Direct chemical evidence for eumelanin pigment from the Jurassic period.

Melanin is a ubiquitous biological pigment found in bacteria, fungi, plants, and animals. It has a diverse range of ecological and biochemical functions, including display, evasion, photoprotection, detoxification, and metal scavenging. To date, evidence of melanin in fossil organisms has relied entirely on indirect morphological and chemical analyses. Here, we apply direct chemical techniques to categorically demonstrate the preservation of eumelanin in two > 160 Ma Jurassic cephalopod ink sacs and to confirm its chemical similarity to the ink of the modern cephalopod, Sepia officinalis. Identification and characterization of degradation-resistant melanin may provide insights into its diverse roles in ancient organisms.

Near-infrared excited state dynamics of melanins: the effects of iron content, photo-damage, chemical oxidation, and aggregate size.

Ultrafast pump-probe measurements can discriminate the two forms of melanin found in biological tissue (eumelanin and pheomelanin), which may be useful for diagnosing and grading melanoma. However, recent work has shown that bound iron content changes eumelanin's pump-probe response, making it more similar to that of pheomelanin. Here we record the pump-probe response of these melanins at a wider range of wavelengths than previous work and show that with shorter pump wavelengths the response crosses over from being dominated by ground-state bleaching to being dominated by excited-state absorption. The crossover wavelength is different for each type of melanin. In our analysis, we found that the mechanism by which iron modifies eumelanin's pump-probe response cannot be attributed to Raman resonances or differences in melanin aggregation and is more likely caused by iron acting to broaden the unit spectra of individual chromophores in the heterogeneous melanin aggregate. We analyze the dependence on optical intensity, finding that iron-loaded eumelanin undergoes irreversible changes to the pump-probe response after intense laser exposure. Simultaneously acquired fluorescence data suggest that the previously reported "activation" of eumelanin fluorescence may be caused in part by the dissociation of metal ions or the selective degradation of iron-containing melanin.

High-performance liquid chromatography estimation of cross-linking of dihydroxyindole moiety in eumelanin.

Eumelanin pigments consist of various ratios of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and 5,6-dihydroxyindole (DHI). On alkaline hydrogen peroxide oxidation, these indole moieties give rise to pyrrole-2,3,5-tricarboxylic acid (PTCA) and pyrrole-2,3-dicarboxylic acid (PDCA), respectively. In a recent study, we detected considerable amounts of other pyrrole acids, pyrrole-2,3,4,5-tetracarboxylic acid (PTeCA) and pyrrole-2,3,4-tricarboxylic acid (isoPTCA), among the oxidation products of fossil ink sacs more than 160 million years old. PTeCA and isoPTCA arise from the cross-linking of the DHI moiety of eumelanin at the C2 and/or C3 positions. We mimicked the process of cross-linking by heating synthetic eumelanins prepared from various ratios of DHICA and DHI at 100 °C for 18 days (or at 40 °C for 180 days). The heated eumelanins were analyzed after alkaline peroxide oxidation as PTCA, PDCA, PTeCA, and isoPTCA by high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection. On heating, PTCA decreased rapidly due to decarboxylation, whereas PDCA decreased gradually. Concurrently, PTeCA increased gradually to levels close to PTCA. IsoPTCA also increased gradually at lower levels. Similar changes were observed at 40°C at a much slower rate. These findings suggest that the PTeCA/PTCA ratio may serve as a good indicator of aging (cross-linking) of eumelanin.

The effect of hydration on the UV absorption coefficient of intact melanosomes.

The physical properties of melanosomes have been shown to depend on water content. Herein, the ultraviolet absorption coefficient at λ = 244 nm for intact bovine choroidal melanosomes is determined from photoemission electron microscopy images recorded as a function of vacuum exposure. The dehydration of the melanosome under ultra-high vacuum manifests itself by a decrease in the absorption coefficient to about 60% of its initial value, and a concomitant increase in its image brightness. This change in the absorption of the melanosome is consistent with the influence of solvent polarity on the UV absorption coefficient of model systems for the pigment eumelanin, the predominant UV absorber contained in the choroid melanosomes.

