Insights into Melanosomes and Melanin from Some Interesting Spatial

J. Phys. Chem. B 2008, 112, 13201–13217

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CENTENNIAL FEATURE ARTICLE Insights into Melanosomes and Melanin from Some Interesting Spatial and Temporal Properties† John D. Simon,* Lian Hong, and Dana N. Peles Department of Chemistry, Duke UniVersity, Durham, NC ReceiVed: May 13, 2008; ReVised Manuscript ReceiVed: July 20, 2008

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 ∼1 µm diameters and ellipsoids with lengths of up to ∼2 µm 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. N2 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. 1. Introduction The history of research on pigmentation reaches back over 400 years. However, despite a considerable effort, general aspects of the molecular structure of pigments found in human tissue have been defined only recently and detailed structural information is still lacking. The gap in our knowledge is exacerbated by the very terminology still used. In this article, we first outline the history of the discovery of melanin and melanosomes with the goal to present our emerging understanding of their properties, along with the difficulties created by their names. Following this historical development, the article presents current research within the context of asking the question: what spatial and temporal scales are most important for understanding the structure and functions of naturally occurring pigment assemblies? We argue that there is a need to understand the physical and chemical properties of the surface of natural pigment structures and how these properties change † This year marks the Centennial of the American Chemical Society’s Division of Physical Chemistry. To celebrate and to highlight the field of physical chemistry from both historical and future perspectives, The Journal of Physical Chemistry is publishing a special series of Centennial Feature Articles. These articles are invited contributions from current and former officers and members of the Physical Chemistry Division Executive Committee and from J. Phys. Chem. Senior Editors. * Corresponding author. Phone: (919) 660-0330. Fax: (919) 684-4421.

over the human life span. Research focused along these lines is only beginning. We review recent studies that apply surfacesensitive methodologies to the study of intact melanosomes, and draw connections between the results of these studies and the function and age-dependent behavior exhibited by melanosomes. We conclude with a discussion of future research challenges. 2. A Brief History of the Past 400 Years of Melanin Research A historical perspective on melanin research reveals a fascination with human pigmentation for many centuries. In 1614, Santorio Santorius (1561-1636), an Italian physician, published De statica medicina (“On Medical Measurement”sthe first systematic study of basal metabolism) in which he attributed black skin’s complexion to the presence of bile.1 This work was generally supported by the Italian anatomist Marcello Malpighi (1628-1694), who reported in 1665 that the dermis and stratum corneum are colorless in both blacks and whites and that the blackness of Africans must originate in the underlying mucous and reticular bodyscolored by bile.2 Experiments by the French anatomist Alexis Littre´ (1658-1726) failed to find black gelatinous bile in skin but instead discovered an insoluble black pigment adhered to the reticular membrane.3 Claude-Nicolas Le Cat (1700-1768), a French physician,

10.1021/jp804248h CCC: $40.75  2008 American Chemical Society Published on Web 09/26/2008

13202 J. Phys. Chem. B, Vol. 112, No. 42, 2008 John D. Simon received his B.A. from Williams College and Ph.D. from Harvard University. He was a postdoctoral fellow with Professor M. A. El-Sayed at UCLA and held faculty appointments at UCSD before joining the Department of Chemistry at Duke University as the George B. Geller Professor in 1998. He is currently the Vice Provost of Academic Affairs at Duke. He is the Editor of Photochemistry and Photobiology, and the coauthor (with Don McQuarrie) of Physical Chemistry: A Molecular Approach. His research interests currently focus on the molecular structure, assembly, and aerobic reactivity of human pigments and amyloidogenic peptides. Lian Hong received her B.S. degree in Chemical Engineering from Nanjing University, M.S. degree in Physical Chemistry from Peking University, and Ph.D. degree in Physical Chemistry from Duke University. She is currently a Research Associate working in the Department of Chemistry at Duke University. Her research is focused on physical studies on human pigments and amyloidogenic peptides. Dana Peles received her B.S. degree in Chemistry and Mathematics from Randolph-Macon College. She is currently a Ph.D. candidate working in Dr. John Simon’s lab in the Department of Chemistry at Duke University. Her research is focused on the applications of photoemission electron microscopy to pigments and nanomaterials.

claimed that skin pigment was not bile but black deposits he called “æthiops”, which were secreted directly into the skin through the tips of the cutaneous nerves.4 The 20th century brought forth a more molecular picture of pigments and their formation. In 1901, Otto v. Furth and Hugo Schneider proposed that formation of the pigment now commonly called “melanin” (derived from the Greek word melas, meaning black) resulted from the reaction of an intercellular oxidase with aromatic groups in certain proteins.5 A Swiss dermatologist, Bruno Bloch (1878-1933), was inspired by this hypothesis and tried to substantiate it with experimental evidence. Bloch found that melanin granules were deposited in the cytoplasm of cells located in the basal layer of the epidermis, and called these specialized cells melanoblasts.6 He further put forth evidence that the catalytic effect of cells to oxidize dopa to melanin was due to an enzyme, which he called dopa-oxidase. However, at that point in time, dopa had never been observed in mammalian skin and the enzyme had not been isolated. In 1895, Emile Borquelot and George Betrand discovered tyrosinase in fungi,7 and subsequently, Bertrand further established that it converted tyrosine into a black pigment similar to that found in mammalian tissues.8 In 1927, Henry Stanley Raper isolated dopa from the first oxidation process of the action of tyrosinase on tyrosine.9 Providing evidence that tyrosine is present in all tissues, including the skin, Raper used tyrosinase from plants and mealworms to establish the primary chemical steps for the enzymatic oxidation of tyrosine to melanin. In doing so, he identified a red intermediate known as dopachrome and isolated derivatives of 5,6-dihydroxyindole (DHI) and 5,6dihydroxyindole-2-carboxylic acid (DHICA). This seminal work by Raper continues to serve as the foundation upon which molecular models for the biosynthesis of melanins are built. In 1948, Howard Mason extended Raper’s molecular scheme and introduced the “polymer” model of melanin, involving selfcondensation of indole quinones.10 In 1954, Barry Commoner and co-workers recognized the free radical character of melanins.11 Four years later, Laurens White demonstrated that squid and mammalian melanins exhibited cation-exchange properties,12 and in 1978, Tadeusz Sarna and co-workers established that metal ions bind to the o-semiquinone radical centers within melanin polymers to yield chelate complexes.13 These works fostered the hypothesis linking the free radical character and metal chelation abilities of melanins to their functional roles in human tissue. Specifically,

Simon et al. the free radical character of the pigment spurred research that examined the oxidation and reduction reactions of melanin.14,15 The results revealed the ability of the pigment to serve as a scavenger of reactive oxygen species, thus attributing a protective functional role to melanin present in tissues experiencing high oxidative stress.16,17 The ability of melanins to coordinate metal cations was recently reviewed,18 and currently, several functional ramifications of these interactions are being discussed in the literature. These range from the sequestration of transition metal cations in the substania nigra of the human brain19,20 to Ca2+ homeostasis21,22 and the release of intracellular Ca2+ that accompanies the transfer of melanosomes from melanocytes to keratinocytes in the skin.23,24 In 1978, Sarna and co-workers established that photoexcitation of synthetic melanins resulted in the reduction of oxygen to superoxide and hydrogen peroxide.25 This process was later shown to occur for natural pigments26 and since then has fostered extensive research aimed at understanding how different types of melanins and natural changes affect the balance between its photoprotective properties and its potentially deleterious sideeffect of photoinduced oxidative stress. Although the precise molecular structure of melanins remain unknown, powerful tools for probing their molecular composition were developed on the basis of oxidative and reductive degradation of the pigments, followed by identification and quantification of the molecules produced. In 1952, Luigi Panizzi and Rodolfo Nicolaus identified pyrrole 2-3-5-tricarboxylic acid (PTCA) as the most significant fragment from the degradation of sepia melaninsmelanin granules isolated from the ink sac of the cuttlefish sepia officinalis.27 During the 1960s, melanin was classified into two major typesseumelanin (eu, a Greek prefix for “good” or “well”) and phaeomelanin (phaeo, a Greek prefix meaning “dark”; the current literature generally reports this pigment as pheomelanin)sreflecting different molecular precursors in melanogenesis. Between 1967 and 1968, Guiseppe Prota, Nicolaus, and collaborators isolated melanin from red feathers, and established the centrality of 5-S-cysteinyldopa as a precursor to the pheomelanins.28-33 5-S-Cysteinyldopa was then first detected in melanoma tissue and urine by Hans Rorsman and co-workers in 1972.34 In 1968, Ernesto Fattorusso and Luigi Minale and their associates identified degradation fragments from the pheomelanins, including aminohydroxyphenylalanine (AHP).35 In fact, the late 1960s through the 1980s witnessed the development of a variety of chemical analyses whereby oxidative degradation and reductive hydrolysis of natural pigments produced a set of “molecular markers” unique to the type of pigment being examined.36,37 In addition to chemical analyses, Roger Sealy and co-workers demonstrated that electron spin resonance spectroscopy, a nondestructive technique, could be used to determine the relative amounts of pheomelanin and eumelanin present in natural samples.38 Extensive developments by Shosuke Ito refined these chemical approaches, introducing methods to quantify the eumelanin and pheomelanin pigments.39 The methods advanced by Ito are currently the best approaches available for deriving molecular information about melanins, and are routinely used to determine the molecular composition of melanins. Specifically, the yields of PTCA, pyrrole-2,3-dicarboxylic acid (PDCA), and 4-AHP produced by degradation techniques are used to infer the relative contributions of specific molecular monomerssbut not their chemical connectivitysto the overall architecture of the pigment.40-45 Figure 1 shows examples of such analyses on ocular melanosomes reported by Ito and Kazumasa Wakamatsu

