Luận án tiến sĩ: Physical and chemical analyses of melanins

PHYSICAL AND CHEMICAL ANALYSES OF MELANINS By Lian Hong Department of Chemistry Duke University Date:_ April 24 2006 Approved: l£-=. Simon, Supervisor ⁄2 322 Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Chemistry in the Graduate School of Duke University 2006 UMI Number: 3244835 INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted.

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ProQuest Information and Learning Company 300 North Zeeb Road P. Box 1346 Ann Arbor, MI 48106-1346 ABSTRACT PHYSICAL AND CHEMICAL ANALYSES OF MELANINS By Lian Hong Department of Chemistry Duke University Date:__ April 24 2006 Approved: An abstract of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Chemistry in the Graduate School of Duke University 2006 Abstract Melanin is a pigment that is ubiquitous in the biological world. In addition to its obvious function of adornment, melanin is believed to serve as a metal reservoir and sunscreen. This work examined the metal binding behavior, the chemical structures and the interaction with light of natural melanins isolated from several different sources.

Sepia eumelanin was used as a model to study the metal binding behavior of eumelanins. Infrared spectroscopic analysis suggests that Mg(II), Ca(II) and Zn(II) are mainly bound to carboxyl groups and Cu(II) binds to hydroxyl groups at pH ~4. The effect of metal content on the aerobic reactivity was examined by analyzing the ability of melanin to nick DNA. Of the metals studied, Cu(II)- and Fe(III)-loaded melanin showed the most damaging effects to DNA.

The photoreactivity of melanin is most important in two parts of the body: the skin and the eye. We first studied melanosomes from hair, which correlate well with the pigment in the skin. Chemical analysis showed that eumelanin was the major pigment in the melanosomes in black hair, while the melanosomes from red hair contained both pheomelanin and eumelanin. Photoelectron emission microscopic (PEEM) analyses on the two melanosomes revealed that the photoionization threshold of eumelanin was 4.4 eV and that pheomelanin was 3.

The low threshold of pheomelanin may contribute to the high incidence of skin cancer in redheads. 11 We then examined the physical and chemical properties of bovine ocular melanosomes. The collective data showed that iris melanosomes had a higher degree of crosslinking and conjugation than choroid and RPE melanosomes of the same age group. The content of carboxyl groups and the degree of conjugation in melanosomes were smaller in older samples.

These tissue and age-related differences are likely to result in variation of efficiency in melanin functions such as metal binding and light absorption. Finally, we examined the photoionization properties of human RPE melanosomes and lipofuscin, another important pigment in the RPE. RPE melanosomes became more pro-oxidant with aging and lipofuscins were more pro- oxidant than melanosomes. iv Contents Page Abstract ili List of Tables vi List of Figures Vii Abbreviations 1X Acknowledgements xii Chapter One: Background Chapter Two: Insights to the Metal Binding to Melanin and its Effects on the 11 Aerobic Reactivity of Melanin Chapter Three: Physiochemical Analysis of Melanosomes Extracted from 35 Human Black and Red Hair Chapter Four: Physiochemical Analysis of Melanosomes Extracted from 57 Bovine Choroid, Iris and Retinal Pigment Epithelium Chapter Five: Age-Dependent Ionization Potentials of Melanosomes and 99 Lipofuscin Isolated from Human Retinal Pigment Epithelium Cells References 121 Biography 132 List of Tables Table Description Page The pH values of salt solutions after suspension of EDTA-washed 19 melanin.

2-2 Summary of the changes in the IR spectra of melanin upon loading of 22 different metals. 3-1 Chemical degradation analyses of human hair melanosomes. 47 3-2 Metal ion contents of human hair melanosomes. 49 Summary of the size analysis of the melanosomes isolated from 68 mature bovine eyes.

Summary of the size analysis of the melanosomes isolated from 69 newborn bovine eyes. 4-3 The contents of Na(I), Mg(II), K(I), Ca(ID and Zn(II) in bovine ocular 72 melanosomes determined by ICP-MS. 4-4 Summary of amino acid analyses for bovine ocular melanosomes. 74 4-5 Summary of chemical degradation analyses of bovine ocular 76 melanosomes.

