and incorporating contents and membrane components from inside the cell and the cell surface. In the process, melanosomes become acidified. The thesis pursued in this review explores the importance of the melanosome as the final common pathway of regulation of melanin biosynthesis.     

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.     

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.     

Retinal pigment epithelium pigment granules stimulate the photo-oxidation of unsaturated fatty acids

The cellular pigments of the retinal pigment epithelium (RPE) have been shown to catalyze free radical activity, especially when illuminated with visible or ultraviolet light. This activity is sufficient to cause photooxidation of several major cellular components. The present investigation determined the relative ability of melanin, lipofuscin, and melanolipofuscin granules isolated from human and bovine eyes to oxidize polyunsaturated fatty acids, specifically linoleic and docosahexaenoic acids. The dark reactivity as well as the light-stimulated reactions were determined. The production of hydroperoxide derivatives of the linoleic and docosahexaenoic acids were determined by NADPH oxidation coupled to the activity of glutathione peroxidase, and also by production of thiobarbituric acid reactive substances. All RPE pigment granules stimulated fatty acid oxidation when irradiated with short wavelength (< 550 nm) visible light, with the melanosomes exhibiting the greatest light-induced activity. Only lipofuscin granules, however, caused peroxidation of fatty acids in the dark. These findings provide additional support for the role of RPE pigments in "blue light toxicity" as well as indicating that accumulation of lipofuscin may contribute to increased photooxidation in the aging RPE.     

Morphological changes in hair melanosomes by aging

Various changes appear in hair by aging, and graying is the most remarkable one. Changes in melanocytes have been well studied as the cause; however, little is known about the change in melanosomes which have a role of carrying melanin pigments into hair shafts. Using pigmented hairs of Japanese females from their age of 4–75, I isolated melanosomes and observed them. As a result, I found a significant change in the morphology of hair melanosomes with age. They were ellipsoidal on the whole and there was no age dependence in the major axis, while the minor axis significantly increased and its frequency distribution broadened with age. The anticipated volume of the melanosome of the oldest person hairs was about twice larger than that of child hairs. This enlargement of melanosome seems to be a cause of the age‐related color change in pigmented hairs from brown to black.     

An estimate of melanosome concentration in pigment tissues.

Concentration of melanosomes in various tissues has been unknown because of the impracticability of their direct quantification. Using an indirect approach comprising the estimation of melanin both in freeze-dried tissue samples and in isolated melanosomes, we obtained data on the amount of melanosomes in various pigment tissues. The concentrations of melanosomes found in the tissues were relatively high, not only reflecting the dark color of pigment tissues but also explaining their capacity to perform various functions ascribed to the presence of melanin.     

Fungal pigments inhibit the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of darkly pigmented fungi

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS) has been used to discriminate moniliaceous fungal species; however, darkly pigmented fungi yield poor fingerprint mass spectra that contain few peaks of low relative abundance. In this study, the effect of dark fungal pigments on the observed MALDI mass spectra was investigated. Peptide and protein samples containing varying concentrations of synthetic melanin or fungal pigments extracted from Aspergillus niger were analyzed by MALDI–TOF and MALDI–qTOF (quadrupole TOF) MS. Signal suppression was observed in samples containing greater than 250 ng/μl pigment. Microscopic examination of the MALDI sample deposit was usually heterogeneous, with regions of high pigment concentration appearing as black. Acquisition of MALDI mass spectra from these darkly pigmented regions of the sample deposit yielded poor or no [M+H]⁺ ion signal. In contrast, nonpigmented regions within the sample deposit and hyphal negative control extracts of A. niger were not inhibited. This study demonstrated that dark fungal pigments inhibited the desorption/ionization process during MALDI–MS; however, these fungi may be successfully analyzed by MALDI–TOF MS when culture methods that suppress pigment expression are used. The addition of tricyclazole to the fungal growth media blocks fungal melanin synthesis and results in less melanized fungi that may be analyzed by MALDI–TOF MS.     

Neuromelanin of the Human Substantia Nigra: A Mixed-Type Melanin

Abstract: Model melanins, synthesized with different cysteinyldopamine/dopamine ratios in the incubates, were oxidized with KMnO₄ and the resulting compounds were analyzed by HPLC. The ratios between a phaeomelanin-derived compound, thiazole-4,5-dicarboxylic acid (TDCA), and a compound derived from eumelanin, pyrrole-2,3,5-tricarboxylic acid (PTCA), reflected the composition of the model melanins. The neuromelanin of the human substantia nigra was isolated, and the pigment, as well as intact brain tissue from human substantia nigra was oxidized with KMnO₄ and the TDCA/PTCA ratios were determined. Analysis of the isolated neuromelanin showed it to contain 2.3% sulfur and 8.1% nitrogen. The sulfur content indicates the pigment is a mixed-type melanin, and the TDCA/PTCA ratio indicates that it consists of units derived from benzothiazines and from indoles in about equal amounts.     