Centennial Feature Article

Figure 1. Quantity and ratio of melanin in cultures of senescent uveal melanocytes from eyes with various colored irises. These data show that the amount and types of melanin vary with iris color. The amount of pheomelanin (PM) remains fairly constant for different colored irides, but the total amount of melanin (TM) present (pheomelanin and eumelanin (EM)) is greater in dark-colored irides. Reprinted from ref 46 with permission. Copyright 2007 Blackwell.

in 2007, indicating that there is variability in the composition of melanins from different colored irides.46 Melanin in situ is generally produced in melanosomes, specialized organelles in pigment generating cells. (Neuromelanin differs in that its biosynthesis does not take place within defined melanosomes, although the pigment is found in membrane-bound organelles.) The isolation of melanosomes from their surrounding tissue was pioneered by Makoto Seiji in 1961.47 Ultrastructural differences in the formation of melanosomes containing eumelanin and pheomelanin were reported in the mid 1960s by a variety of groups. In 1968, John A. Brumbaugh reported the following from his scanning electron micrographs of ocular melanosomes from fowls:48 The longitudinal strands of the rod-shaped eumelanin premelanosomes are coupled by cross-links that occur eVery 200 Å. The resulting network is a matting of connected components showing a repeating hexagonal configuration. Each side of a hexagon is 115 Å long. This matting appears rolled up inside the premelanosome membrane when Viewed in cross section. In the oVoid or spherical pheomelanin premelanosomes, the zigzag longitudinal strands form but cross-links are few or absent, resulting in a disorganized matrix. Lighter-than-normal pheomelanin granules show eVen shorter longitudinal strands and less organization. The isolation of melanosomes has been extensively refined by Vince Hearing.49-51 Rorsman isolated neuromelanin of the human substantia nigra in 1991,52 and in the past few years, Luigi Zecca has provided significant data on the role that neuromelanin plays in neuronal vulnerability and neurodegenerative diseases.20,53,54 Quantification of the amount of red pigment present in neuromelanin is based in part on the important degradation product thiazole-2,4,5-tricarboxylic acid (TTCA), reflecting that the pigment is derived from reactions with dopamine, not with dopa as in other tissues.55,56 Like most cellular organelles, melanosomes are membranebound structures. While research efforts largely focus on the melanin contained in these structures, melanosomes also contain lipids, enzymes, and proteins. In 2003, Ettore Appella and his collaborators reported a proteomic analysis of melanosomes at

J. Phys. Chem. B, Vol. 112, No. 42, 2008 13203 an early stage of development, identifying 6 of the known melanosome-specific proteins, 56 proteins that were shared with other organelles, and 6 new proteins that are specific to the melanosome.57 This analysis was followed in 2006 by the characterization of the complete melanosome proteome, identifying ∼1500 proteins associated with the different stages of pigmentation exhibited by melanosomes.58 In 2006, we described the phospholipids present in melanosomes isolated from the iris, choroid, and retinal pigment epithelium (RPE) of bovine eyes.59 The eye is unique in that the pigmented tissues have differing embryonic origins. Melanosomes in the iris and choroid originate in the neural crest, while those of the RPE originate in the outer neuroectodermal sheath of the eyecup.60-62 These origins are reflected in the lipid composition: the lipid profiles found in the choroid and iris melanosomes are similar but differ from the lipids found in the RPE. In uveal melanosomes, the major lipid species is sphingomyelin, while, in the RPE, it is glycerophosphoethanolamine (GPEtn), which is not present in the uveal melanosomes. There are more than 100 different lipids present in each type of melanosome. In 2007, we discovered a new class of lipids, concentrated in human neuromelanin.63 These recent reports point to the complexity of the molecular composition of the melanosome and suggest that understanding its structure and function involves more than just the properties of melanin. 3. MelaninsWhat’s in a Name? In 1950, Aaron Lerner and Thomas Fitzpatrick made the following statement in their review on the biochemistry of melanin formation:64 It is amazing that one term melanin has been used for many years to describe these natural and synthetic pigments eVen though there are a Variety of melanins and eVen though no exact definition for the term can be giVen. The two major types of melaninseumelanin and pheomelaninsreflect different molecular precursors in melanogenesis: eumelanins are defined as those pigments derived from the oxidation of tyrosine by tyrosinase, while pheomelanins entail subsequent reaction of the oxidized product(s) with cysteine. In 1966, Prota, Mario Piattelli, and Rodolfo Nicolaus proposed that pheomelanin resulted from the specific reaction of cysteine with quinones produced by tyrosine or dopa oxidation.65 In 1976, Prota and R. H. Thompson summarized the use of this terminology by stating the following:66 The main reason for defining these pigments on the basis of their biogenetic origin lies in the inadequacy of our knowledge of their complex chemical structure which haVe challenged many generations of chemists. This statement was published 32 years ago and remains true today despite significant efforts by researchers from a range of disciplines. Figure 2 presents the current understanding of the initial steps of the biosynthetic pathways of eumelanin and pheomelanin.67 As indicated, eumelanin is composed of oligomers of 5,6dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA), and pheomelanin is derived from benzothiazine units. While the potential role of DHICA in melanogenesis was recognized by Raper and Mason, melanins were largely viewed as homopolymers of DHI until the 1980s. This model stemmed largely from the assumption that the rearrangement of dopach-

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Figure 2. The early steps of melanogenesissthe biosynthetic pathway to melanin. The first step of melanogenesis is the oxidation of tyrosine by tyrosinase. Dopaquinone is the branch point between formation of eumelanin and pheomelanin. In the production of eumelanin, a second enzyme, dopachrome tautomerase, has been identified; it converts dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA). No additional enzymes in the pheomelanin pathway have been discovered. Reprinted from ref 67 with permission.

rome occurred spontaneously, and so decarboxylation to DHI would occur. A foreshadowing of the importance of DHICA in natural melanins was the report by Ito and Nicol, who isolated oligomers of DHICA from pigment from the tapetum lucidum of the sea catfish.68 In 1980, Ann Ko¨rner and John Pawelek provided evidence that the conversion of dopachrome to DHICA was under regulatory control, reporting the finding of a conversion factor in three different melanomas that catalyzed this reaction.69 In a pivotal paper published in 1986, Ito demonstrated that natural eumelanins are not homopolymers of DHI but rather copolymers of DHI and DHICA.70 Their results clearly establish there are fundamental molecular differences between natural and DHI melanins prepared synthetically or by enzymatic oxidation of dopa. In 1992, Hearing and coworkers established that tyrosine-related protein 2 (tyrp2), a unique protein in the melanosome, functioned as the dopachrome tautomerase to produce DHICA.71 Proof of the incorporation of DHICA into mammalian melanin was then confirmed by Hearing and co-workers at this time.72 These works established that natural eumelanins are derived from DHI and DHICA. One of the important implications of these works is establishing that natural melanins were different from synthetic models, which were and remain almost exclusively derived from decarboxylated DHI precursors. In 1997, Ito and co-workers provided standards for the analysis of mixed DHI/DHICA eumelanins

and used these to examine the amount of DHICA present in a variety of natural melanins.45 Ito and co-workers applied data from the pulse radiolysis studies reported by Edward Land and Patrick Riley73-75 to examine the kinetic rates for several steps of the Raper-Mason scheme for melanogenesis and thereby established the pivotal role of dopaquinone in controlling melanogenesis, and in particular the branching between eumelanin and pheomelanin production.67 Figure 3 summarizes this important result by presenting the kinetic information for the four important steps that take place early in melanogenesis. Note that the rate constants r1 through r4 are all controlled by the intrinsic chemical reactivity of dopaquinone. Only the initial oxidation of tyrosine by tyrosinase is needed to promote these reactions. These kinetic data suggest a three-step process of melanogensis. Initially, cysteinyldopa formation will occur as long as the concentration of cysteine is greater than 0.13 µM. Second, oxidation of cysteinyldopa to pheomelanin occurs for concentrations of cysteinyldopa greater than 9 µM. Finally, eumelanin is generated, occurring after most of the cysteinyldopa and cysteine levels are depleted. Such a kinetic model predicts a structural motif with pheomelanin at the core and eumelanin at the surface (Figure 4). This “casing” model was originally proposed in 1982 by Rorsman based on biochemical evidence76 and has received indirect support by several other studies.77-79 Later in this article,


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Figure 3. The kinetics of the early steps of melanogenesis involving dopaquinone. These kinetic data suggest a three-step process of melanogenesis. Initially, cysteinyldopa formation occurs as long as the concentration of cysteine is greater than 0.13 µM. Then, oxidation of cysteinyldopa to pheomelanin occurs for concentrations of cysteinyldopa greater than 9 µM. Finally, eumelanin is formed after most of the cysteinyldopa and cysteine levels are depleted. Reprinted from ref 67 with permission.

