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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.

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.

Melanins are polymeric phenolic pigments classified into two groups based on their chemical structures and molecular precursors: eumelanin (brown-black) and pheomelanin ( yellow-red). Eumelanin is highly resilient and has a proven fossil record, extending back at least similar to 200 Ma. It is widespread in the biological world, occurring in fungi, the ink sacs of cephalopods, the feathers of birds, and the hair, skin, eyes, brain and inner ears of mammals. Although the presence and chemical attributes of fossil eumelanin have been documented, there are few data constraining its long term survival. Here we use a diversity of analytical techniques to compare the chemistry and morphology of fossilized cephalopod ink from three deposits of similar age and lithology, but different maturation histories. We demonstrate that the chemistry of eumelanin begins to alter at the onset of the oil window and is largely independent of age. The decrease in surviving melanin is accompanied by an increase in the relative abundance of organic macromolecular material (kerogen) but, critically for the correct interpretation of fossils, is not accompanied by a consistent change in granule morphology. (C) 2013 Elsevier Ltd. All rights reserved.

Melanins are biological pigments found throughout the animal kingdom that have many diverse functions. Pump-probe imaging can differentiate the two kinds of melanins found in human skin, eumelanin and pheomelanin, the distributions of which are relevant to the diagnosis of melanoma. The long-term stability of the melanin pump-probe signal is central to using this technology to analyze melanin distributions in archived tissue samples to improve diagnostic procedures. This report shows that most of the pump-probe signal from eumelanin derived from a Jurassic cephalopod is essentially identical to that of eumelanin extracted from its modern counterpart, Sepia officinalis. However, additional classes of eumelanin signals found in the fossil reveal that the pump-probe signature is sensitive to iron content, which could be a valuable tool for pathologists who cannot otherwise know the microscopic distributions of iron in melanins.

Qualitative and quantitative insights into the capacity and association constant for the binding of chymotrypsin to polyacrylate-coated gold nanoparticles is determined using fluorescence quenching, optical absorption and circular dichroism spectroscopy, isothermal calorimetry, and gel electrophoresis. The collective data reveal a binding capacity and constant for this particular system of ~7 and ~2 × 10(6) M(-1), respectively. These values vary among the individual techniques, and not all techniques are able to provide quantitative information. The present study demonstrates that accurately quantifying the association between nanoparticles and biological materials requires using multiple approaches to ensure consistency among the binding parameters determined.

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.

Central to understanding the photochemical properties of melanosomes is a direct measurement of their absorption coefficients. Herein, the absorption spectra of intact melanosomes of varying molecular compositions and embryonic origins were measured and compared over the spectral range from 245 to 310 nm. The absorption spectra of melanosomes predominately comprised of the eumelanin pigment were found to differ significantly from their constituent precursor molecules, 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA). This difference was most notable in the UV-A region and indicates that the electronic structures of the monomeric building blocks, DHICA in particular, are significantly modified upon polymerization to the melanin pigment. Furthermore, in comparing embryonic differences, the absorption coefficients of melanosomes isolated from bovine retinal pigment epithelial (RPE) cells (originating from the primitive forebrain) were greater than those for bovine choroid or iris melanosomes (originating from the neural crest). This difference suggests that either the pigment is present in greater density in RPE melanosomes or that there is an underlying difference in molecular structure.

This review examines recent studies on the thermodynamics of copper association with amyloid-β, α-synuclein and prion protein, with an eye towards using this information to understand the etiology of associated neurodegenerative diseases. A variety of binding affinities and binding sites, which are essential to understand the function and consequence of copper-protein interaction, have been reported for copper to these three neurobiologic systems. This current review reconciles the disparate models presented in the literature.

The accumulation of lipofuscin has previously been implicated in several retinal diseases including Best's macular dystrophy, Stargardt's disease and age-related macular degeneration (AMD). Previously one of the major fluorophores of lipofuscin was identified as a bis-retinoid pyridinium salt called A2E, which is known to photochemically cause damage. In addition to A2E, there are numerous components in RPE lipofuscin that are unidentified. These compounds were determined to be structurally related to A2E by their fragmentation pattern with losses of 106, 190, 174 and/or 150 amu from the parent ion and the formation of fragments of ca 592 amu. The vast majority consists of relatively hydrophobic components corresponding to derivatized A2E with molecular weights in discrete groups of 800–900, 970–1080 and > 1200 m/z regions. In order to determine the mechanism of these modifications, A2E was chemically modified by; (1) the formation of specific esters, (2) reaction with specific aldehydes and (3) spontaneous auto-oxidation. The contribution of ester formation to the naturally occurring components of lipofuscin was discounted since their fragmentation patterns were different to those found in vivo. Alternatively, reactions with specific aldehydes result in nearly identical products as those found in vivo. Artificial aging of RPE lipofuscin gives a complex mixture of structurally related components. This results from the auto- and/or photooxidation of A2E to form aldehydes, which then back react with A2E giving a series of higher molecular weight products. The majority of these modifications result in compounds that are much more hydrophobic than A2E. These higher molecular weight materials have increased values of log P compared to A2E. This increase in hydrophobicity most likely aids in the sequestering of A2E into granules with the concomitant diminution of its reactivity. Therefore, these processes may serve as protective mechanisms for the RPE. Copyright © 2010 John Wiley & Sons, Ltd.