大鲵皮肤色素提取工艺及抗氧化研究

为优化大鲵皮肤黑色素的提取工艺条件,探讨大鲵皮肤黑色素组成成分及体外抗氧化活性,采用酶法和碱溶酸沉法提取大鲵皮肤黑色素,以氢氧化钠浓度、液料比、提取温度为影响色素提取率因素,优化黑色素提取工艺条件,用紫外-可见光谱仪、红外光谱仪和超高效液相质谱仪测定黑色素的光谱特性,测定其抗氧化性。结果表明:大鲵皮肤黑色素最佳提取工艺条件为氢氧化钠浓度1.5 mol/L、液料比1∶15、提取温度45℃,黑色素提取率达0.65%。大鲵皮肤黑色素的紫外最大吸收波长为214 nm,由真黑色素和脱黑色素两种色素组成,其对超氧阴离子自由基的清除率为30.51%,对羟基自由基的清除率为54.17%。大鲵皮肤黑色素具有一定的体外抗氧化能力。     

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.     

Photobleaching of retinal pigment epithelium melanosomes reduces their ability to inhibit iron‐induced peroxidation of lipids

Melanin in the human retinal pigment epithelium (RPE) is believed to play an important photoprotective role. However, unlike in skin, melanosomes in the RPE are rather long‐lived organelles, which increases their risk of modifications resulting from significant fluxes of light and high oxygen tension. In this work, we subjected purified bovine RPE melanosomes to prolonged aerobic exposure with intense visible and near ultraviolet radiation and studied the effects of irradiation on the melanosome's capacity to inhibit peroxidation of lipids induced by iron/ascorbate. We found that control, untreated melanosomes show a concentration‐dependent inhibition of the accumulation of lipid hydroperoxides and the accompanying consumption of oxygen, but photolysed melanosomes lose their antioxidant efficiency and even became prooxidant. The prooxidant action of partially photobleached melanosomes was observed for pigment granules with a melanin content reduced by about 50% compared with untreated melanosomes, as determined by electron spin resonance spectroscopy. We have previously shown that a similar loss in the content of the RPE melanin occurs during human lifetime, which may suggest that the normal antioxidant properties of human RPE melanin become compromised with aging.     

Pigment types in selected color genotypes of Asiatic sheep

The types and amounts of pigments in fibers from variously colored Tajik, Hissar, and Caracul sheep were determined by three methods: high-performance liquid chromatography, electron spin resonance spectroscopy, and light microscopic evaluation of melanosomes. In both dominant and recessive black lambs the color is due to eumelanin pigment. Brown and red phenotypes are the result of interaction of AWt and EBl, EBr, or EY alleles, and these colors are caused by mixtures of eumelanin and pheomelanin in varying ratios. The HPLC and ESR measurements detected these differences in melanin type, while direct characterization of melanosomes generally failed to distinguish between melanin type or relative ratio of melanin type.     

Photobleaching of retinal pigment epithelium melanosomes reduces their ability to inhibit iron-induced peroxidation of lipids

Melanin in the human retinal pigment epithelium (RPE) is believed to play an important photoprotective role. However, unlike in skin, melanosomes in the RPE are rather long-lived organelles, which increases their risk of modifications resulting from significant fluxes of light and high oxygen tension. In this work, we subjected purified bovine RPE melanosomes to prolonged aerobic exposure with intense visible and near ultraviolet radiation and studied the effects of irradiation on the melanosome's capacity to inhibit peroxidation of lipids induced by iron/ascorbate. We found that control, untreated melanosomes show a concentration-dependent inhibition of the accumulation of lipid hydroperoxides and the accompanying consumption of oxygen, but photolysed melanosomes lose their antioxidant efficiency and even became prooxidant. The prooxidant action of partially photobleached melanosomes was observed for pigment granules with a melanin content reduced by about 50% compared with untreated melanosomes, as determined by electron spin resonance spectroscopy. We have previously shown that a similar loss in the content of the RPE melanin occurs during human lifetime, which may suggest that the normal antioxidant properties of human RPE melanin become compromised with aging.     

FT-IR spectroscopy of natural melanins isolated from Harding-Passey mouse melanoma

The alterations caused on the structure of melanins by acid and alkaline extraction methods were studied by means of techniques such as Fourier Transform Infrared spectroscopy and thermogravimetry. Acid extraction results in the hydrolysis of aromatic monomers of the pigment, leading to uncyclicized residues and to a less conjugated polymer. Alkaline methods catalyse the covalent binding of proteins present in the melanin extract, thus leading to melanoprotein artifacts. A new method based on the delipidation with ether and deproteinization with SDS of previously isolated melanosomes is proposed. This method shows that melanins present in Harding-Passey mouse melanoma have a bound protein moiety, so that they should exist "in vivo" as melanoprotein complexes. Thermogravimetric data suggest that the chromogen moiety of the melanoproteins in Harding-Passey mouse melanoma is a mixture of eu- and pheomelanins.     