4-6 C, N and H mass percentages in melanosomes obtained by elemental 78 analysis are corrected according to their amino acid and metal contents. 4-7 C/N and O/N molar ratios on the surface of bovine ocular 82 melanosomes determined by XPS. 4-8 Composition of carbon functional groups. 85 Composition of oxygen functional groups.

86 4-10 Composition of nitrogen functional groups. 87 4-11 The peak positions of the aromatic rings and carbonyls in the IR 91 spectra of bovine ocular melanosomes. Summary of the size analysis of human RPE melanosomes from three 107 vi different patients. List of Figures Description Page An abbreviated illustration of biogenesis of eumelanin and pheomelanin.

1-2 Proposed typical structures of eumelanin and pheomelanin based on biosynthetic pathway and chemical degradation analysis of melanin. IR spectrum of Ca(II)-enriched melanin as a function of the solution 23 concentration of Ca(ID). IR spectrum of Zn(I])-enriched melanin as a function of the solution 25 concentration of Zn(II) in the spectral region of a) 900-1900 cm” and b) 2000-4000 cm”. 2-3 IR spectrum of Cu(II)-enriched melanin as a function of the solution 26 concentration of Cu(II) in the spectral region of a) 900-1900 cm” and b) 2000-4000 cm”.

2-4 Image of a typical gel of pUC18 DNA with bands of two forms. 30 Aerobic reactivity of melanins loaded with different metal ions in a) 31 dark and b) light reactions. 2-6 The effects of [Fe(III] on the ability of melanin to break supercoiled 33 DNA in a dark reaction. 3-1 AFM images of melanosomes isolated from a) black hair and b) red 44 hair.

3-2 High magnification AFM images of black-hair melanosomes (a, b) 45 and red-hair melanosomes (c, d). 3-3 Integrated wavelength-dependent FEL-PEEM data for a) human black 51 hair and b) human red hair melanosomes. 3-4 Time-dependent absorption of Fe(II)-Cyt ¢ following the mixing of 33 Fe(II)-Cyt c with equal masses of eumelanosomes and pheomelanosomes. vii SEM images of melanosomes isolated from a) mature bovine RPE, b) newborn bovine RPE, c) mature bovine choroid, d) newborn bovine choroid, e) mature bovine iris and f) newborn bovine iris.

The distributions of the lengths of the long axis of a) newborn bovine 66 RPE melanosomes and b) mature bovine RPE melanosomes. The high-resolution X-ray photoelectron spectra of Cls in (a) 83 newborn choroid and (b) mature choroid melanosomes. 4-4 The high-resolution X-ray photoelectron spectra of Ols in (a) 84 newborn choroid and (b) mature choroid melanosomes. 4-5 IR spectra of a) newborn bovine choroid, iris and RPE melanosomes 90 b) mature bovine choroid, iris and RPE melanosomes in the range of 800-2000 cm `.

4-6 The !C CP/MAS solid-state NMR spectra of a) mature choroid 95 melanosomes, b) EDTA-washed mature iris melanosomes, c) EDTA- washed mature choroid melanosomes, d) EDTA-washed newborn iris melanosomes and e) EDTA-washed newborn choroid melanosomes. 5-1 SEM images of RPE melanosomes from patients a) 14, b) 59, and c) 105 76, years old, respectively. AFM micrographs of RPE melanosomes: a, b) a smooth granule and 109 c, d) a granule with a rough surface and e, f) a “transient” granule. 5-3 SEM images of lipofuscin granules from donors a) 14, b) 59, and c) 110 76 years old.

5-4 The emission spectra of RPE lipofuscin granules from donors a) 14, b) 112 59, and c) 76 years old. 5-5 Integrated wavelength-dependent FEL-PEEM data for bovine ovoid 115 and rod-shaped melanosomes. 5-6 Integrated wavelength-dependent FEL-PEEM data for melanosomes 116 isolated from human RPE cells from a) 14- and b) 76-year-old specimens. 5-7 Integrated wavelength-dependent FEL-PEEM data for lipofuscin 117 isolated from human RPE cells from a) 14- and b) 76-year-old specimens.