Figure 4. Schematic of the casing modelspheomelanin encased in eumelaninsfor melanosomes containing both pigments. In this model, pheomelanin is produced first, consistent with the relative rate data given in Figure 3. Once formed, eumelanin is deposited on the surface; the thickness of the layer depends on the degree of eumelanin production, which is ultimately linked to the continued oxidation of tyrosine by tyrosinase. In this scheme, part of the eumelanin coating is cut away to reveal the pheomelanin interior. Within this model, the chemical analysis of different iris melanosomes, Figure 1, indicates that dark-colored irides have an extensive eumelanin surface while lightcolored irides have a thin eumelanic exterior. Therefore, a small amount of surface damage could result in exposure of the pheomelanin. (DQ, dopaquinone; DAQ, dopaminequinone). Reprinted from ref 67 with permission.

we will examine direct evidence in support of a casing model for neuromelanin. Motivated in part by the hypothesis that the epidemiological data revealing fair skin individuals are more susceptible to skin cancers is linked to greater phototoxicity than pheomelanin,80-82 there has been extensive interest in comparing the aerobic reactivity of eumelanin and pheomelanin. Because pure pheomelanin is not found in nature, the majority of studies on this pigment are on synthetic systems. Ito has reported on the optimized conditions for preparing the synthetic pigment.83 Miles Chedekel and co-workers reported on the photodynamic properties of pheomelanins,84,85 reporting the activation of molecular oxygen through photoionization by UV-A radiation. Action spectra for photoinduced oxygen consumption have been reported for a variety of synthetic and natural melanins,86,87 and these action spectra generally differ significantly from the absorption spectrum of the pigment. In 1999, we put forth an explanation for this disparity between the absorption and action spectra by demonstrating for sepia melanin the action spectrum matches the absorption spectra of oligomeric constituents of the natural melanins.88 We extended this to other melanin systems,89,90

established that aggregation of these constituents affects their aerobic reactivity,91 and most recently demonstrated that different molecular constituents in synthetic pheomelanin are responsible for the emission, transient absorption, and oxygen photoconsumption exhibited by this pigment.92 These later studies confirmed the observed increased aerobic reactivity of pheomelanin in the UV-A, originally reported by Chedekel. In exploring the hypothesized link between pheomelanin photoreactivity and skin cancer, Prota and co-workers reported that UV sensitivity is associated with high pheomelanin and low eumelanin levels.93 In contrast, based on an examination of eumelanin and pheomelanin concentrations in human skin before and after exposure to UV radiation, Jonathan Rees and co-workers speculated that factors other than the amount of pheomelanin may be important in determining cancer susceptibility.94 Douglas Brash and co-workers examined the induction of DNA lesions and apoptosis upon UV exposure of congenic mice of with black, yellow, and albino coats.95 In their experiments, UV-B-induced cyclobutane dimerization and apoptosis measured by sunburn cells or keratinocytes containing active caspase-3 was strain independent. However, the UV irradiation of melanin photosensizes adjacent cells to caspase-3 independent apoptosis, and this occurs at a frequency greater than the apoptosis induced by UV absorption by DNA. In particular, pheomelanin exhibited a 3-fold greater activity than eumelanin, supporting the hypothesis that melanin-induced apoptosis may contributed to the observed epidemiology of skin cancer for black-, blond-, and red-haired populations. At present, surprisingly, molecular knowledge only exists for a length scale of the simple organic molecules described above (∼1 nm). No details are currently known about the size of the resulting molecular structures that comprise “melanin” in the micrometer-sized organelles. The lack of progress in defining the molecular structure is emphasized by the continued indiscriminate use of generic terms (melanin, eumelanin, pheomelanin) to refer to fungi, mammalian, invertebrate, and synthetic pigments. Such general use highlights one of the major challenges of the field: a tendency to compare “melanins”, to draw conclusions and implications from observations made on different systems, and to infer the structure and function of mammalian pigments from studies on synthetic pigments and


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Figure 5. (A) Transmission electron microscopy image of a section of human RPE tissue. The image reveals the presence of melanosomes (black), lipofuscin (L), and melanosomes encased in lipofuscin called melanolipofuscin; image courtesy of Ulrich Schraermeyer. (B) Scanning electron microscopy image of isolated human RPE melanosomes. (C) Atomic force microscopy image revealing the substructure of the surface of the RPE melanosome. (D) Transmission electron microscopy image of a section of human substantia nigra; image courtesy of Luigi Zecca. (E) Transmission electron microscopy image of isolated organelles from human substantia nigra; image courtesy of Luigi Zecca. (F) Scanning electron microscopy image of isolated neuromelanin granules. The images represent different spatial scales. In the case of RPE melanosomes, the average length of the ellipsoidal organelle is ∼1.6 µm. Image C is 1 µm × 1 µm. For the brain pigments, the average diameter of an organelle is ∼1 µm. Image F is 4 µm × 4 µm.

model melanogenesis reactions. While in part a result of the limitations of the “melanin” vocabulary, the state of the current literature also reflects the real situation that there is a lack of molecular understanding of natural melaninssboth at the levels of the pigment molecules themselves and their structure within the intact organelle. Therefore, it is unclear whether the range of synthetic systems studied are appropriately designed for generating insight into the structure and function of natural pigments or if various mammalian and invertebrate melanins are accurate models for the human pigment. Finally, it is important to mention that neuromelanin differs from the pigments described above and does not follow the general outline of the reactions given in Figure 2.96,97 Our understanding of the biochemistry of neuromelanin is even more rudimentary than for the melanins isolated from many other tissues (eye, hair), and the current evidence indicates that the chemistry of dopaminequinine and cysteinyl catechols are central to the formation of neuromelanin. Over the past few years, review articles on melanin have appeared covering a wide range of chemical and biological topics, including the chemistry of melanogenesis,98 the chemical and physical properties of synthetic and natural eumelanins,99 the chemistry of mixed melanogenesis,67 the regulation of melanin synthesis in vivo,100 the morphology of natural melanosomes,101 the regulation of skin pigmentation and the effects of UV exposure,102 the physiological roles of ocular melanosomes,103 the role of iron and neuromelanin in brain aging,104 the physiological and pathogenic aspects of neuromelanin,105 and the role of neuronal pigmented autophagic vacuoles in aging and disease.96 It is not our intention to cover the same topics as these recent reviews but rather to offer a perspective on the importance of understanding the surface chemistry of the melanosome.

4. Framing the Research in Terms of Spatial and Temporal Scales Let us consider framing research on melanins by asking the question: what spatial and temporal scales are most important in understanding its structure and function? The concept of spatial scales is exemplified in Figure 5 for the retinal pigment epithelium (RPE) of the human eye (Figure 5A-C) and the substantia nigra of the human brain (Figure 5D-F). Figure 5A shows a transmission electron microscopy cross-sectional image of human RPE cells. Melanosomes are clearly present within these cells, and it is reasonable to conclude that their function is tied, in part, to their interactions with other molecules present in the cells; Figure 5B shows a scanning electron microscopy image of melanosomes isolated from RPE cells. Finally, Figure 5C shows an atomic force image of the surface, revealing the presence of subunits that are ∼30 nm in diameter. Our second example of human brain pigment shares some features of that observed for RPE cells but also presents some distinct features. Figure 5D shows a transmission electron microscopy image of intact tissue from the substantia nigra. Neuromelanin organelles are clearly embedded in the tissue. The organelles are enclosed by a double lipid bilayer and contain lipid droplets and melanin granules. Figure 5E reveals isolated organelles, where the surrounding tissue has been removed. These organelles clearly differ from the melanosome structures observed in the RPE. Figure 5F shows the isolated neuromelanin granules, after removal of the bilayer and lipid droplets, and similar to RPE pigment, the AFM image reveals a substructure of diameter of a few tens of nanometers. The images presented in Figure 5 reflect different levels of spatial analysis that can be exploited to study pigments. In moving from part A of Figure 5 to part C or D to F, we move from the intact biological tissue (mm) to the intact organelle


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Figure 6. (left) Melanin content of human RPE cells with age. The melanin content is normalized to 1 mg of protein/mL of buffer. A regression analysis of these data indicates that ∼50% of the melanin is lost over 90 years. Reprinted from ref 124 with permission. Copyright 2003 Elsevier. (right) Photoconsumption of oxygen for different age cohorts (in years) of human RPE cells: 80 (]). The data show that the rate of oxygen uptake increases by a factor of 2.4 between the 80 cohorts. Reprinted from ref 125 with permission. Copyright 2002 Lippincott Williams & Wilkins.