"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.

There is no consensus on the coordinating ligands for Cu(2+) by Abeta. However, the differences in peptide sequence between human and rat have been hypothesized to alter metal ion binding in a manner that alters Cu(2+)-induced aggregation of Abeta. Herein, we employ isothermal titration calorimetry (ITC), circular dichroism (CD), and electron paramagnetic resonance (EPR) spectroscopy to examine the Cu(2+) coordination spheres to human and rat Abeta and an extensive set of Abeta(16) mutants. EPR of the mutant peptides is consistent with a 3N1O binding geometry, like the native human peptide at pH 7.4. The thermodynamic data reveal an equilibrium between three coordination spheres, {NH(2), O, N(Im)(His6), N(-)}, {NH(2), O, N(Im)(His6), N(Im)(His13)}, and {NH(2), O, N(Im)(His6), N(Im)(His14)}, for human Abeta(16) but one dominant coordination for rat Abeta(16), {NH(2), O, N(Im)(His6), N(-)}, at pH 7.4-6.5. ITC and CD data establish that the mutation R5G is sufficient for reproducing this difference in Cu(2+) binding properties at pH 7.4. The substitution of bulky and positively charged Arg by Gly is proposed to stabilize the coordination {NH(2), O-, N(Im)(His6), N(-)} that then results in one dominating coordination sphere for the case of the rat peptide. The differences in the coordination geometries for Cu(2+) by the human and rat Abeta are proposed to contribute to the variation in the ability of Cu(2+) to induce aggregation of Abeta peptides.

Central to understanding the photochemical function(s) of melano- somes is the determination of their absorption properties and an understanding of how the absorption varies with the molecular composition of melanin. Herein, the absorption coefficients for melanosomes containing predominantly eumelanin, a polymeric pigment derived from the molecular precursors 5,6-dihydroxyindole (DHI)and5,6-dihydroxyindole-2-carboxylicacid(DHICA),arereportedfor λ=244nm. The absorption coefficient varies with the DHICA/DHI ratio, determined from chemical degradationanalyses.WithincreasingDHICAcontent,theabsorptioncoefficientof the melanosome increases. This observation is consistent with reported extinction coefficients, which reveal that at 244 nm, the extinction coefficient of DHICA is a factor of ∼2.1 greater than that of DHI. The melanosome absorption coefficients are compared to predicted values based on a linear combination of the absorption of the constituent monomers.

Uveal melanosomes from the iridal stroma contain both eumelanin and pheomelanin, the ratio of which varies with iris color. Herein, we report the absorption coefficient at lambda = 244 nm for individual human iridal stroma melanosomes from dark brown and blue-green irides. The melanosomes are nearly identical in size, but differ in the relative concentration composition, ranging from a eumelanin/pheomelanin ratio of 14.8:1 (dark brown) to 1.3:1 (blue-green or hazel). The absorption coefficient of the melanosome decreases as its pheomelanin content increases. The origin of this decrease is attributed to a corresponding decrease in the number of UV-absorbing chromophores, reflecting the different molecular volumes of the monomeric building blocks of the two pigments. In agreement with reported data on synthetic pigments, the absorption coefficient of pheomelanin is found to be slightly larger than that for eumelanin at lambda = 244 nm (by a factor of 1.2). On the basis of the reported optical properties of synthetic models, this result suggests that the absorption of pheomelanin is less than eumelanin at wavelengths of biological relevance ( approximately 315-400 nm).