Vili Abbreviations AEM atomic force microscopy aminohydroxyphenylalanine age-related macular degeneration ChNS chloroform-insoluble ChS chloroform-soluble COOH carboxyl CP/MAS cross-polarization magic angle spinning CV coefficient of variation Cyt Cytochrome DHI 5,6-dihydroxylindole DHICA 5,6-dihydroxylindole-2-carboxylic acid DOPA dihydroxylphenylalanine DTT dithiothreitol EB ethidium bromide EDTA ethylenediaminetetraacetic acid FEL free electron laser HI hydriodic acid HPLC high performance liquid chromatography ICP-MS inductively coupled plasma mass spectrometry IPE iris pigment epithelium infrared 1X the length of the long axis minimal erythemal dose amine normal hydrogen electrode national institute of standards and technology nuclear magnetic resonance neuromelanin OH hydroxyl PBS phosphate buffered saline PBSA Dulbecco’s phosphate buffered saline without calcium and magnesium PD Parkinson’s disease PEEM photoelectron emission microscopy PTCA 2,3,5-pyrroletricarboxylic acid ROS reactive oxygen specie RPE retinal pigment epithelium RSF relative sensitivity factor the length of the short axis SEM scanning electron microscopy SN substantia nigra TEM transmission electron microscopy TDCA thiazole-4,5-dicarboxyl acid ™ total melanin X TMS tetramethylsilane XPS X-ray photoelectron spectroscopy xi Acknowledgements First I would like to thank my thesis advisor Dr. Simon for his guidance, support, encouragement and training throughout these past years. His broad connections with research groups and scientists outside Duke are essential for the completion of my dissertation. I gratefully acknowledge our collaborators: Prof.

Kazumasa Wakamatsu, and Shosuke Ito for carrying out chemical degradation analysis; Bhavin B. Adhyaru, Chi- Yuan Cheng and Prof. Bowers for collecting '3C solid state NMR spectra; and Jacob Garguilo, Robert J. Nemanich and Glenn S.

Edwards for performing photoelectron emission microscopic analyses. I also appreciate the past and current members of the Simon Lab. In particular, I would like to thank Dr. Yan liu for stimulating discussions, help of experiments and sharing of her experience and knowledge with me.

I acknowledge her invaluable help and her important inputs in chapter 2 and chapter 3 of this thesis. Alexander Samokhvalov for employing PEEM and analyzing black/red hair melanosomes and Laura Anzaldi for help on the size analysis of human RPE melanosomes. I also thank Jenny Perry, Laura Lamb, Valerie Kempf, William Derek Bush, Weslyn Ward and Jean Hatcher for various help. I owe special thanks to my husband Xiangqian for his patience, love, and encouragement during these years of my studies.

Finally, I would like to thank my family and friends for their support. xii Chapter One. Background Occurrence of Melanin and Melanogenesis Melanin is an omnibus term describing a large range of natural and synthetic phenolic-quinonoid pigments. Since this current work is focusing on the characterization of natural melanins, only the properties of natural melanins will be discussed in this section.

Natural melanins are classified into two groups according to their chemical structures and molecular precursors: the insoluble black to brown eumelanin derived from dihydroxyphenylalanine (DOPA), and the alkaline-soluble yellow to red pheomelanin derived from DOPA and cysteine (Crippa 1989; Ito 1993; Riley 1997; Ito 1998). Eumelanin is widespread in the biological world. It occurs in the skin, hair, eye, brain and inner ear of mammals, the feathers of birds, the ink sac of cuttlefish, and in some reptiles, amphibians and fungi. Pheomelanin, on the other hand, is mainly found in the feathers of birds and the hair and skin of mammals.

Melanin pigmentation occurs in a specialized organelle, the melanosome, in most pigment generating cells (Prota 1992). In addition to the melanin content, melanosomes contain structural proteins, enzymes, lipids, and metals (Prota 1992). Eumelanosomes, with eumelanin as the major pigment, are mostly integrated granules, uniformly and densely packed with pigments (Parakkal 1967). Pheomelanosomes, however, with pheomelanin as the major pigment, are unevenly packed with pigments and are loosely aggregated small granules (Parakkal 1967).