(µm) to molecular components (nm). Each step is a reduction of the spatial scale, and with each step, there is the potential for isolating a “purer” pigment. However, along with this “purification” comes the removal of the neighboring biological components, thus eliminating their interactions with the pigment. Such a reduction likely affects both structure and function. Thus, one must exercise great care in translating the findings across these spatial scales. The integrity of the melanosome is necessary for melanin to carry out its biological function, and it is the surface of the melanosomes where melanin interacts with molecules within tissue cells. Thus, the surface properties of natural pigments are a key spatial scale and the next section of this paper examines in detail what is known about surface structure and function. Now consider the temporal scale. One of the most cited functions for melanin is that of photoprotection. The ability to synthesize model pigments, combined with advances in timeresolved spectroscopies, has enabled studies to elucidate the rates and mechanisms of relaxation following the absorption of ultraviolet radiation. Melanin undergoes rapid nonradiative relaxation with high quantum efficiency, supporting its ascribed function as a photoprotector.92,106-113 While pertinent to a detailed understanding of the photophysics of melanins, we argue, however, that this is not the most important time scale associated with pigment function. Let us revisit the two types of pigments discussed abovesRPE and substantia nigrasto determine the temporal scale of interest. In the case of the human RPE, formation of melanosomes occurs early in fetal development, ceasing within a few weeks.114 Polymerization of melanin within these melanosomes continues until, at approximately two years of age, the RPE contains only mature melanosomes.115 Whether melanogenesis occurs in the RPE after approximately this point in time has not been definitely established. Premelanosomes, or partially melanized melanosomes, which are indicative of ongoing melanogenesis, have never been observed in adult RPE cells. In addition, very little or no tyrosinase activity can be detected in adult bovine RPE cells.116,117 Therefore, melanin biosynthesis either is absent in adult human RPE cells or occurs only at a very slow rate, and whether there is turnover of RPE melanosomes also remains unknown. Melanin in the RPE can act as a scavenger or quencher of reactive oxygen species (ROS) and thereby protect the neural retina.118,119 With age, the constant exposure of the RPE to high

levels of oxygen and light may diminish melanin’s antioxidant properties.120-123 Under these conditions, melanin may become less protective. Figure 6 reproduces work reported by Sarna and co-workers revealing two important observations. First, the melanin content of the RPE decreases significantly in aged human eyes.124 Second, there is an increased ability of these melanosomes to photoconsume oxygen with age,125 a feature that is detrimental to the cell because of the consequent production of ROS that can damage cellular components. Thus, we argue that the temporal scale of greatest interest is that of the human life span, and the challenges presented are to determine how the melanosome changes along this temporal scale, to determine whether these changes affect function, and to determine whether damage to the melanosome increases the risk of or directly causes degenerative diseases in pigmented tissue. A similar argument can be made about the pigment in the substantia nigra. Figure 7 shows results published by Zecca and co-workers demonstrating two important changes that occur over the human life span. First, in contrast to RPE melanosomes, the amount of melanin in the substantia nigra increases with age.126 Second, the concentration of iron contained within the substantia nigra increases early in life and then remains essentially constant after 20 years.53 As one of the main functions ascribed to neuromelanin is to buffer iron concentrations during aging, this result is of great interest as the concentration of neuromelanin continues to increase over the next 7 decades of life. Understanding age-dependent metal binding to neuromelanin is important in developing a full picture of pigment function. 5. The Centrality of the Surface of the Melanosome to Function Discussions of the function(s) of melanin generally fall into three areas: photoprotection and the balance between its photoprotective properties and its potentially deleterious sideeffects of photoinduced oxidative stress, scavenging reactive oxygen species (ROS) and thereby mitigating their damaging effects on cellular processes, and binding and sequestering of metal cations. While broad in scope, these properties are not mutually exclusive. For example, the ability of melanin to sequester metals such as iron and copper protects the cell from Fenton chemistry, which would otherwise be a source of


Simon et al. must occur between a surface site and the metal. Furthermore, the surface structure must allow the cation to access the interior of the melanosome. Thus, the surface plays a significant role in the metal chelation properties of the melanosomes. Despite the clear centrality of the surface of the melanosome to their range of ascribed behaviors, there are few studies that probe the structure and chemical properties of this surface. Over the past decades, our group has used surface-sensitive analytic, spectroscopic, and imaging techniques to examine the structural and chemical properties of many types of pigmentsssepia melanin granules, human, and bovine ocular melanosomes, human hair melanosomessand neuromelanin. Such studies provide new insights into the structure and chemical properties of these pigments. Furthermore, studying surface properties of melanosomes as a function of age is supporting the development of new models for their age-dependent behaviors. 6. Probing Physical and Chemical Properties and Age-Related Changes of the Surface of Melanosomes

Figure 7. (top) Neuromelanin content in the substantia nigra pars compacta with age. The melanin is normalized to protein content. The data indicate that the amount of neuromelanin increases by a factor of ∼6 between 20 and 90 years of life. Reprinted from ref 126 with permission. Copyright 2002 Elsevier. (bottom) Age-dependent concentration of iron in the substantia nigra of the human brain. The iron concentration increases during the first ∼2 years of life and then remains constant. Reprinted from ref 53 with permission. Copyright 2001 Blackwell.

oxidative stress. Consider what features of the melanosomes are central for its ability to carry out this range of functions. Melanosomes are highly colored. Under normal solar illumination, the absorption of UV radiation (the region most important for the function of photoprotection) occurs throughout the melanosome.127,128 However, as mentioned above, accompanying the absorption of UV light, ROS are generated with a quantum efficiency of ∼10-4 to 10-5.26,86 Thus, UV photoexcitation also generates species that can be detrimental to the cell. To cause damage, the photogenerated reactive species must be able to diffuse into the surrounding cellular matrix. This most readily occurs for reactions taking place at the surface of the melanosome. Fortunately, the majority of the light is absorbed by the interior of the melanosome, mitigating the undesired surface photochemistry. The quenching of ROS (e.g., O2-•, OH•, RO•) takes place through chemical reactions between the ROS and the melanosome. ROS first encounter the surface of the melanosome, and hence, it is reasonable to assert that the most important aspect of the melanosome in terms of mitigating ROS is the chemical properties of its surface. Finally, consider metal chelation. While melanosomes have the ability to sequester large concentrations of metal cations (up to ∼8% by weight in the case of sepia),129,130 initial binding

Studies of the surface of melanosomes to date have been carried out chiefly with four experimental approaches described below. We first examine the surface area and porosity of intact melanosomes obtained from measurements of the adsorption/ desorption isotherms of N2. For sepia, the experimentally determined surface area is larger than that calculated for a solid sphere using the diameter obtained from SEM images. These results suggest the surface is not smooth and characterized by significant structure. Second, we consider further the “smoothness” of the surface using atomic force microscopy (AFM). Conventional AFM imaging provides direct measurement of the surface morphology. Recent work probing various specific interactions between the surface and the AFM tip provide novel insights into the structure near the surface of the melanosome as well as changes in the surface that occur with age. In an effort to develop a more chemical understanding of the surface, we will discuss results from X-ray photoelectron spectroscopy (XPS) studies. XPS measurements enable determination of the different oxidation states of carbon, oxygen, and nitrogen present on or near the surface. The results can be compared to complementary techniques used to discern information about the bulk melanosomes, providing insights into whether the molecular groups present on the surface differ from the bulk. The sample requirements of XPS limit such studies to systems where melanosomes can be obtained in large quantities. In keeping with our interest in understanding the effects of aging, we will focus our discussion on results obtained on two different age populations of bovine ocular melanosomes isolated from the iris and choroid. The fourth application we discuss is photoemission electron microscopy (PEEM). PEEM enables direct determination of the oxidation potential, and quantifying the electrochemical potential of the surface is central to the thermodynamic understanding of the reaction between melanosomes and oxygen/ROS. Intact melanosomes are not amenable to conventional electrochemical techniques, and thus, knowledge of the electrochemical properties of melanins has relied on measurements of synthetic models. As mentioned above, it is not clear that such models accurately reflect the properties of native systems. PEEM uniquely addresses this issue for the surface oxidation potential. The results of PEEM studies on the surface properties of melanosomes provide unique insights into the structure and agedependent properties of human melanosomes. Herein, we focus on two important insights: first, evidence for a casing model