The morphological and chemical changes associated with the exposure of melanosomes to methyl iodide are assessed by a variety of analytical, imaging and spectroscopic methods. Scanning electron microscopy, light scattering and N(2) adsorption measurements all indicate significant changes in the morphology of the pigment following methylation. Solid-state nuclear magnetic resonance (SS-NMR) spectroscopy and chemical degradation analysis reveals the methylation results in the introduction of ester groups into the pigment structures. Amino acid analysis further reveals that Arg, Cys, His, Ser and Tyr undergo methylation; the SS-NMR data provide additional evidence for the methylation of the sulfur of Cys. Methylation results in increased solubility of the melanosome; the absorption properties of the dissolved material are characterized by an absorption maximum at 225 nm, with a long tail throughout the UV-A and UV-B, indicating that the solubilized material is a combination of protein and pigment. The methylation-induced decomposition of the melanosomes provides new insights into both the observed increase in O-methyl derivatives of the indolic precursor to eumelanin in the urine of melanoma patients and how increased levels of biologic methylating agents in the brain induce symptoms that resemble Parkinson's disease.

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.

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.

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 μg/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.

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.

An accurate data analysis method for determining stoichiometry and thermodynamic parameters from isothermal titration calorimetry data for the binding of macromolecules to metal cations that are solubilized through an association with a weak ligand is presented. This approach is applied to determine the binding constant for the association of Cu(II) to the first 16 residues of the Alzheimer's amyloid beta peptide, Abeta(1-16) under conditions where Cu(II) is rendered soluble through weak binding to glycine. At pH 7.2 and 37 degrees C, a binding constant of 1.5 x 10(9) M-1 (Kd = 0.7 nM) is determined for the association of Cu(II) with Abeta(1-16).

The binding of tetra(4-N,N,N,N-trimethylanilinium)porphyrin (TAP) to melanins quenches the porphyrin emission. Time-resolved femtosecond absorption spectroscopy reveals that the mechanism behind this quenching is ultrafast nonradiative energy transfer (τET < 100 fs) from electronically excited TAP to melanin. Similar dynamics are observed for both dopa and cysteinyldopa melanins. Steady-state emission studies demonstrate that the emission from melanin increases upon excitation of bound TAP, thereby confirming that rapid energy transfer occurs. These results are consistent with previous photoacoustic studies, which revealed that the TAP–melanin complex behaves like a supermolecular system liberating heat as a whole.

The presence of lipofuscin in postmitotic cells is considered a hallmark of the aging process. In the retinal pigment epithelium (RPE), lipofuscin is found as micrometer-sized spherical particles and characterized by its yellow autofluorescence when exposed to blue light. This exposure to light is also known to produce reactive oxygen intermediates (ROI), but the particular molecular constituent(s) responsible for this phototoxicity have yet to be completely identified. Resulting mostly from the autophagocytosis of intracellular organelles, the composition of lipofuscin is poorly defined but known to contain protein, lipids and several fluorophores. The subsequent identification of one of the fluorophores in lipofuscin, A2E, generated much interest and resulted in a variety of studies to understand its potential role in the phototoxicity of lipofuscin. Several modes of toxicity have been suggested through which A2E can affect the health of RPE cells. These modes include photoinduced production of ROI, which places additional oxidative stress on RPE cells, the disruption of membrane integrity through its natural role as an amphiphilic detergent and inhibition of key cellular functions. This article presents the current understanding of the photochemistry of A2E and its involvement as a phototoxic agent in RPE cells.

Bovine iris and choroid melanosomes at two ages (1 week and2 years) were examined by inductively coupled plasma mass spectrometry (ICP-MS), elemental analysis, infrared spectrometry (IR) and X-ray photoelectron spectrometry (XPS). When iris and choroid melanosomes at the same age were compared, the quantification of metal elements by ICP-MS revealed that choroid melanosomes had a higher binding capacity for the carboxylate-binding metal ions (e.g. Na+ K+, Mg2+, Ca2+ and Zn2+). Elemental analysis showed a higher O:N ratio in choroid melanosomes. Both observations suggested that choroid melanosomes have a higher content of carboxylate-containing monomer than iris melanosomes. IR spectrometric analysis showed a red shift (approximately 8 cm(-1)) of the absorption peak of aromatic C=C, C=N and C=O at approximately 1630 cm(-1) in the IR spectrum of iris melanosomes relative to choroid melanosomes. Increased conjugation in the molecular structure of the pigment is proposed to contribute to this peak shift. It is also notable that although the elemental analysis showed different C, N and O contents in the two types of melanosomes, XPS showed almost the same elemental compositions on the surface of two types of iris and choroid melanosomes studied. When the melanosomes from the same tissues at different ages were compared, ICP-MS analysis suggested that the number of carboxylate groups in the melanosomes decreased with age. Both elemental analysis and XPS showed that C:N ratio decreased with age, which was proposed to be due to both a decrease in carboxylate groups in mature samples and to the fissure of phenol rings caused by age-associated oxidation. Such age-related oxidative damage diminishes conjugation and is manifested by blue shifts of absorption peaks for aromatic double bonds in the IR spectra of mature melanosomes. XPS analysis showed that the ratio of C-O:C=O decreased with age. These tissue-related and age-related chemical differences between samples affected the optic density and metal binding properties of melanosomes, which are believed to be closely associated with the biological functions of melanins.