The red and the black.

"Pigmentation, which is primarily determined by the amount, the type, and the distribution of melanin, shows a remarkable diversity in human populations, and in this sense, it is an atypical trait."--E. J. Parra. Melanin is found throughout the human body, skin, eye, brain, hair, and inner ear, yet its molecular structure remains elusive. Researchers have characterized the molecular building blocks of melanin but have not been able to describe how those components fit together in the overall architecture of the pigment. Melanin is categorized into two distinct classes, pheomelanin (red) and eumelanin (black). Although these classes share a common biosynthetic origin, specific molecular reactions occurring early in pigment production differentiate these two types. Pure eumelanin is found throughout nature, which has allowed researchers to characterize and quantify its chemical properties. However, pure pheomelanin is not observed in nature and rarely makes up more than ~25% of the total melanin present. In this Account, we explore our current understanding of the structure and reactivity of the red and black pigments. Epidemiological studies of skin and ocular cancers suggest that increasing relative proportions of pheomelanin correlate with increased risk factors for these diseases. Therefore, understanding the factors that control the relative abundance of the two pigments has become increasingly important. Consequently, researchers have worked to elucidate the chemistry of pheomelanin to determine whether the pigment could cause these cancers and, if so, by what mechanisms. The photoactivation of oxygen by pheomelanin in the UV-A range could contribute to the development of UV-induced cancers: recent measurement of the surface photoionization threshold of intact melanosomes reveals a lower photoionization potential for pheomelanin than eumelanin. A complementary study of intact human melanosomes isolated from different colored irides reveals that the absorption coefficient of the melanosome decreases with increasing pheomelanin content. These results suggest that the epidemiological data may simply result from an increased exposure of the underlying tissues to UV light.

New melanic pigments in the human brain that accumulate in aging and block environmental toxic metals.

Neuronal pigments of melanic type were identified in the putamen, cortex, cerebellum, and other major regions of human brain. These pigments consist of granules 30 nm in size, contained in organelles together with lipid droplets, and they accumulate in aging, reaching concentrations as high as 1.5-2.6 microg/mg tissue in major brain regions. These pigments, which we term neuromelanins, contain melanic, lipid, and peptide components. The melanic component is aromatic in structure, contains a stable free radical, and is synthesized from the precursor molecule cysteinyl-3,4-dihydroxyphenylalanine. This contrasts with neuromelanin of the substantia nigra, where the melanic precursor is cysteinyl-dopamine. These neuronal pigments have some structural similarities to the melanin found in skin. The precursors of lipid components of the neuromelanins are the polyunsaturated lipids present in the surrounding organelles. The synthesis of neuromelanins in the various regions of the human brain is an important protective process because the melanic component is generated through the removal of reactive/toxic quinones that would otherwise cause neurotoxicity. Furthermore, the resulting melanic component serves an additional protective role through its ability to chelate and accumulate metals, including environmentally toxic metals such as mercury and lead.

Neuromelanins in various regions of human brain are associated with native and oxidized isoprenoid lipids.

Neuromelanin (NM) isolated from seven regions of the human brain is found to contain series of natural and oxidized isoprenoid lipids. Specifically, dolichols (dol) and dolichoic acids (dol-CA) with 14-22 and 14-21 isoprene units are identified. Standards of nor-dolichol and nor-dolichoic acid were used to determine the relative amounts of dol and dol-CA compared to the total lipids present in NM for each region. The cerebellum, putamen, globus pallidus, and premotor cortex contained similar amounts of dol, comprising approximately 8-9.5% of the total lipid weight. Interestingly, the corpus callosum contains substantially lower quantities of both dol and dol-CA compared to the other regions-less than 4% dol relative to the total lipid weight. Oxidized and reduced dolichol-related species were identified and determined to be region-dependent.

Binding of Cu(II) to human alpha-synucleins: comparison of wild type and the point mutations associated with the familial Parkinson's disease.