Centennial Feature Article for human neuromelanin, and second, the age-dependent photoinduced aerobic reactivity of human RPE melanosomes. Surface Area Measurements. The surface area of a solid can be determined from the analysis of low-temperature (77 K) nitrogen (N2) adsorption to the surface of interest. Adsorption is usually described through isotherms, which express the amount of N2 present on the surface as a function of its pressure. In 1938, Stephan Brunauer, Paul Emmett, and Edward Teller (BET) developed an isotherm that can account for multilayer absorption.131 In 1951, E. P. Barret, L. G. Joyner, and P. P. Halenda (BJH) developed a scheme for calculating the mesopore distribution of material from its nitrogen adsorption data.132 BET and BJH analyses have been applied to the pressure dependence of N2 adsorption on sepia pigment granules.133 The data depend on the preparative procedure used to isolate the pigment, specifically the drying process, and comparative studies indicate that supercritical drying produces unaggregated granules that best reflect the natural pigment. Analysis of this sample reveals a surface area of 37.5 m2 g-1. Sepia granules are spherical with an average diameter of ∼150 nm and a density of 1.68 g cm-3.134 It is of interest to compare the surface area determined from the adsorption isotherm to that calculated for a smooth nonporous sphere. Such a calculation yields a surface area to mass ratio of 24 m2 g-1, about 65% of the measured value. BET surface area analysis was also conducted on a variety of metal-saturated sepia samples, where the metal concentrations are on the order of 8% by weight. Specifically natural, EDTAtreated, Fe(III)-, Ca(II)-, Cu(II)- and Zn(II)-saturated sepia granules have the same surface areas,129 indicating that metal binding or removal does not change the surface area available for N2 adsorption. This result is also consistent with SEM imaging studies, where no significant changes in morphology are observed among these samples. Given that the “pores” likely play a role in the transport of the metals within the melanosome, at saturation, these pores should not be available for adsorption of N2. The lack of any effect on the surface area of type and concentration of bound metals suggests that the difference between the measured surface area and that for the corresponding spherical structure is not a result of granule porosity but reflective of a rough surface. BET analysis has also been carried out on melanosomes isolated from human black hair.135 Hair melanosomes exhibit a surface area of 6.3 m2 g-1. The differences between the surface areas for sepia granules and human hair melanosomes reflect their different geometric shapes. Spatial imaging experiments indicated that hair melanosomes can be approximated by the geometrical solid of a prolate ellipsoid. If the average ellipsoidal shape determined from images of isolated melanosomes is used and the density of the hair melanosomes is assumed to be the same as that for sepia, a calculated surface area per gram of 7.43 m2 g-1 is found, which is more than that determined experimentally. However, the validity of this estimate is not clear. The distribution of ellipsoidal dimensions for the isolated hair melanosomes varies significantly, in contrast to the narrow size distribution observed for sepia. Using individual melanosome dimensions to calculate surface area gives results ranging from 5 to 8 m2 g-1, which span that determined from adsorption studies. In addition, it is not clear whether the density of the hair melanosome is equal to that for sepia, which unfortunately is the only intact melanin structure for which a density is known. Thus, in the case of melanosomes from hair, it is not possible to unambiguously conclude whether the predicted surface area

J. Phys. Chem. B, Vol. 112, No. 42, 2008 13209 assuming a smooth surface is smaller or larger than that measured from N2 adsorption. Fortunately, the “smoothness” of the surface can be directly addressed using atomic force microscopy. Atomic Force Microscopy. More details of the surface structure can be obtained from atomic force microscopy (AFM). In AFM, a sharp tip located near the end of a cantilever beam is scanned across the sample surface using a piezoelectric positioner. Images are generated by monitoring changes in the tip-sample interaction. Of the three operating modes commonly used to collect images (contact, noncontact, and tapping), tapping mode is the optimal approach for measuring the surface morphology of melanosomes. In tapping mode, the cantilever oscillates close to its first bending mode resonance frequency with an oscillation amplitude in the range 20-200 nm. The height is adjusted so that the tip contacts the sample for a short duration for each oscillation. As the tip approaches the sample, the tip-sample interactions alter the amplitude, resonance frequency, and phase angle of the oscillating cantilever. During scanning, the amplitude at the operating frequency is maintained at a constant level, by adjusting the distance between the tip and the sample. The image of the phase changes that occur during the scanning of the tip provides significantly more contrast than the topographic image. Phase images are sensitive to material properties of the surface, e.g., chemical composition, and thus, changes in phase can be used to reveal structural features that are often not observed in the corresponding height image. The N2 adsorption/desorption data in the previous section suggest that the surface has substantial roughness. AFM imaging of sepia, black hair, and ocular melanosomes (uveal and retinal) show that the surface indeed has substructure.135-140 Images of all these types of melanosomes reveal a surface that can be described by the packing of structures whose lateral dimension is a few tens of nanometers. This substructure appears in all cases despite the fact that the natural melanosomes have different shapes, e.g., spherical vs ellipsoidal, and different dimensions, roughly 150 nm (diameter of sepia) vs 450 nm × 1 µm and ∼900 nm × 2 µm (ellipsoidal axes of black hair and bovine RPE melanosomes, respectively). The corrugated surface structure is consistent with the conclusion drawn from the N2 adsorption data. The spatial scale of the melanosome surface was further refined in an experiment where the AFM tip was used to cut individual pigment granules (Figure 8).136 The AFM tip was fixed at a certain height above the mica substrate and then programmed to draw a straight line across the sample. The cut creates a trough ∼30 nm deep and ∼30 nm wide through the granules and a ridge of the displaced material of height ∼20 nm along the edge of the trough. The cut line is not straight but reflects the asymmetry of material force acting on the tip due to the spherical structure of the granules. The ability to displace material in this manner indicates that the molecular constituents comprising the region near the surface of the granule are small compared to the dimension of the tip (and trough). This invasive measurement was the first experiment to establish that the molecular constituents of the surface have a maximum spatial scale of ∼10 nm and are held together by noncovalent bonds. The above imaging studies established the presence of ∼30 nm substructures in melanosomes from various sources. This type of substructure is consistent with the cross-linked strands reported by Brumbaugh in 1968 for ocular melanosomes from fowls.48 We also have used AFM to examine age-dependent


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Figure 8. (left) AFM height image of a eumelanin aggregate. (right) Height image of the aggregate shown in the left panel after using the AFM tip to scribe a line across the sample. These data established that the molecular constituents comprising the surface of sepia have a maximum spatial scale of ∼10 nm and are held together by noncovalent bonds.

Figure 9. Four different types of nanoindentation force curves exhibited by RPE melanosomes: (A) continuous (51, 63), (B) discontinuous (15, 17), (C) penetration (31, 0), and (D) adhesion (3, 20). The numbers in the parentheses are the percentage of this type of curve observed for young (14-year-old) and old (76-year-old) melanosomes, respectively. The penetration and adhesion curves show a strong age dependence and reflect substantial age-dependent changes in the surface structure. The penetration force curves are attributed to the presence of lipid deposits on part of the young melanosomal surface, likely a remnant from the original organelle membrane prior to pigmentation, which disappears over life. The adhesion sites are attributed to deposits of lipofuscin on the RPE surface, which occur with aging. Reprinted from ref 137 with permission.

properties, performing imaging and nanoindentation measurements on human RPE melanosomes of different ages in aqueous suspension.137 The imaging data show surface topology similar to that observed on dry melanosomes. However, new information was revealed by the force-indentation measurements. RPE melanosomes exhibited four different types of force-indentation responses: continuous, discontinuous, penetration, and adhesion, exemplified by Figure 9A-D, respectively. When the force-indentation response was a continuous curve, values for Young’s moduli could be extracted (Figure 9A), revealing a distribution of values ranging from a few megapascals to a few tens of megapascals and a small dependence on age of the average value (12 and 16 MPa for 14- and 76year-old RPE melanosomes). Discontinuous force curves (Figure 9B) revealed the presence of hard structures encountered as the melanosome was penetrated. These structures serve to impede the motion of the AFM tip, and the data suggest that they are moved aside as a result of the increased force, relaxing back to their original position upon withdrawal of the tip.

Figure 9C shows the case where the tip indents or penetrates the melanosome and the force then decreases, as indicated by the spiky force pattern. This behavior indicates that this region of the melanosome has a loose structure on the outermost top surface with a thickness in the range of ∼20 nm. This behavior is unique to young RPE melanosomes, indicating that there are significant changes in the composition of the outermost layer of the melanosome with age. This loose structure in the young melanosomes has been attributed to the presence of lipid deposits on part of the melanosomal surface, likely a remnant from the original organelle membrane prior to pigmentation, which disappear over life.137 Finally, in some cases (Figure 9D), there is significant adhesion (approximately 450 pN) when the AFM tip is withdrawn from the surface. During the adhesion events, the tip sticks to melanosomes and stretches material by a distance of 10-20 nm. This type of behavior is rarely observed in the 14-year-old melanosome specimen but constitutes a larger percentage, ∼20%, of the older specimen, indicating that its

Centennial Feature Article origin is age related. Adhesion is attributed to the age-dependent accumulation of lipofuscin on the surface of the melanosome.137 Evidence supporting this conclusion is provided below in our discussion of PEEM experiments on these same melanosomes. AFM has also been used to examine changes in surface morphology of porcine RPE melanosome following photodegradation.141 Spectroscopic and chemical techniques reveal that the illumination of porcine melanosomes by broadband visible light (420-630 nm) results in loss of melanin, reduced ability of the melanosome to bind iron, increased efficiency for the photogeneration of superoxide radical anion, and reduced ability to inhibit iron-catalyzed free radical decomposition of H2O2. AFM images clearly show that illumination results in the fragmentation of the organelles and changes in surface morphology. These studies suggest that, with aging, light-induced changes in RPE melanosomes may compromise their ability to function. The melanin content of human RPE decreases by a factor of 2.5 over 90 years of life.124 The in vitro fragmentation and disruption of the melanosome structure that accompany illumination by visible light provide important insights into how the disruption of melanosome structure upon exposure to ambient light leads to the loss of melanin observed in human RPE cells with aging. X-ray Photoelectron Spectroscopy. X-ray photoelectron spectroscopy (XPS) is widely used to analyze functional groups present on or near the surface of solids (∼5 nm in depth depending on the apparatus and experimental conditions). In the case of melanosomes, XPS would reveal insights into the functional groups present near the surface. XPS data could then be compared to elemental analysis to ascertain whether there are significant differences between the distributions of functional groups on the surface and in the bulk. Such a study has been performed on the bovine uveal melanosomes (iris and choroid) isolated from two different age cohorts.142 The three elements C, N, and O account for more than 97% of the elements present (excluding H) on the surface. From highresolution XPS spectra of the 1s orbitals of C, N, and O, the relative amounts of these elements were determined. Compared to elemental analysis of the bulk melanosome, the C/N and O/N ratios on the surface differ from those of the bulk. This result reveals that these melanosomes are not of uniform composition and structure from the surface to their center. Iris and choroid melanosomes from the same age cohort reveal similar C, N, and O content on the surface, with distributions of oxidation states for each element. However, both properties (elemental composition and distribution of oxidation states) vary with age. Most of the nitrogen present in these melanosomes is tied up in an indole structure, and did not show tissue- or age-related differences. However, both C1s spectra and O1s spectra show that the surface of mature melanosomes contains significantly fewer CsO groups versus CdO groups than newborn melanosomes. The CsO/CdO ratios are the same for choroid and iris melanosomes from the same age cohort, and decrease with age. These results reveal that the surface of the fully melanized melanosomes contains indole-derived and quinone-derived monomers. On the basis of the similarities of the CsO/CdO ratios for choroid and iris melanosomes within the same age cohort, it is reasonable to hypothesize that the content of indolederived monomer and quinone-derived monomer are well regulated in vivo. The decreased CsO/CdO ratio and decreased total amount of CsO and CdO in adult melanosomes also support the conclusion that surface oxidation occurs with age.