Calcium regulation in melanocytes affects numerous biological pathways including protecting the redox balance in the cell and regulating the supply of substrate, l-tyrosine, for melanogenesis. The pigment contained in the melanocytes, melanin, has been implicated in maintaining calcium homeostasis in the cell and is known to be involved with calcium ion regulation in the inner ear. Herein, the association constant for Ca(2+) binding to Sepia melanin is determined by isothermal titration calorimetry to be 3.3 (+/-0.2) x 10(3)/M. This value is comparable with other well-established intracellular calcium-binding proteins that serve to buffer calcium concentrations, lending further support to the hypothesis that melanosomes serve as intracellular mediators of calcium homeostasis in melanocytes. Using this binding constant and the data from a fluorescent Ca(2+) displacement assay, the pK(a) of the carboxyl group coordinated to Ca(2+) is determined to be 3.1 +/- 0.1.

A technique for measuring the photoionization spectrum and the photoelectron emission threshold of a microscopic structured material is presented. The theoretical underpinning of the experiment and the accuracy of the measurements are discussed. The technique is applied to titanium silicide nanostructures and melanosomes isolated from human hair, human and bovine retinal pigment epithelium cells, and the ink sac of Sepia officinalis. A common photothreshold of 4.5 ± 0.2 eV is found for this set of melanosomes and is attributed to the photoionization of the eumelanin pigment. The relationship between the photoionization threshold and the electrochemical potential referenced to the normal hydrogen electrode is used to quantify the surface oxidation potential of the melanosome. The developed technique is used to examine the effect of iron chelation on the surface oxidation potential of Sepia melanosomes. The surface oxidation potential is insensitive to bound Fe(III) up to saturation, suggesting that the metal is bound to the interior of the granule. This result is discussed in relation to the age-dependent accumulation of iron in human melanosomes in both the eye and brain.

We correct an error to our previous article. In Figure 5 and the text description of that data; iron concentrations were incorrectly labeled as μM, but they should have been mM. No conclusions of the paper are changed. The corrected text and figure are: Figure 5 Average spectra of iron loaded S. officinalis eumelanin obtained by varying the initial concentration of iron(III) chloride. “Figure 5 shows the pump–probe response of EDTA washed S. officinalis eumelanin loaded with different initial concentrations of iron(III) chloride. The eumelanin is saturated with iron when the initial concentration of iron is greater than 1 mM.9 Increasing the initial iron concentration causes the negative signal when the pulses are overlapped (at t = 0) to appear and the positive signal when the pump precedes the probe (t > 0) to disappear; the 0.25 mM is similar to the S. officinalis eumelanin spectrum given in Figure 1. This value is approximately in agreement with the reported concentrations found in natural S. officinalis melanin.9”

© 2015 John Wiley & Sons A/S. During the past decade, melanins and melanogenesis have attracted growing interest for a broad range of biomedical and technological applications. The burst of polydopamine-based multifunctional coatings in materials science is just one example, and the list may be expanded to include melanin thin films for organic electronics and bioelectronics, drug delivery systems, functional nanoparticles and biointerfaces, sunscreens, environmental remediation devices. Despite considerable advances, applied research on melanins and melanogenesis is still far from being mature. A closer intersectoral interaction between research centers is essential to raise the interests and increase the awareness of the biomedical, biomaterials science and hi-tech sectors of the manifold opportunities offered by pigment cells and related metabolic pathways. Starting from a survey of biological roles and functions, the present review aims at providing an interdisciplinary perspective of melanin pigments and related pathway with a view to showing how it is possible to translate current knowledge about physical and chemical properties and control mechanisms into new bioinspired solutions for biomedical, dermocosmetic, and technological applications.