The Cu(II)-alpha-synuclein interaction has been invoked as an important process in the pathogenesis of Parkinson's disease. Herein, we report binding constants and stoichiometry under near-physiological conditions for the binding of Cu(II) to human alpha-synuclein. Specifically, we compare the binding of Cu(II) to wild-type (WT) protein and two separate single mutation proteins that are associated with familial Parkinson's diseases: A30P and A53T. Cu(II) binds to all three alpha-synuclein proteins with a 1:1 stoichiometry. The Cu(II) binding constants, however, vary among the proteins studied. Cu(II) binding to WT and A53T at 37 degrees C is similar with a pH-dependent binding constant (K) of approximately 2.4 x 10(9) and approximately 4.8 x 10(9) M(-1) at pH 7.2 and 7.4, respectively. Cu(II) binding to A30P, however, exhibits two binding constants. The major binding site of A30P, characteristic of >90% of the bound Cu(II), has binding constants of 1.6 x 10(9) and 3.6 x 10(9) M(-1) at pH 7.2 and 7.4, respectively, slightly lower ( approximately 70%) than that characteristic of WT or A53T at the corresponding pH. The second less populated binding exhibited by A30P has a large binding constant, approximately 10(10) M(-1). Our size exclusion analysis ruled out the contribution of protofibrils to the strong Cu(II) binding. Previous studies indicated that A30P had a larger proportion of intermediate species (e.g., small oligomeric species, such as dimers and trimers) relative to WT and A53T. Thus, we propose that the high affinity site is attributed to the binding of Cu(II) to those small oligomeric species.

Human iridal stroma melanosomes of varying pheomelanin contents possess a common eumelanic outer surface.

Uveal melanosomes originating in the iridal stroma contain both black (eumelanin) and red (pheomelanin) pigment. Recent studies reveal that the eumelanin/pheomelanin ratio varies with iris color, with lower ratios being observed for lighter color (hazel, blue) irides. This is of great interest because the epidemiology of uveal melanomas also indicates an increased incidence for lighter-colored irides. Herein, we examine human iridal stroma melanosomes from dark brown and blue-green irides, which are characterized by a eumelanin/pheomelanin ratio of 14.8 and 1.3, respectively. Atomic force microscopy reveals that the melanosomes extracted from these different colored irides have a similar size and overall morphology. Studies of the surface ionization potentials reveal that the surface of these melanosomes is pure eumelanin, despite the significant difference in their overall pigment composition. These data indicate that the pheomelanin present in the melanosome is encased by eumelanin, providing support for the "casing model" architecture of mixed melanins advanced from kinetic studies of the early steps in the melanogenesis pathway. Because of the different bulk composition, these results indicate that the thickness of the outer eumelanin coating decreases as the iride color lightens. Oxidative damage to the melanosome surface is therefore more likely to enable access to the photoreactive pheomelanin in the lighter irides than that in the eumelanin-rich dark irides. This provides new insights into the potential contribution of iridal stroma melanosomes both to inducing oxidative stress and to accounting for the observed iris-color-dependent epidemiology of uveal melanoma.

Challenges in applying photoemission electron microscopy to biological systems.

Photoemission electron microscopy (PEEM) is a unique surface-sensitive instrument capable of providing real-time images with high spatial resolution. While similar to the more common electron microscopies, scanning electron microscopy and transmission electron microscopy, the imaging technology relies on the photogeneration of electrons emitted from a sample through light excitation. This imaging technique has found prominence in surface and materials sciences, being well suited for imaging flat surfaces, and changes that occur to that surface as various parameters are changed (e.g. temperature, exposure to reactive gases). Biologically focused PEEM received significant attention in the 1970s, but was not aggressively advanced since that pioneering work. PEEM is capable of providing important insights into biological systems that extend beyond simple imaging. In this article, we identify and establish important issues that affect the acquisition and analysis of biological samples with PEEM. We will briefly review the biological impact and importance of PEEM with respect to our work. The article also concludes with a discussion of some of the current challenges that must be addressed to enable PEEM to achieve its maximum potential with biological samples.

