Melanin

ht.

J. i?iochem.

Pergamon

Cdl Bid. Vol. 29, No. II, pp. 1235-1239, 1997 ‘CT 1997 Elsevier Science Ltd. All rights reserved Printed in Great Britain

PII: S1357-2725(W)OOO13-7

MOLECULES

1357-2725197

$17.00 c 0.00

IN FOCUS

Melanin P. A. RILEY Department of Molecular Pathology, University College London Medical School, 46 Cleveland Street, London, WlP 6DB, U.K. Melanin is an irregular tight-absorbing polymer contaluiug iudoles and other lutermedlate products derived from tbe oxidation of tyrosine. Melanin is widely dispersed in tbe animal and plant kingdoms. It is the major pigment present in the surface structures of vertebrates. The critical step in melanin biogenesis is tbe oxidation of tyrosine by tbe enzyme tyroslnase. In vertebrates this enzyme is active only in specializedorgauelles in retinal pigment epitbellum and melanocytes. In mammals melanin is formed as intracellular granules. Melanin granules are transferred from melanocytes to epithellal cells and form tbe predomluaut plgmeut of hair and epidermis. Melauin has many biological functions. Reactive quluoue intermediates in the melanin biosynthetic pathway exhibit antibiotic properties aud the polymer is an important stre element of plant cell walls and insect cuticle. Light absorptiou by melanla has several functions, including photoreceptor sldeldlng, thermoregulation, pbotoprohtlou, display. Melanin is a powerful cation chelator and may act as a free radical sink. Melah is used commercially as a component of photoprotective creams, although maiuly for its free radical scavenging rather thau its tight absorption properties. Tbe p&neat is also a potential target for anti-melanoma therapy. 0 1997 Elsevler Science Ltd. All rights reserved Keywords: melanin pbotoprotectlon

free radicals redox reactions metal chelation

Int. J. Biochem. Cell Biol. (1997) 29, 1235-1239

INTRODUCTION

STRUCTURE

Melanin, a predominantly indolic polymer, is the major pigment present in surface structures of vertebrates. The origin of the name, from melanos (Greek dark), is not clear, but is usually attributed to the Swedish chemist Berzelius (1840). The term “melanin” has been used fairly indiscriminately to mean any dark pigment but the nomenclature has been refined in the case of mammalian melanin to include eumelunins, which, on permanganate oxidation, yield a small amount (about 1%) of pyrrole tricarboxylic acid (PTCA), and phaeomelanins, which are sulphurcontaining melanins that, on degradation by hydroiodic acid, give rise to about 20% aminohydroxyphenylalanine (AHP) (Ito and Fujita, 1985).

The basic structural unit of melanins is usually represented by covalently linked indoles (Fig. 1). Although it must be emphasized that the overall structure is not known, most melanins appear to be mixed polymers based on indoles but containing variable amounts of other pre-indolic products of the synthetic pathway. A simplified schematic outline of the melanogenic pathway is shown in Fig. 2. The indolic domains may be stacked by van der Waal’s interactions giving approximately 3.4 A interlayer spacing in X-ray diffraction (Thathachari, 1976), but the irregular interposition of other residues makes many regions of the polymer essentially amorphous. PROPERTIES

Light absorbance Received

8 October

1996; accepted

13 January

The melanin polymer has many interesting properties, among which the most conspicuous

1997. 1235

1236

P. A. Riley Conjugated PHOTON

polymer @-SORPTION

0

orthoquinone ELECTRON

hydroquinone ELECTRON

decarboxylated indole moiety

ACCEPTOR

-0/1iI,coo:zg 2carhoxyl indole moieties

DONOR

. CATION

BINDING

0

semiquinones DONOR or ACCEPTOR

.