Melanosomes and lipofuscin were isolated from 14-, 59-, and 76-year-old, human retinal pigment epithelium specimens and examined. The morphological features of these samples were studied by scanning electron microscopy and atomic force microscopy, and the photoionization properties were examined by photoelectron emission microscopy. Ovoid- and rod-shaped melanosomes were observed. The size of the granules and the distribution between the two shapes show no significant age-dependent change. However, there is a higher occurrence of irregularly shaped aggregates of small round granules in older samples which suggests degradation or damage to melanosomes occurs with age. The melanosomes from the 14-year-old donor eye are well characterized by a single photoionization threshold, 4.1 eV, while the two older melanosomes exhibit two thresholds around 4.4 and 3.6 eV. Lipofuscin from both young and old cells show two thresholds, 4.4 and 3.4 eV. The similarity of the potentials observed for aged melanosomes and lipofuscin suggest that the lower threshold in the melanosome sample reflects lipofuscin deposited the surface of the melanosome. The amount, however, is not sufficient to alter the density of the melanosome, and therefore these granules do not separate in a sucrose gradient at densities characteristic of the typical melanolipofuscin granule. These data suggest that thin deposits of lipofuscin on the surface of retinal pigment epithelium melanosomes are common in the aged eye and that this renders the melanosomes more pro-oxidant.

The application of UV-free electron laser photoelectron emission microscopy (UV-FEL PEEM) to measure the threshold photoelectron spectrum and photoionization potential for human eumelanosomes is described. The origin of potential artifacts and the limitations of the technique are discussed and their potential effects on the measured photoionization potential are quantified. The UV-FEL-PEEM images collected on human eumelanosomes isolated from black hair show that the organelle is photoionized by UV-B radiation. The photoionization threshold is determined to be 4.6 ± 0.2 eV. This result provides new insight into the origin of the differences between the photoionization and oxygen photoconsumption action spectra for eumelanins.

Ochratoxins are a class of naturally occurring compounds produced by several fungi. The most toxic is ochratoxin A (OTA), and occurrence of some human nephropathies and tumors correlate with enhanced OTA exposure. In this Account, the following areas are examined: molecular details of the binding of OTA to human ser-um albumin (HSA), the influences of binding to HSA on the trans-port of OTA across epithelial cell membranes by organic anion transport proteins, the oxidative activation of OTA, and the for-mation of OTA adducts with biological molecules. These studies are beginning to provide a detailed chemical model for the trans-port, accumulation, and genotoxic and carcinogenic effects of OTA.

The binding site of ochratoxin A (OTA) within domain 2A of human serum albumin (HSA) is examined by theoretical simulations and site-directed mutagenesis experiments. The calculated binding constant, based on docking experiments and theoretical affinity constants derived from the empirical free energy of binding as implemented in AutoDock 3.0, for the OTA dianion (3.7 × 106 M-1) is in good agreement with experimental value of 5.2 × 106 M-1. The carboxy terminus of OTA associates with R218 and R222 of the protein. Binding is reduced by over an order of magnitude for the mutant R218A in both experiments and theoretical simulations. The carbonyl of the lactone and the phenolic group of OTA are in close proximity to R257. The experimental binding constant of OTA to the R257A mutant is 1.6 × 105 M-1, over an order of magnitude smaller than for the wild-type protein. The predicted binding constant based on a comparison of the lowest-energy conformer from docking studies performed in AutoDock 3.0 of OTA to the R...

purpose. Lipofuscin accumulates in human retinal pigment epithelium (RPE) cells with age and may be the main factor responsible for the increasing susceptibility of RPE to photo-oxidation with age. As the composition, absorption, and fluorescence of lipofuscin undergo age-related changes, the purpose of this study was to determine whether photoreactivity of lipofuscin granules also changes with the donor age.\ud \ud methods. To determine whether the photoreactivity of lipofuscin itself is age related, lipofuscin granules were isolated from human RPE and pooled into age groups. Photoreactivity was assessed by measuring action spectra of photo-induced oxygen uptake and photogeneration of reactive oxygen species. Separation of chloroform-soluble (ChS) and -insoluble (ChNS) components by Folch’s extraction was used to determine the factors responsible for the age-related increase in lipofuscin photoreactivity.\ud \ud results. The observed rates of photo-induced oxygen uptake and photo-induced accumulation of superoxide-derived spin adducts indicated that when normalized to equal numbers of lipofuscin granules, aerobic photoreactivity of lipofuscin increased with age. Both ChS and ChNS mediated photogeneration of singlet oxygen, superoxide radical anion, and photo-oxidation of added lipids and proteins. Although both ChS and ChNS exhibited substantial photoreactivities, neither exhibited significant age-related changes when normalized to equal dry mass. In contrast, ChNS contents in lipofuscin granules significantly increased with aging.\ud \ud conclusions. Aerobic photoreactivity of RPE lipofuscin substantially increases with aging. This effect may be ascribed to the increased content of insoluble components.