Photoaging of human retinal pigment epithelium is accompanied by oxidative modifications of its eumelanin

Although photodegradation of the retinal pigment epithelium (RPE) melanin may contribute to the etiology of age‐related macular degeneration, the molecular mechanisms of this phenomenon and the structural changes of the modified melanin remain unknown. Recently, we found that the ratio of pyrrole‐2,3,4,5‐tetracarboxylic acid (PTeCA) to pyrrole‐2,3,5‐tricarboxylic acid (PTCA) is a marker for the heat‐induced cross‐linking of eumelanin. In this study, we examined UVA‐induced changes in synthetic eumelanins to confirm the usefulness of the PTeCA/PTCA ratio as an indicator of photo‐oxidation and compared changes in various melanin markers and their ratios in human melanocytes exposed to UVA, in isolated bovine RPE melanosomes exposed to strong blue light and in human RPE cells from donors of various ages. The results indicate that the PTeCA/PTCA ratio is a sensitive marker for the oxidation of eumelanin exposed to UVA or blue light and that eumelanin and pheomelanin in human RPE cells undergo extensive structural modifications due to the life‐long exposure to blue light.     

Proteomic and bioinformatic characterization of the biogenesis and function of melanosomes

Melanin, which is responsible for virtually all visible skin, hair, and eye pigmentation in humans, is synthesized, deposited, and distributed in subcellular organelles termed melanosomes. A comprehensive determination of the protein composition of this organelle has been obstructed by the melanin present. Here, we report a novel method of removing melanin that includes in-solution digestion and immobilized metal affinity chromatography (IMAC). Together with in-gel digestion, this method has allowed us to characterize melanosome proteomes at various developmental stages by tandem mass spectrometry. Comparative profiling and functional characterization of the melanosome proteomes identified approximately 1500 proteins in melanosomes of all stages, with approximately 600 in any given stage. These proteins include 16 homologous to mouse coat color genes and many associated with human pigmentary diseases. Approximately 100 proteins shared by melanosomes from pigmented and nonpigmented melanocytes define the essential melanosome proteome. Proteins validated by confirming their intracellular localization include PEDF (pigment-epithelium derived factor) and SLC24A5 (sodium/potassium/calcium exchanger 5, NCKX5). The sharing of proteins between melanosomes and other lysosome-related organelles suggests a common evolutionary origin. This work represents a model for the study of the biogenesis of lysosome-related organelles.     

Melanosomal pH controls rate of melanogenesis, eumelanin/phaeomelanin ratio and melanosome maturation in melanocytes and melanoma cells.

The skin pigment melanin is produced in melanocytes in highly specialized organelles known as melanosomes. Melanosomes are related to the organelles of the endosomal/lysosomal pathway and can have a low internal pH. In the present study we have shown that melanin synthesis in human pigment cell lysates is maximal at pH 6.8. We therefore investigated the role of intramelanosomal pH as a possible control mechanism for melanogenesis. To do this we examined the effect of neutralizing melanosomal pH on tyrosinase activity and melanogenesis in 11 human melanocyte cultures and in 3 melanoma lines. All melanocyte cultures (9 of 9) from Caucasian skin as well as two melanoma cell lines with comparable melanogenic activity showed rapid (within 24 h) increases in melanogenesis in response to neutralization of melanosomal pH. Chemical analysis of total melanin indicated a preferential increase in eumelanin production. Electron microscopy revealed an accumulation of melanin and increased maturation of melanosomes in response to pH neutralization. In summary, our findings show that: (i) near neutral melanosomal pH is optimal for human tyrosinase activity and melanogenesis; (ii) melanin production in Caucasian melanocytes is suppressed by low melanosomal pH; (iii) the ratio of eumelanin/phaeomelanin production and maturation rate of melanosomes can be regulated by melanosomal pH. We conclude that melanosomal pH is an essential factor which regulates multiple stages of melanin production. Furthermore, since we have recently identified that pink locus product (P protein) mediates neutralization of melanosomal pH, we propose that P protein is a key control point for skin pigmentation. We would further propose that the wide variations in both constitutive and facultative skin pigmentation seen in the human population could be associated with the high degree of P-locus polymorphism.     

Melanocytes: the new Black

Melanocytes, pigment-producing cells residing primarily in the hair follicle, epidermis and eye, are responsible for skin hair and eye pigmentation. Pigmentation is achieved by the highly regulated manufacture of the pigment melanin in specialised organelles, melanosomes that are transported along dendritic processes before being transferred to growing hair, or keratinocytes where melanin protects from UV-induced DNA damage. Because loss of melanocytes gives a clear pigmentation phenotype yet is non-lethal, over 130 genes implicated in the development or function of this cell type have been identified to date, and in humans the loss of melanocytes or their ability to produce pigment, or transport or transfer melanosomes is associated with several diseases such as vitiligo, albinism and Hermansky-Pudlak syndrome. Importantly, the effective combination of genetics, cell and molecular biology possible with this cell type is attracting an increasing number of researchers focussed on understanding how cells coordinate survival, proliferation, differentiation and stem cell maintenance.