J. Phys. Chem. B, Vol. 112, No. 42, 2008 13211 Unfortunately, because of the sample requirements of XPS, this approach has not been extended to the study of RPE melanosomes or neuromelanins. Photoemission Electron Microscopy. PEEM provides spatially resolved images of materials or molecules whose work function is less than the energy of the incident light.143 PEEM is inherently a surface technique. In the case of biomolecular assemblies, the incident ionizing light can penetrate the sample, and thus, electrons are actually generated from molecules both on and near the surface. The penetration depth depends on the optical properties of the material being imaged. The escape yield of the photoionized electron depends on its point of origin, with the highest yield occurring from molecules on the surface and a decreasing yield with depth into the material. The depth from which electrons can escape is dependent on the material and is estimated to be in the range of a few nanometers for organic structures.144 For materials that are effective electron scavengers (e.g., melanosomes), it is likely that electrons photogenerated below the surface are efficiently trapped, and the image is dominated by the ionization properties of the surface. For photon energies exceeding the work function(s) of the material of interest, the intensity of a point in the image depends on the difference between individual work functions and the photon energy, and so image contrast can reveal regions of different ionization potentials. PEEM has been successful at imaging biological systems such as viruses, eukaryotic cells, cultured cancer cells, and cytoskeletons.145-148 A number of studies detailing the photoemission quantum yields of biological samples have also appeared.148,149 The photoionization threshold can be determined from a fit of the nonlinear Fowler equation to the intensity dependence of the PEEM image on the wavelength of incident radiation.150 Biological structures exhibit ionization thresholds in the UV region, and thus, determination of this value requires a tunable UV light source. The Duke OK-5 free electron laser (FEL) is tunable from 193 to 400 nm and has proven to be an ideal light source for performing wavelength-dependent PEEM.151 In collaboration with Robert Nemanich’s group, we used FEL-PEEM to determine the photoionization thresholds of different types of melanosomes.138,152-156 Figure 10 shows the experimental data for melanosomes isolated from human black and red hair. The solid line is the fit to the nonlinear Fowler equation. From this fit, we find that black hair melanosomes have a surface photoionization threshold of 4.4 eV (282 nm) and red hair melanosomes exhibit two threshold potentials, 4.4 and 3.8 eV (326 nm).154 (We first reported the thresholds for black and red hair melanosomes to be 4.6 and 3.9 eV, respectively.153 This was done using a linearized version of the Fowler equation to fit the experimental data. Using the full equation150 and perfoming a nonlinear fit gives the values mentioned above.154) Chemical degradation analyses of these melanosomes show that the pigment in black hair melanosomes is eumelanin while that in red hair is 68% eumelanin and 32% pheomelanin.139 Thus, the 4.4 and 3.8 eV thresholds are attributed to eumelanin and pheomelanin, respectively. We find that all melanosomes studied that contain only eumelanin (sepia, bovine, and human RPE) have a threshold potential of ∼4.4 eV. A lower ionization threshold for pheomelanin is not unexpected and is supported by both femtosecond transient absorption spectroscopy and electron paramagnetic resonance oximetry experiments.154 The lower ionization potential observed for pheomelanin could be an important part of the explanation for the greater incidence rate of UV-induced skin cancers in the


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Figure 10. Integrated brightness of the PEEM image for black (left) and red (right) hair melanosomes divided by the square of the sample temperature, S/T2, plotted as a function of the excitation energy (hν/kBT). The solid lines are the best fit of the nonlinear Fowler equation to the data points. For black hair melanosomes, the data can be described by a single ionization threshold of ∼4.4 ( 0.2 eV (282 nm). To fit the data for red hair melanosomes requires two ionization thresholds of ∼4.4 ( 0.2 and 3.8 ( 0.2 eV (282 and 326 nm). Reprinted from ref 154 with permission. Copyright 2006 American Society for Photobiology.

populations whose melanins contain increased concentrations of pheomelanin. The connection between the vacuum threshold potential and the electrochemical potential vs NHE has also been explored in great depth.157-161 The relationship between the two scales has largely focused on comparing electrochemical and work function data on Hg, and the data indicate that 0 V vs NHE corresponds to -4.44 V in vacuum. While this is the IUPAC recommended value, it is worth mentioning that the different studies cited above report a range of values corresponding to 0 V (vs NHE) of ∼ -4.4 V to ∼ -4.8 V. Generally, measurements incorporating the contact potential difference of the uncharged metal and the work function of the clean surface result in the higher values in this range while work on immersed electrodes, which are believed to retain their interfacial region, results in lower values. The relationship between vacuum and electrochemical scales means that the PEEM data reflected by Figure 10 uniquely provide information about the electrochemical potential of molecules on the surface of the melanosome. The oxidation potentials of eumelanin and pheomelanin on the surface of the melanosome are 0 V and +0.6 V vs NHE, respectively. This result further underscores the concern of using synthetic melanins to model natural pigments. The oxidation potential of thin films of DHI-melanin is E1/2 ) +125mV vs Ag/AgCl,162 and cyclic voltammetric measurements of synthetic dopamine melanin at pH 5.6 reveal two main peaks in oxidation at +460 and +525 mV versus SCE.163 These values differ from that for the pigment on the surface of the natural melanosome, most likely reflecting differences in the molecular structure of the pigment. Implication of PEEM Studies of the Surface of Melanosomes: Evidence for a Casing Model for Melanosomes Containing Eumelanin and Pheomelanin and Its Consequences. The case of mixed melanosomes containing both eumelanin and pheomelanin offers intriguing opportunities to investigate the heterogeneity of their structure, with a result that suggests several important consequences of that structure. We used the photoionization threshold of eumelanin and pheomelanin to examine two mixed melanin systems: bovine iris melanosomes and human neuromelanin. Chemical degradation analyses reveal bovine iris melanosomes and human neuromelanin contain 1.2 and 25% pheomelanin, respectively.55,142,164 Measurement of the photoionization threshold found that both bovine iris melanosomes and human neuromelanin exhibited a common threshold of 4.4 eV, characteristic of eumelanin alone.156 In the case of the iris melanosomes, 1.2% pheomelanin content is close to the detection limits of the technique, and

one could propose that pheomelanin is on the surface but not detected because of its low concentration. However, the pheomelanin in neuromelanin is quantitatively similar to that of the human red hair melanosomes. This result reveals that the surfaces of both neuromelanin and iris melanosomes are composed of eumelanin. Consequently, if pheomelanin is not detected on or near the surface of the granules, it must sit within the pigment assembly. This result is not totally surprising. As described in section 2, the rate constants in Figure 3 support a mechanism for mixed melanogenesis that can result in formation of a pheomelanin core encased by eumelanin (Figure 4). In light of this conclusion, it is perhaps surprising that the pheomelanin signal was observed for the red hair melanosomes. Are these melanosomes different? After all, the melanin is produced by mixed melanogenesis and so one can ask why they do not present themselves as structures with a pheomelanin core and eumelanin exterior. In neuromelanin, production of pheomelanin competes with the cyclization of dopaminequinone. In the case of iris or red hair melanosomes, production of pheomelanin competes with the cyclization of dopaquinone. Dopaminequinone cyclizes 2 orders of magnitude more slowly than dopaquinone and hence pheomelanin production will dominate until cysteine levels are nearly exhausted (as discussed in section 3).73,165 The increase in rate of cyclization for dopaquinone means that eumelanin formation can more effectively compete with pheomelanin production, and thus, mixed melanins would be expected closer to the surface than in the dopaminequinonederived pigments. However, we also need to consider the effect of the isolation procedure on the integrity of the melanosome present naturally in the tissue. Our studies indicate that red hair melanosomes tend to fall apart under the same isolation procedure that produces intact melanosomes from black hair. Furthermore, the red hair melanosomes do not exhibit the well-defined ∼30 nm substructure that is characteristic of pure eumelanin systems (sepia, RPE). Once again, this is consistent with the difference in the cross-linked substructure reported for eumelanin and pheomelanin premelanosomes by Brumbaugh in 1968.48 Thus, the interior of the red hair melanosomes is likely exposed upon extraction from the keratin matrix of the hair, so we cannot unambiguously state whether pheomelanin is on the surface or encapsulated in eumelanin, as found for iris melanosomes and neuromelanin. It is possible that the casing model is generally applicable to melanosomes containing mixed melanin (both from dopaminequinone and dopaquinone), but it remains to be validated for dopaquinone-derived melanins. Let us consider the potential implications of this structural model. In the brain, dopamine is cytotoxic to neuronal cells through oxidation to dopaminequinone.166 Kinetic data show