An enhanced photoreactivity of the red melanin, pheomelanin compared to the black melanin, eumelanin is commonly invoked to explain why fair-skinned individuals are more susceptible to skin cancers. Ultrafast pump−probe spectroscopy is used to examine the primary photodynamics of eumelanin from Sepia officinalis and synthetic pheomelanin following excitation at 303 nm. Both melanins reveal a transient absorption throughout the visible spectrum that rises within the instrument response. On the picosecond time scale, the transient absorption signal decays to a constant nonzero value, and the dynamics to nonexponential. Thus, both melanins do not exhibit complete ground-state recovery, suggestive of the formation of long-lived intermediates. Comparison to the initial transient absorption indicates the yield of such species is identical for the two pigments. The shape of the transient spectrum for the two melanins differ; however, global fits to the 13 different probe wavelengths used to construct each spectr...

Ochratoxin A (OTA), a fungal metabolite of strains of Penicillium and Aspergillus, binds in its dianion form to Sudlow site I of human serum albumin (HSA) with high affinity. In this study, isothermal calorimetry (ITC) is used to study the binding of OTA and its O-methyl derivative (MOA). Calculations of the equilibrium geometry of the monoanion and dianion of OTA reveal only small structural changes among the lowest energy conformers. The ITC data show the binding of MOA, which lacks the phenolic proton of OTA, is accompanied by the uptake of a proton from the surrounding solvent. At pH 7.13, the binding of OTA is accompanied by uptake of 0.43 ± 0.15 protons from the solvent. At this pH, the monoanion (0.54) and dianion (0.46) forms of OTA are both present in solution. However, the pKa of the phenolic group of OTA decreases by more than three units upon protein binding, and so all available OTA is bound to the protein as the dianion. To account for the ITC data, a model is proposed in which the proton is...

Computation methods were used to examine the tautomerization equilibria for 5,6-dihydroxyindole (DHI, 2) and 5,6-indolequinone (IQ, 3). Relative energies were calculated at the B3LYP and PBE0 level of theory; solvent effects were modeled by using the CPCM method. Nine tautomers of 2 were examined. Our data showed that the generally accepted molecular structure of 2 corresponds to the most stable tautomer in both gas phase and aqueous solution. In aqueous solution, the quinone methide tautomer was the second most stable structure, being destabilized by 6 kcal mol-1. In contrast, gas-phase DFT calculations on four tautomers of 3 suggest this compound exists as a mixture of two tautomers, the quinone and the quinone methide. The relative concentration of the quinone methide is predicted to be sufficient to be detected experimentally. The energy difference between these two tautomers increases in solution so concentration of quinone methide should be negligible in polar solvents. Vertical excitation energies ...

Ochratoxins are fungal metabolites known to contaminate human and animal feed. Ochratoxin A (OTA) is the most widespread form of the toxins and is believed to be responsible for human renal diseases. For the majority of its lifetime within the body, OTA remains bound to the plasma protein human serum albumin (HSA). In this paper, the binding of three OTA derivatives (ochratoxin B (OTB), ochratoxin hydroquinone (OHQ), and O-methylated OTA (MOA)) to HSA is examined using optical spectroscopy. The binding constants decrease as follows: OTA2- (5.2 × 106 M-1) > OTB2- (1.8 × 106 M-1) > OHQ2- ∼ OHQ- (2.2 × 105 M-1) > MOA- (3 × 104 M-1). Studies of the binding of OTB, OHQ, and MOA to recombinant proteins corresponding to the domains of HSA reveal binding to all domains but with different affinities. Similar to OTA, all derivatives exhibit the largest binding constant for domain 2. These ligands are displaced by 2,3,5-triiodobenzoate (TIB), indicating they share a common binding site and bind to Sudlow Site I wit...

Gold nanoparticles (2.2 nm) protected by a densely packed monolayer of 9-(9-fluorenyl)-nonane-1-thiol (MPC-b) or a mixed monolayer of 9-(9-fluorenyl)-alkane-1-thiol with nonane-1-thiol (MPC-c and MPC-d) were studied by transient spectroscopy upon 305 nm excitation with subpicosecond laser pulses. Electronic relaxation of the gold core took place through a fast electron−phonon interaction within 2.0 ps, followed by a slow phonon−phonon interaction over a period longer than 15 ps. Electronic coupling of the excited fluorenyl groups with the gold core in these composite clusters affected the observed gold electron relaxation dynamics, likely by adding an excitation path for gold electrons by working as a sensitizer. The efficiency of energy transfer from the excited fluorene to the gold particles was affected by surface composition, with the ratio of the fast decay component to the slow decay component decreasing upon increasing the fraction of fluorenyl groups present at the surface. Thus, energy transfer f...