Neuromelanins isolated from different regions of the human brain exhibit a common surface photoionization threshold.

Neuromelanin isolated from the premotor cortex, cerebellum, putamen, globus pallidus and corpus callosum of the human brain is studied by scanning probe and photoelectron emission microscopies and the results are compared with previously published work on neuromelanin from the substantia nigra. Scanning electron microscopy reveals common structure for all neuromelanins. All exhibit spherical entities of diameters between 200 and 400 nm, composed of smaller spherical substructures, approximately 30 nm in diameter. These features are similar to that observed for many melanin systems including Sepia cuttlefish, bovine eye, and human eye and hair melanosomes. Photoelectron microscopy images were collected for all neuromelanins at specific wavelengths of ultraviolet light between 248 and 413 nm, using the spontaneous emission output from the Duke free electron laser. Analysis of the data establishes a common threshold photoionization potential for neuromelanins of 4.7 +/- 0.2 eV, corresponding to an oxidation potential of -0.3 +/- 0.2 V vs the normal hydrogen electrode (NHE). These results are consistent with previously reported potentials for neuromelanin from the substantia nigra of 4.5 +/- 0.2 eV (-0.1 +/- 0.2 V vs NHE). All neuromelanins exhibit a common low surface oxidation potential, reflecting their eumelanic component and their inability to trigger redox processes with neurotoxic effect.

Chemical characterization of pterosaur melanin challenges color inferences in extinct animals.

Melanosomes (melanin-bearing organelles) are common in the fossil record occurring as dense packs of globular microbodies. The organic component comprising the melanosome, melanin, is often preserved in fossils, allowing identification of the chemical nature of the constituent pigment. In present-day vertebrates, melanosome morphology correlates with their pigment content in selected melanin-containing structures, and this interdependency is employed in the color reconstruction of extinct animals. The lack of analyses integrating the morphology of fossil melanosomes with the chemical identification of pigments, however, makes these inferences tentative. Here, we chemically characterize the melanin content of the soft tissue headcrest of the pterosaur Tupandactylus imperator by alkaline hydrogen peroxide oxidation followed by high-performance liquid chromatography. Our results demonstrate the unequivocal presence of eumelanin in T. imperator headcrest. Scanning electron microscopy followed by statistical analyses, however, reveal that preserved melanosomes containing eumelanin are undistinguishable to pheomelanin-bearing organelles of extant vertebrates. Based on these new findings, straightforward color inferences based on melanosome morphology may not be valid for all fossil vertebrates, and color reconstructions based on ultrastructure alone should be regarded with caution.

Neuronal pigmented autophagic vacuoles: lipofuscin, neuromelanin, and ceroid as macroautophagic responses during aging and disease.

The most striking morphologic change in neurons during normal aging is the accumulation of autophagic vacuoles filled with lipofuscin or neuromelanin pigments. These organelles are similar to those containing the ceroid pigments associated with neurologic disorders, particularly in diseases caused by lysosomal dysfunction. The pigments arise from incompletely degraded proteins and lipids principally derived from the breakdown of mitochondria or products of oxidized catecholamines. Pigmented autophagic vacuoles may eventually occupy a major portion of the neuronal cell body volume because of resistance of the pigments to lysosomal degradation and/or inadequate fusion of the vacuoles with lysosomes. Although the formation of autophagic vacuoles via macroautophagy protects the neuron from cellular stress, accumulation of pigmented autophagic vacuoles may eventually interfere with normal degradative pathways and endocytic/secretory tasks such as appropriate response to growth factors.

Insights into melanosomes and melanin from some interesting spatial and temporal properties.

