Lian Hong

Comparison of Structural and Chemical Properties of Black and Red Human Hair Melanosomes

Abstract Melanosomes in black and red human hair are isolated and characterized by various chemical and physical techniques. Different yields of 4-amino-hydroxyphenolanaline by HI hydrolysis (a marker for pheomelanin) and pyrrole-2,3,5-tricarboxylic acid by KMnO4/H+ oxidation (a marker for eumelanin) indicate that the melanosomes in black hair are eumelanosomes, whereas those in red hair are mainly pheomelanosomes. Atomic force microscopy reveals that eumelanosomes and pheomelanosomes have ellipsoidal and spherical shapes, respectively. Eumelanosomes maintain structural integrity upon extraction from the keratin matrix, whereas pheomelanosomes tend to fall apart. The black-hair eumelanosomes have an average of 14.6 ± 0.5% amino acids content, which is attributed to the internal proteins entrapped in the melanosomes granules. The red-hair melanosomes contain more than 44% of amino acid content even after extensive proteolytic digestion. This high content of amino acids and the poorly reserved integrity of red-hair melanosomes suggest that some proteins are possibly covalently bonded with the melanin constituents in addition to those that are entrapped inside the melanin species. Soluene solubilization assay indicates the absorbance of melanin per gram of sample, adjusted for the amino acid content, is a factor of 2.9 greater for the black-hair melanosomes than the red-hair melanosomes. Metal analysis reveals significant amounts of diverse heavy metal ions bound to the two types of melanosomes. The amount of Cu(II) and Zn(II) are similar but Fe(III) content is four times higher in the red-hair melanosomes. 13C solid-state nuclear magnetic resonance spectra and infrared spectra are presented and are shown to be powerful techniques for discerning differences in the amino acid contents, the 5,6-dihydroxyindole-2-carboxylic acid:5,6-dihydroxyindole ratio, and the degree of cross-linking in the pigment. Excellent agreement is observed between these spectral results and the chemical degradation data.

Insights into Melanosomes and Melanin from Some Interesting Spatial and Temporal Properties

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

Comparisons of the Structural and Chemical Properties of Melanosomes Isolated from Retinal Pigment Epithelium, Iris and Choroid of Newborn and Mature Bovine Eyes¶

Abstract Melanosomes were isolated from the retinal pigment epithelium (RPE), iris and choroid of mature (age >2 years) and newborn (age <1 week) bovine eyes. Scanning electron microscopy was utilized to analyze the morphology of the melanosomes, which were found to vary among different tissues and different ages. While the total content of amino acids differs slightly (ranging from 9% to 15% by mass), the distributions of the amino acids are similar. The pheomelanin content is low in the choroid and the RPE (0.1–0.5%), and moderate in the iris (<2%); therefore, the major melanin component of bovine eye melanosomes is eumelanin, independent of the shape of the melanosomes. The yields of pyrrole-2,3,5-tricarboxylic acid from melanosomes decrease in the following order: choroid > iris > RPE, and exhibit decreasing yields with age. 13C solid-state nuclear magnetic resonance (NMR) spectroscopic analysis of iris and choroid melanosomes indicates the same trends. These observations suggest that the 5,6-dihydroxyindole-2-carboxylic acid contents decrease in the following order: choroid > iris > RPE, and decrease with age. Moreover, the 13C solid-state NMR spectra show (1) for the same age samples, the CH:Cq ratio for choroid is larger than that for iris melanosomes; and (2) an increase in the concentration of carbonyl groups with age within each type of melanosome.

Photoionization Thresholds of Melanins Obtained from Free Electron Laser‐Photoelectron Emission Microscopy, Femtosecond Transient Absorption Spectroscopy and Electron Paramagnetic Resonance Measurements of Oxygen Photoconsumption

Abstract Free electron laser–photoelectron emission microscopy (FEL-PEEM), femtosecond absorption spectroscopy and electron paramagnetic resonance (EPR) measurements of oxygen photoconsumption were used to probe the threshold potential for ionization of eumelanosomes and pheomelanosomes isolated from human hair. FEL-PEEM data show that both pigments are characterized by an ionization threshold at 282 nm. However, pheomelanosomes exhibit a second ionization threshold at 326 nm, which is interpreted to be reflective of the benzothiazine structural motif present in pheomelanin and absent in eumelanin. The lower ionization threshold for pheomelanin is supported by femtosecond transient absorption spectroscopy. Unlike photolysis at 350 nm, following excitation of solubalized synthetic pheomelanin at 303 nm, the transient spectrum observed between 500 and 700 nm matches that for the solvated electron, indicating the photoionization threshold for the solubalized pigment is between 350 and 303 nm. For the same synthetic pheomelanin, EPR oximetry experiments reveal an increased rate of oxygen uptake between 338 nm and 323 nm, narrowing the threshold for photoionization to sit between these two wavelengths. These results on the solubalized synthetic pigment are consistent with the FEL-PEEM results on the human melanosomes. 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 red-haired individuals.