Centennial Feature Article that in neurons, cysteinyldopamines are produced until the levels of available cysteine are depleted (similar to the scheme shown in Figure 3 for dopaquinone). In fact, among the various brain regions, the highest levels of 5-S-cysteinyldopamine were detected in substantia nigra, although still in trace concentrations.167 The formation of cysteinyldopamines is hypothesized to be a protective mechanism, preventing formation of the neurotoxic dopaminequinone. A casing model for the structure of neuromelanin therefore provides a mechanism to capture dopaminequinone as part of the pheomelanic core, removing this neurotoxic precursor, and then shielding it from the neuron by encasing it in a eumelanin coat. It is also interesting to consider this result in terms of the pathology of Parkinson’s disease (PD). In Parkinson’s disease, there is a selective loss of pigmented neurons in the substantia nigra.168 From a thermodynamic perspective, an oxidation potential of 0 V vs NHE for the surface of neuromelanin is not sufficiently reductive to generate a high level of oxidative stress. Therefore, neuromelanin’s surface electrochemical potential is less reductive than might be anticipated given neuromelanin’s complicated but unresolved role in the selective loss of pigmented neurons of the substantia nigra associated with PD.55,169-173 Neuromelanin can be oxidized and degraded by H2O2,55 and this process likely occurs also in the human brain during PD, where an extensive depletion of neuromelanin occurs after neuronal death. Degradation of neuromelanin would expose the pheomelanic core, which would then cause oxidative stress. Thus, a slow loss of pigment could actually result in a constant level of inflammation. With regard to iris melanosomes, consider epidemiology data on uveal melanomas, which are the most common intraocular malignant tumor in human adults.174-178 A population based study on the relationship between racial/ethnic group and incidence of uveal melanoma found that the incidence of uveal melanoma is highest in non-Hispanic whites, followed by Hispanics, Asians, and blacks, with a white/black incidence ratio of uveal melanoma of 18:1.174 These epidemiological data suggest that the light-colored eye is at higher risk for the occurrence of uveal melanoma. In fact, several studies have shown that light-colored irises (blue, hazel, etc.) have a higher incidence of uveal melanoma.175-177 Recently, a meta-analysis based on 10 studies (1732 cases) revealed that a blue or gray iris is a statistically significant risk factor for the development of uveal melanoma.178 Earlier this year, Ito and collaborators reported the quantity and types of melanin in iridal melanocytes (Figure 1), finding that the amount and type of melanin present vary with iris color.46 Relevant to this discussion, the amount of pheomelanin remains fairly constant for different colored irises, but the total amount of melanin present (the other component being eumelanin) is greater in dark-colored irises. These results suggest that dark-colored irides have an extensive eumelanin surface while light-colored irides have a thin eumelanic exterior where a small amount of surface damage could result in exposure of the pheomelanin core. We are currently examining this issue by obtaining PEEM data on melanosomes from different colored human irides, and comparing the data to the eumelanin/ pheomelanin content determined from chemical analysis. Coupled to a lower total amount of melanin in lighter-colored irises, this model could be a contributor to the observed epidemiology of uveal melanoma. Implication of PEEM Studies of the Surface of Melanosomes: Photoinduced Aerobic Reactivity and Age-Dependent Changes in the Surface Composition of Human RPE

J. Phys. Chem. B, Vol. 112, No. 42, 2008 13213 Melanosomes. Finally, we reconsider the observation that, with increasing age, the photoinduced uptake of oxygen by RPE melanosomes increases (Figure 5).125 This increased aerobic reactivity suggests that aging melanosomes contribute to oxidative stress. Central to understanding the mechanism underlying this behavior is knowing the age-dependent surface oxidation potential(s) of RPE melanosomes. This is precisely the information provided by PEEM. We now consider the two proposed models to account for this behavior and describe PEEM experiments designed to test whether they contribute to the observed behavior. First, with age, there could be a lowering of the surface oxidation potential through oxidative/ photochemical damage and/or iron binding.123,179-181 The oxidation of DHI and especially DHICA are accelerated in the presence of iron,182 suggesting that the binding of iron to the surface of the melanosome should affect the local oxidation potential, and perhaps increase the capacity of the melanosome to induce oxidative stress. To address the potential role of iron, the surface oxidation potential of sepia was determined as a function of added Fe(III) up to the saturation limit of 86 000 ng/mg melanin.138 It is interesting to note that this saturation limit corresponds to about one Fe(III) for every four monomeric building blocks in the pigment. SEM images show that the diameter and surface characteristics are unaffected by the added iron.129 Changes of pH in the solutions reveal that the binding of an individual Fe(III) cation results in the release of ∼3 H+ ions from the pigment granule, suggesting that binding occurs to catechol groups,130 which is consistent with recently reported Raman studies.183 PEEM studies reveal that the threshold photoionization energy is not affected by added Fe(III). Therefore, if there are changes in the surface structure that accompany iron binding, which would need to be at a level unresolvable by SEM or AFM, such changes do not produce measurable differences in the electrochemical properties. Second, the increased aerobic reactivity may arise from lipofuscin deposits on the surface of RPE melanosomes.115,184-186 Lipofuscin is a heterogeneous group of lipid-protein aggregates with a characteristic fluorescence emission.187 With age, RPE cells lose melanosomes and gain lipofuscin.188-190 PEEM studies of age-dependent RPE melanosomes and lipofuscin reveal that young (14-year-old) human RPE melanosomes exhibit a single surface photoionization threshold of 4.1 eV, while older (g59-year-old) melanosomes have two thresholds, ∼4.4 and 3.6 eV. Lipofuscin, however, reveals two ageindependent thresholds of 4.4 and 3.4 eV.138 These collective results support the conclusion that lipofuscin is present on the surface of the isolated melanosomes and increases in coverage with age. While RPE melanosomes exhibit an increase in their reactivity with oxygen by a factor of 2.4 between young (80-year-old) specimens, the rate of oxygen uptake for lipofuscin is ∼13-fold greater than that in the young RPE melanosomes.125 Thus, if lipofuscin covers ∼2.4/13 ) 0.18 of the surface area of the melanosome, it could account for the observed age-dependent increase. It is interesting to recall that AFM force-indentation experiments suggested that 20% of the surface area of old (76-year-old) RPE melanosomes was coated by lipofuscin.137 With aging, RPE melanosomes can become totally encased in lipofuscin, forming a material commonly referred to as melanolipofuscin. It requires a significant buildup of lipofuscin to see these modified melanosomes in TEM imaging.115 Thus,

13214 J. Phys. Chem. B, Vol. 112, No. 42, 2008 in studying age-dependent behavior, it is necessary to separate melanosomes from melanolipofuscin and lipofuscin. The current accepted practice is to separate pigments on a discontinuous sucrose gradient, taking advantage of the different densities of melanosomes, melanolipofuscin, and lipofuscin. Melanosomes collect in a sucrose layer with a density of 1.255 g cm-3, while lipofuscin collects at the interface between layers with densities of 1.178 and 1.153 g cm-3. Melanolipofuscin can be separated in a number of layers between these two values, reflecting varying thicknesses of the lipofuscin coating. It then becomes important to determine whether this difference is sufficient to separate partially coated RPE melanosomes from uncoated melanosomes using the sucrose gradient method, which is the common approach for generating samples of “purified” melanosomes. Taking the density of melanosomes to be 1.255 g cm-3 and that of lipofuscin to be the midpoint of the two layers at the interface, 1.166 g cm-3, then as lipofuscin adheres to the surface of the melanosome, the density of the organelle will decrease. Consider the amount of lipofuscin that must adhere to the melanosome to move the organelle into the interface between 1.255 and 1.178 g cm-3. If we take the desired density to be the midpoint between the two layers, 1.216 g cm-3, and model the RPE melanosome as an ellipsoid (axes of 800 and 365 nm), this change of density would require that lipofuscin encase the entire melanosome with a thickness of 90 nm. If we restrict the coverage to 20%, then the lipofuscin deposits would need to be 450 nm tall. AFM images do not show obvious changes in surface morphology at these “adhesion” sites. The force-indentation experiments suggest a thickness of ∼10 nm, and for coverage of 20% of the surface, the density of the organelle changes by 0.1%, clearly less than can be resolved using a discontinuous sucrose gradient isolation procedure. Thus, the melanosomes are not pure but would contain varying levels of lipofuscin adhesion, which are likely the precursors to isolated melanolipofuscin. The important point remains that the presence of thin deposits of lipofuscin on the surface of the RPE melanosome would affect both the surface oxidation potential and the overall photoreactivity of the melanosome. The collective data from surface-sensitive microscopies and spectroscopies support the conclusion that the accumulation of such thin randomly located deposits of lipofuscin are responsible for the increased aerobic reactivity observed with age. 7. Future Perspectives While much is known about melanosomes and the melanin pigment, there remain many significant unanswered questions. There will continue to be great interest in determining the molecular structure of the different melanins found in nature with an eye to associating the similarities and differences between those structures with function(s) in different tissues. Several groups are working on structure elucidation for natural and synthetic systems. In addition, there is an expanding literature on the computation properties of model systems.191-198 However, as highlighted in the historical review of this field, it remains important to exercise great care in relating the findings on model and synthetic systems to the natural pigment. Not only do model systems differ on a molecular level, but they lack the structural organization present in the melanosome or neuromelanin organelle, and it is reasonable to believe that this organization is central to functional properties. Hopefully a clear nomenclature will be developed in the near future so that these distinctions are clearly evident to the reader, much like what