Melanosomes are organelles found in a wide variety of tissues throughout the animal kingdom and exhibit a range of different shapes: spheres of up to approximately 1 mum diameters and ellipsoids with lengths of up to approximately 2 mum and varying aspect ratios. The functions of melanosomes include photoprotection, mitigation of the effects of reactive oxygen species, and metal chelation. The melanosome contains a variety of biological molecules, e.g., proteins and lipids, but the dominant constituent is the pigment melanin, and the functions ascribed to melanosomes are uniquely enabled by the chemical properties of the melanins they contain. In the past decade, there has been significant progress in understanding melanins and their impact on human health. While the molecular details of melanin production and how the pigment is organized within the melanosome determine its properties and biological functions, the physical and chemical properties of the surface of the melanosome are central to their range of ascribed functions. Surprisingly, few studies designed to probe this biological surface have been reported. In this article, we discuss recent work using surface-sensitive analytic, spectroscopic, and imaging techniques to examine the structural and chemical properties of many types of natural pigments: sepia melanin granules, human and bovine ocular melanosomes, human hair melanosomes, and neuromelanin. N 2 adsorption/desorption measurements and atomic force microscopy provide novel insights into surface morphology. The chemical properties of the melanins present on the surface are revealed by X-ray photoelectron spectroscopy and photoemission electron microscopy. These technologies are also applied to elucidate changes in surface properties that occur with aging. Specifically, studies of the surface properties of human retinal pigment epithelium melanosomes as a function of age are stimulating the development of models for their age-dependent behaviors. The article concludes with a brief discussion of important unanswered research questions in this field.

Quantification of the binding constant of copper(II) to the amyloid-beta peptide.

The amyloid beta (A beta) peptide of Alzheimer's disease binds copper(II), and the peptide-bound metal may be a source of reactive oxygen species and neurotoxicity. To circumvent peptide aggregation and reduce redox activity, there is growing interest in using metal chelates as drug therapeutics for AD, whose design requires accurate data on the affinity of A beta peptides for copper(II). Reports on Cu2+ binding to A beta range from approximately 10(5) to approximately 10(9); these values' being obtained for different peptide lengths (1-16, 1-28, 1-40, 1-42) at varying pH. Herein, we report that Cu2+'s binding to A beta(1-40) at 37 degrees C occurs in a 1:1 stoichiometry with a pH-dependent binding constant: 1.1 (+/-0.2) x 10 (9) M (-1) and 2.4 (+/-0.2) x 10 (9) M(-1) at pH 7.2 and 7.4, respectively. Under identical conditions, A beta(1-16) reveals a comparable binding constant, confirming that this portion of the peptide is the binding region. Several previously reported values can be reconciled with the current measurement by careful consideration of thermodynamics associated with the presence of competing ligands used to solubilize copper.

Probing skin pigmentation changes with transient absorption imaging of eumelanin and pheomelanin.

As some of the most ubiquitous and biologically important natural pigments, melanins play essential roles in the photoprotection of skin. Changes in melanin production could potentially be useful for clinical diagnosis of the progression stage of melanoma. Previously we demonstrated a new method for imaging melanin distribution in tissue with two-color transient absorption microscopy. Here we extend this study to longer wavelengths and show that we are able to image melanin in fixed thin skin slices with higher signal-to-noise ratios (SNRs) and demonstrate epimode imaging. We show that both photothermal effects and long-lived excited states can contribute to the long-lived signal. Eumelanin and pheomelanin exhibit markedly different long-lived excited state absorption. This difference should enable us to map out their respective distribution in tissue samples with subcellular resolution. This technique could provide valuable information in diagnosing the malignant transformation of melanocytes.

Ultra-low temperature oxidation of 5,6-dihydroxyindole: a novel approach to study synthetic melanogenesis.

The detailed structure of melanin remains elusive due to the complexity and insolubility of the pigment. Herein we describe a novel oxidation of 5,6-dihydroxyindole (DHI) as a means to characterize soluble intermediates formed prior to oligomerization. The approach entails the use of a biomimetic copper-peroxo oxidant, at ultra-low temperature (-78 degrees C). DHI oxidized by [LCuII(O2)CuIIL]B(C6F5)4 (L=2,6,10-trimethyl-2,6,10-triazaundecane) under argon produces the one electron oxidation product, semiquinone radical, which is spectroscopically observed at -78 degrees C. MS analysis of the reaction mixture reveals the DHI dimer as well as other extensively oxidized DHI units.