Oxidation Potentials of Human Eumelanosomes and Pheomelanosomes

Abstract Eumelanosomes and pheomelanosomes isolated from black and red human hair, respectively, were studied by photoelectron emission microscopy (PEEM). PEEM images were collected at various wavelengths between 207 and 344 nm, using the spontaneous emission output of the Duke OK-4 free electron laser (FEL). Analysis of the FEL-PEEM data revealed ionization thresholds of 4.6 and 3.9 eV corresponding to oxidation potentials of −0.2 and +0.5 V vs normal hydrogen electrode for eumelanosomes and pheomelanosomes, respectively. The difference in oxidation potential is attributed to the pigment content of the melanosome, namely whether it contains primarily eumelanin and pheomelanin. The effect of added melanosomes on the reduction of Fe(III)-cytochrome showed pheomelanosomes are stronger reducing agents than eumelanosomes, consistent with the measured oxidation potentials. The FEL-PEEM experiment offers to be an important new approach for quantifying the effects of age, oxidation and metal accumulation on the oxidation potentials of intact melanosomes.

Quantification of the Binding Properties of Cu2+ to the Amyloid Beta Peptide: Coordination Spheres for Human and Rat Peptides and Implication on Cu2+-Induced Aggregation

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.

Effects of photodegradation on the physical and antioxidant properties of melanosomes isolated from retinal pigment epithelium

Abstract Melanosomes of the retinal pigment epithelium (RPE) are relatively long-lived organelles that are theoretically susceptible to changes induced by exposure to visible light. Here melanosomes were isolated from porcine RPE cells and subjected to high intensity visible light to determine the effects of illumination on melanosome structure and on the content and antioxidant properties of melanin. As compared to untreated melanosomes, illuminated granules showed morphologic changes consistent with photodegradation, which included variable reductions in electron density demonstrated by transmission electron microscopy (TEM), and particle fragmentation and surface disruption revealed by scanning electron microscopy (SEM) and atomic force microscopy. Illuminated melanosomes had lower melanin content, indicated by measures of absorbance and electron spin resonance (ESR) signal intensity, and reduced ability to bind iron, shown by chemical and ESR analyses. Compared to untreated melanosomes, ESR–spin trapping analyses further indicated that illuminated melanosomes show increased photogeneration of superoxide anion and reduced ability to inhibit the iron ion–catalyzed free radical decomposition of hydrogen peroxide. It appears therefore that visible light irradiation can disrupt the structure of RPE melanosomes and reduce the amount and antioxidant properties of melanin. Some of these changes occur in human RPE melanosomes with aging and the results obtained here suggest that visible light irradiation is at least partly responsible. The consequence of light-induced changes in RPE melanosomes may be a diminished capacity of melanin to help protect aged cells from oxidative damage, perhaps increasing the risk of diseases with an oxidative stress component such as age-related macular degeneration.

Binding of Metal Ions to Melanin and Their Effects on the Aerobic Reactivity

Abstract The binding of Mg(II), Ca(II), Zn(II), Cu(II) and Fe(III) to ETDA-washed Sepia melanin is quantified by inductively coupled plasma mass spectrometry. By monitoring the solution pH change associated with metal uptake, it is concluded that Mg(II), Ca(II) and Zn(II) bind to carboxylic acid groups in melanin, Cu(II) binds to hydroxyl (OH) groups and Fe(III) binds to OH or amine groups. The aerobic reactivity of melanins with different metal contents is analyzed by examining their ability to cause strand breaks in supercoiled pUC18 DNA. Cu(II)- and Fe(III)-enriched melanins induce the most damage. Hydroxyl radical, ·OH, is proposed to be one of the reactive oxygen species responsible.

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

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

Insight into the binding of divalent cations to Sepia eumelanin from IR absorption spectroscopy.

Abstract IR absorption spectroscopy is used to examine the binding of the divalent cations Mg(II), Ca(II), Zn(II) and Cu(II) to melanin granules isolated from the ink sacs of Sepia officinalis. The functional groups of the melanin granules interacting with the bound metal ions are deduced by examining the effect of metal concentration on transition frequencies associated with the COOH, NH and OH moieties of the pigment. The coordinating groups vary with metal ion and with concentration. For the experimental conditions used (initial solution pH of 4, ionic strength of 100 mM and a melanin concentration of 1 mg mL−1) Mg(II), Ca(II) and Zn(II) bind to carboxylate groups and Cu(II) binds predominantly to phenolic (catechol) groups However, at a concentration of 10 mM Cu(II) also shows evidence of binding to carboxylate and amine groups, reflecting a secondary binding site that becomes populated as the catechol sites are depleted.