Melanins protect tissue by absorption and rapid nonradiative, nonreactive dissipation of ultraviolet (UV) light. However, melanins also produce reactive oxygen species (ROS) upon UV illumination. A chemical understanding of this dichotomy of photoprotection and phototoxicity has not been established. Herein this issue is examined by studying the UV-B induced oxidation and reduction of cytochrome c by ROS generated by different aggregation states of eumelanin. The quantum yield for superoxide anion by unaggregated oligomers is 7.4 x 10(-3), an order of magnitude greater than that characteristic of the bulk pigment. The quantum efficiency of hydrogen peroxide production by oligomers is 5.7 x 10(-3), and its production is attributed to reaction between superoxide anion and hydroquinone groups on eumelanin oligomers. Aggregation of oligomers results in a reduction of these quantum yields, having a significantly greater effect on the efficiency of hydrogen peroxide production. This effect is attributed to the decrease in surface concentration of hydroquinone sites upon aggregation. The effect of aggregation on the photogeneration of ROS serves to provide a foundation for the understanding of the dichotomy of photoprotective and phototoxic properties of melanin.

Melanosomes have the capacity to bind significant concentrations of calcium, suggesting there are surface binding sites that enable cations to access the interior of fully pigmented melanosomes. The surface of melanosomes is known to contain significant concentrations of carboxylate groups which likely are the initial biding sites for calcium, but their arrangement on the surface of the melanosome is not known. In various calcium proteins, a bidentate coordination by two carboxylate groups is the most common structure. In this study, we determine the distance between neighboring surface carboxylic acid groups by examining the binding of a series of diamines (+)H3N(CH2)mNH3(+) (m = 1-5) to melanosomes isolated from the ink sacs of Sepia officinalis and bovine choroid tissue. Of these amines, ethylenediamine (m = 2) shows optimal bidentate binding, revealing a narrow distribution of distances between neighboring carboxylic acid groups, ∼480 pm, similar to that found in proteins for calcium binding motifs involving two carboxylate groups.

Ochratoxin A (OTA), a fungal metabolite produced by several strains of Aspergillus and Penicillium, binds to serum albumin with high affinity only in the completely deprotonated form (dianion). The pKa of the phenolic group of OTA decreased by more than three units when it was bound to human serum albumin (HSA). Optical spectroscopy provided evidence that HSA has at least two binding sites for OTA, each being able to accommodate one dianion. These two sites were characterized by the binding constants of 5.2 × 106 and 1.0 × 105 M-1. The binding constant for the monoanion of OTA was estimated to be ∼103 M-1. Fluorescence polarization spectroscopy confirmed weak interaction of the monoanion with the protein in the F and E forms (pH 8) compared to the N form (pH ∼ 7). Fluorescence anisotropy decay of the dianion of OTA bound to HSA (36.6 ns) was much longer than its emission lifetime (5.2 ns) and was close to reported values for the rota...

Optical spectroscopy was used to examine the binding of ochratoxin A (OTA) and warfarin (WAR) to human serum albumin (HSA). Both molecules in the deprotonated form showed high affinity binding to HSA. The close proximity of the highest affinity binding site of the OTA dianion to that of the WAR monoanion was suggested by depolarization of WAR emission in ternary mixtures with [OTA]/[WAR] ≥ 3. Fluorescence polarization data also showed that both OTA and WAR simultaneously bind to HSA for 0.1 ≤ [OTA]/[WAR] ≤ 1. The failure of WAR to displace OTA under these conditions is in accord with the much smaller binding constant for WAR. In all displacement experiments either the HSA-to-WAR or HSA-to-OTA concentration ratio was kept constant and close to unity. Evidence of energy transfer from electronically excited WAR to OTA when both species are bound to HSA was obtained from fluorescence emission data. The efficiency of this energy transfer provided an estimate of 27 A as the upper limit of the distance between W...

Time-resolved spectroscopic techniques are used to determine the primary photoprocesses of A2E in solution. Comparison of the absorption and excitation spectrum of A2E in methanol solution indicates excitation at 400 nm populates the S2 excited state. Transient absorption signals decaying with a time constant of 0.9 ps were observed probing around 800 nm. These signals are attributed to the S2→Sn transition and reveal the S2→S1 relaxation occurs on the subpicosecond time scale. Transient absorption data probing at shorter wavelengths (480 and 550 nm) are attributed to the S1→Sn absorption. These signals exhibit an exponential decay with a time constant of 11 and 13 ps, respectively. Time-resolved emission measurements of the corresponding S0←S1 decay reveal a nonexponential decay; however, >95% of the signal amplitude is described by an exponential decay with a time constant of 12.4 ps. Both time-resolved emission and absorption experiments therefore indicate repopulation of the ground electronic state oc...