Simon et al. Fitzpatrick and co-workers did for the terminology of vertebrate melanin-containing cells in 1966.199 In the remainder of this section, we pose three grand challenge questions that build on the spatial scales described in this article. Each has its corresponding questions that accompany aging, as the integrity of the molecules on the surface and the associated functional roles played by the melanosome are all influenced by age-dependent changes in surface structure. Solving these questions will not only significantly advance the chemical understanding of melanin in its assembled, natural form but could allow us to develop novel approaches for using the melanosome as a therapeutic platform. Is There Molecular Organization on the Surface of the Melanosome? It is interesting to consider the “molecular” aspects of the surface that occur during formation of the melanosome. Initially, the melanosome is unpigmented and exhibits a clear lipid bilayer membrane. Thus, prior to melanogenesis, the surface of the organelles is certainly structured. Upon complete pigmentation, it is believed that this lipid layer is lost, but it is not known whether there is a spatial scale on which there is well defined molecular organization. The ability of intact melanosomes to act as ion-exchange materials without apparent changes in morphology argues in support of the existence of an organized surface, yet this certainly has not been experimentally established. An approach is needed to map orientational properties of molecules on the surface, with sufficient spatial resolution to be able to determine whether the surface molecules are aligned, and if so, on what spatial scale such alignment may persist. What Are the Details of the Metal Ion Transport and Storage Process? Melanosomes have the ability to store and release large amounts of metal ions. Recall that ∼150-nmdiameter sepia can store ∼8% Fe(III) by weight (that is one iron atom for every four monomersDHI or DHICAsunits), and no changes can be observed by SEM or surface area as probed by N2 adsorption. It is clear from imaging experiments that the metal ions are not stored on the surface, yet their initial binding must occur to surface sites. Nothing is known about the transport processes associated with the binding and release of metals, yet this function is critical to understanding hypothesized adverse effects of metal binding in the retinal pigment epithelium and human brain as well as the potential role of melanosomes in calcium homeostasis. The “chemical potential” is clearly designed so that metal ions bound to the surface enter the melanosome. However, how does this work on the molecular level? For various amounts of metal, are the ions homogeneously distributed throughout the melanosomes or are they concentrated in the center or just below the surface? How do the channels’ structures then enable the ions to move into and out of the melanosomes? Several metals can cobind, with the presence of one metal not affecting the other in reaching its binding sites. How are these different types of binding sites distributed on the surface and within the melanosome? Can the Surface of the Melanosome Be Used for Pharmacology? In 1979, Bengt Larsson and Hans Tja¨lve reported on the mechanism of drug binding to bovine uveal melanin.200 Since that report, Larsson and co-workers extended their studies on molecular binding to melanin and melanosomes.201,202 In 2007, it was reported that lipophilic beta-blockers bind to bovine melanosomes, which prompted hypotheses about their binding to human melanosomes in the choroid and RPE.203 Binding of drugs to melanosomes would negatively impact drug efficacy at least in the short term. At the same time, one could consider

Centennial Feature Article using the melanosome to provide a means to store a drug in a particular region, and then time-release the drug as controlled by the rate constant for the dissociation of the drug from the melanosome surface. Such an approach would enable the targeting of the RPE/choroid region of the eye or the substantia nigra of the brain. At present, many questions remain to be addressed. What are the molecular details of the surface that control drug binding? What functional group(s) or molecular architectures bind to melanin within different affinity ranges? Can molecules be photoreleased from the surface of the melanosome? Are molecules bound on the surface or do they become intercalated into the melanosome structure? There is a large opportunity for systematic studies in the area of molecular binding to melanosomes. Acknowledgment. This work was supported by the MFEL Program administered by the AFOSR and Duke University. We acknowledge the productive contributions and discussion with many who work in the field of melanin research, especially Shosuke Ito, Tadeusz Sarna, and Luigi Zecca. We thank fellow group members who contributed their energy and ideas to the projects described herein. Finally, we thank Ann G. Motten for her careful editing of the manuscript. References and Notes (1) Santorius, S. De statica medicine et de responsione ad static masticem. Sphorismorum sectionibus septem comprehensa; Marcantonio Brogiollo: Venice, Italy, 1614. (2) Malpighi, M. De externo tactus organ anatomica obserVatio; Apud Agidium Longu: Rome, 1665. (3) Littre, A. Histoirede l”academie royale des sciences; Hochereau: Paris, 1720. (4) Le Cat, C. N. Traitede la Couleur de la Peau Humaines en General, de celle des Negros en Particulier, et de la Metamorphose d’une de ces Coleurs en l’autre soit de Naissance, soit Accidentellement; Amsterdam, The Netherlands, 1765. (5) Furth, V.; Schmeider, O.; Schneider, H. Beitr. Chem. Physiol. Pathol. 1902, 1, 229. (6) Bloch, B. In Handbuch der Hait und Gerschlechts Krankeiten; Jadassohn, J., Ed.; Springer: Berlin,1927; Vol. 1, pp 434-451. (7) Bourquelot, E.; Bertrand, G. C. R. Soc. Biol. 1895, 47, 582. (8) Bertrand, G. C. R. Acad. Sci. 1896, 112, 1215. (9) Raper, H. S Biochem. J. 1927, 21, 89–96. (10) Mason, H. S. J. Biol. Chem. 1948, 172, 83–99. (11) Commoner, B.; Townsend, J.; Pake, G. W. Nature 1954, 176, 689–691. (12) White, L. P. Nature 1958, 182, 1427–1428. (13) Felix, C. C.; Hyde, J. S.; Sarna, T.; Sealy, R. C. J. Am. Chem. Soc. 1978, 100, 3922–3926. (14) Rozanowska, M.; Sarna, T.; Land, E. J.; Truscott, T. G. Free Radical Biol. Med. 1999, 26, 518–525. (15) Sarna, T.; Swartz, H. M. The Physical Properties of Melanins. In The Pigmentary System; Nordlund, J. J., Boissy, R. E., Hearing, V. J., King, R. A., Ortonne, J. P., Eds.; Oxford University Press: New York, 1998; pp 333-357. (16) Sarna, T.; Bober, A.; Burke, J. M.; Korytowski, W.; Nilges, M.; Rozanowska, M. InVest. Ophthalmol. Vis. Sci. 1993, 37, S331. (17) Sarna, T.; Swartz, H. Interactions of melanin with oxygen (and related species). In Atmospheric Oxidation and Anti-oxidants; Scott, G. A., Ed.; Elsevier: Amsterdam, The Netherlands, 1993; Vol. 3, pp129-169. (18) Hong, L.; Simon, J. D. J. Phys. Chem. B 2007, 111, 7938–7947. (19) Double, K. L.; Gerlach, M.; Schunemann, V.; Trautwein, A. X.; Zecca, L.; Gallorini, M.; Youdim, M. B. H.; Riederer, P.; Ben-Shachar, D. Biochem. Pharmacol. 2003, 66, 489–494. (20) Zucca, F. A.; Bellei, C.; Giannelli, S.; Terreni, M. R.; Gallorini, M.; Rizzio, E.; Pezzoli, G.; Albertini, A.; Zecca, L. J. Neural. Transm. 2006, 113, 757–767. (21) Bush, W. D.; Simon, J. D. Pigm. Cell Res. 2007, 20, 134–139. (22) Hoogduijn, M. J.; Smit, N. P.; van der Laarse, A.; van Nieuwpoort, A. F.; Wood, J. M.; Thody, A. J. Pigm. Cell Res. 2003, 16, 127–132. (23) Joshi, P. G.; Nair, N.; Begum, G.; Joshi, N. B.; Sinkar, V. P.; Vora, S. Pigm. Cell Res. 2007, 20, 380–384. (24) Goding, C. Pigm. Cell Res. 2007, 20, 333. (25) Felix, C. C.; Hyde, J. S.; Sarna, T.; Sealy, R. C. Biochem. Biophys. Res. Commun. 1978, 84, 335–341. (26) Sarna, T.; Sealy, R. C. Photochem. Photobiol. 1984, 39, 69–74.

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