Different molecular constituents in pheomelanin are responsible for emission, transient absorption and oxygen photoconsumption.

Steady-state absorption and emission spectroscopies, oxygen activation and transient spectroscopy on a single sample of synthetic pheomelanin are compared. The absorption, emission and excitation spectra of pheomelanin depend on the molecular weight (MW) of the dissolved pigment constituents. While weakly-depending on MW, the maximum of the emission excitation spectrum is approximately 400 nm. The electron paramagnetic resonance oximetry measurements show a clear increase in oxygen uptake between 338 and 323 nm, and also reveal a local enhancement around approximately 370 nm. Pump-probe absorption spectroscopy reveals that photoexcitation of pheomelanin by UVA light generates a transient absorption peak in the visible and UV regions within the instrument response. The action spectrum for the formation of the 780 nm transient species peaks at approximately 360 nm. While both transient absorption bands show the same ultrafast decay component, the 780 nm peak completely vanishes on the 10s of picosecond time scale, but the UV band shows a kinetic evolution to a subsequent intermediate. While in a similar wavelength range, the maximum of the action spectrum derived from the transient data, the emission excitation spectrum and the action spectrum for photoconsumption all differ from one another, suggesting that the chromophore responsible for each is not that same. This raises concern about comparing the results from different photochemical methodologies for melanin, which is a specific case of comparing data on systems where molecular constituents are not well defined.

Role of ocular melanin in ophthalmic physiology and pathology.

The mammalian eye consists of several layers of pigmented tissues that contain melanin. The eye is a unique organ for pigment cell research because one can isolate and compare melanosomes from different tissues and embryonic origins. Retinal, iris and ciliary pigment epithelial cells are derived from the neural ectoderm, more specifically from the extremity of the embryonic optical cup, which is also the origin of the retina. In contrast, the pigment-generating cells in the choroid and in the stroma of the iris and ciliary body, uveal melanocytes, are developed from the neural crest, the same origin as the melanocytes in skin and hair. This review examines the potential functions of ocular melanin in the human eye. Following a discussion of the role of melanins in the pigment epithelium and uveal melanocytes, three specific topics are explored in detail-photo-screening protective effects, biophysical and biochemical protective effects, and the biologic and photobiologic effects of the two main classes of melanins (generally found as mixtures in ocular melanosomes)--eumelanin and pheomelanin.

Surface elastic properties of human retinal pigment epithelium melanosomes.

Atomic force microscope (AFM) imaging and nanoindentation measurements in water were used to probe the mechanical properties of retinal pigment epithelium melanosomes isolated from 14-year-old and 76-year-old donors. Topographic imaging reveals surface roughness similar to previous measurements on dry melanosomes. Force-indentation measurements show different types of responses that were catalogued into four different categories. In these measurements no permanent surface damage of melanosomes was observed as revealed by imaging before and after indentation measurements. The indentation measurements that exhibited nearly elastic responses were used to determine the Young's modulus of melanosomes. The average Young's modulus values are similar for 14-year-old and 76-year-old melanosomes with a somewhat narrower distribution for the 14-year-old sample. These elastic modulus values are considerably higher than the modulus of organelles with cytoplasm (<1 MPa) and approaching values of the modulus of protein crystals (approximately 100 MPa) indicating rather high packing density of biologic material in melanosomes. The width of the Young's modulus distributions is considerable spanning from few megapascals to few tens of megapascals indicating large heterogeneity in the structure. A fraction of the force curves cannot be described by the homogeneous elastic sample model; these force curves are consistent with approximately 10 nm structural heterogeneity in melanosomes. The approach-withdraw hysteresis indicates a significant viscoelasticity, particularly in the samples from the 14-year-old sample. Adhesion of the AFM probe was detected on approximately 3% and approximately 20% of the surface of 14-year-old and 76-year-old samples, respectively. In light of previous studies on these same melanosomes using photoelectron emission microscopy, this adhesion is attributed to the presence of lipofuscin on the surface of the melanosomes. This suggestion indicates that part of the difference in photochemical properties between the old and young melanosomes originates from surface lipofuscin.