Steady-state and time-resolved spectroscopies are used to elucidate the primary photoprocesses following the excitation of ochratoxin A (OTA), its dechlorinated derivative ochratoxin B (OTB), and O-methyl ether of OTA (MOA). The excited-state dynamics of OTA and OTB depend on the protonation of the isocoumarin moiety. Fluorescence spectra of the protonated forms reveal anomalously large Stokes shifts that are attributed to the enol tautomer formed via an intramolecular excited-state proton transfer. No evidence for “normal” emission of the keto form of OTA and OTB is found even in aqueous solutions. MOA, which lacks a proton on the phenol moiety and exists, therefore, only in the keto form, exhibits weak fluorescence with a substantially smaller Stokes shift. The deprotonated species show relatively strong emission typical for phenolate anions. OTA decomposes slowly upon UV irradiation in aqueous solutions. The photoreaction quantum yield varies significantly with solution pH and O2 concentration. The hig...

Atomic force microscopy is used to investigate the structural organization of eumelanin isolated from the inks sacs of the cuttlefish Sepia officinalis. Deposits of eumelanin on mica reveal a range of structures. The most prevalent structure is an aggregate comprised of particles with diameters of 100-200 nm. This morphology is consistent with published SEM images of intact granules. Mechanical manipulation of these structures using the AFM tip show that these particles, while stable, are not a fundamental structural unit but are an aggregate of smaller constituents. Images of the bulk pigments also reveal the presence of filament structures that have an average height and width of approximately 5 nm and tens of nanometers, respectively. Taken along with recent X-ray scattering and mass spectrometry experiments, the AFM data provides strong supporting evidence for the conclusion that eumelanin is comprised of small oligomeric units and that the structural morphology observed in imaging experiments reflects aggregation of these oligomeric molecules. On the basis of the types of structures observed in the AFM images, a model is proposed for the assembly of the macroscopic pigment. The diversity of functions attributed to melanin in the literature is proposed to result from the heterogeneity of aggregated structures.

A novel approach to photoemission electron microscopy is used to enable the first direct measurement of the absorption coefficient from intact melanosomes isolated from bovine retinal pigment epithelial cells. The difference in absorption between newborn and adult melanosomes is in good agreement with that predicted from the relative amounts of the monomeric precursors present in the constituent melanin as determined by chemical degradation analyses. The results demonstrate that for melanosomes containing eumelanins, there is a direct relation between the absorption coefficient and the relative 5,6-dihydroxyindole: 5,6-dihydroxyindole-2-carboxylic acid (DHI:DHICA) content, with an increased UV absorption coefficient associated with increasing DHICA content.

This paper addresses the design and applications of a spectrally resolved confocal fluorescence microscope that measures spectral information for each pixel in the image with a fast multi-point approach. The combined spatial and spectral resolution of the device is demonstrated by performing two-photon imaging of mixtures of fluorescently labeled microspheres. Overlapping emission spectra of dye solutions contained in microcapillaries are readily distinguished. Images of a human skin tissue section and a mouse kidney section are presented which demonstrate that the structure and spectra of biologic samples can be resolved.

Novel high-resolution microscopic techniques have been utilized to characterize the spatial distribution of orange emitting fluorophores, e.g., A2E, in lipofuscin granules isolated from human retinal pigment epithelium cells. Granules have been imaged using atomic force microscopy (AFM) and near-field scanning optical microscopy (NSOM). Near-field scanning optical microscopy images of lipofuscin show that the orange fluorophores, including A2E, are not major components of the granule. These results suggest that the orange fluorophores may not be the dominant photoactive species in lipofuscin.

The emission properties of eumelanin from Sepia officinalis are examined following UV-A excitation. The emission decay is nonexponential, exhibiting decay components on the tens of picosecond to several nanosecond time scales. The corresponding depolarization dynamics are also nonexponential and reveal that the emission becomes totally depolarized with an average time constant of ∼80 ps at 20 °C. The depolarization of the emission is found to be activated; a simple Arrhenius fit to the depolarization rate data gives an activation barrier of 21 ± 3 kJ mol-1. The nonexponential emission decay is concluded to be a reflection of the structural disorder of eumelanin. The rapid and nonexponential depolarization dynamics are attributed to energy transfer processes that occur within “spherical” subunits that comprise the eumelanin aggregates.