0

1

-m

oy

\

coo-

#

Fig. 1. Melanin: schematic structure of indolic melanin shown as a region of 4’-7 linked substituted indoles indicating the high degree of conjugation of such domains. Carboxylic acid groups are attached to the C2 and the $6 functionalities are shown (top to bottom) as carbonyls in the orthoquinone form, deprotonated hydroxyls in the catecholic form, and an equilibrium form of linked semiquinones. The three major functions are indicated as electronic reactions, which render the polymer a potential free radical generator or scavenger, photon absorption and cation binding through the carboxyl groups. Some cationic binding may also involve the deprotonated hydroxyl groups or semiquinones.

is the wide spectral absorbance due to the high degree of conjugation in the molecule. The darkness of the pigment is a result of the fact that much of the visible spectrum is absorbed, including radiation with low quanta1 energy. The lowest energy transitions are from nonbonding to anti-bonding pi orbitals (n-4) that occur predominantly in carbonyl bonds (c--O), which are abundant in most melanins. Melanins also absorb in the ultra-violet region of the spectrum, involving transitions from bonding to anti-bonding pi orbitals (rc+$), which occur in unsaturated carbon bonds. Transitions from bonding to anti-bonding orbitals are facilitated by conjugation permitting electronic delocalization. As the degree of conjugation increases lower quanta1 energies are required for absorption; an effect termed bathochromicity. The majority of the visible spectral energy absorbed by melanin is converted into heat through photon-phonon coupling. Melanins with high levels of indole quinones (the eumelanins) appear darker because of the strong absorbance in the red part of the spectrum. This low frequency light absorption is largely through the carbonyls, melanins with fewer carbonyl groups

are paler and appear more yellow or red as is the case in the phaeomelanins. Of these, the most spectacular are the red melanins from chicken feathers, which contain high levels of sulphur in the form of benzothiazoles (Prota, 1992). Redox properties

Melanins, especially eumelanins, exhibit marked redox properties, and electron delocalization between orthoquinone and catecholic moieties of the polymer give rise to semiquinone free radicals, which can be detected by electron spin resonance spectroscopy (Sealy et al., 1980). Melanins can take part in one-electron and two-electron redox reactions and one of the effects of light absorption is photo-oxidation of the pigment, which, by increasing the carbonyl content, changes the absorbance properties of melanin, the so-called immediate pigment darkening (IPD) reaction. This photo-oxidation process generates superoxide radicals (Sarna and Sealy, 1984). Chelating properties

Melanins also have powerful cation chelating properties (Sarna et al., 1976) through the

i 231

Melanin

tyrosine 3,4-dihydroxyphenylalanine 1 TYROS,ME+

(dopa)

7

)

dopaquinone +

/-

dopyhrome

d

cysteinyldopa > cysteine cysteinyldopa

\

5,6-dihydroxyindole @HI) \

quinone

TRP-2

\

5,6-dihydroxyindole-2-carboxylic I

acid (DHICA)

indolequinone + indole-2-carboxylic acid quinone

1

t

J! MELANIN

Fig. 2. Schematic outline of the melanogenic pathway, showing the major intermediates of both eumelanin and phaeomelanin, indicating that all of them may be incorporated into the polymer. The enzymes involved in melanin biogenesis are tyrosinase and the two tyrosinase-related proteins TRP-I and TRP-2. The function of TRP-2 as a dopachrome tautomerase is established but the role of TRP-I is uncertain.

anionic functions such as the carboxyl and the deprotonated hydroxyl groups (see Fig. 1). SYNTHESIS

AND

DEGRADATION

Melanin biogenesis in animals occurs by an oxidation process starting with the amino acid L-tyrosine (Fig. 2). The major step is the oxidation of tyrosine to dopaquinone by the enzyme tyrosinase. Subsequent steps involve the disproportionation of dopaquinone, which entails spontaneous cyclization to yield indolene-2-carboxylic acid-5,6-quinone (dopachrome), followed by tautomerization catalysed by a tyrosinase-related protein (TRP-2) named dopachrome tautomerase, and oxidation of the resultant 5,6-dihydroxylindole-Zcarboxylic acid (possibly catalysed by another tyrosinase-related protein, TRP- 1). In vertebrates these reactions take place in specialized membrane-bound organelles

r

1

(melanosomes) and the current view is that the group of melanogenic enzymes is present as a complex associated with a protein matrix (Winder et al., 1994), all under the control of the tyrosinase promoter. The tyrosinase promoter is only activated in specialized melanogenic cells (Eisen, 1996) that comprise the retinal pigment epithelium and neural crestderived dendritic cells known as melano-cytes. Melanocytes are widely distributed in the body and are responsible for pigmenting surface structures. In mammals melanin granules are distributed to hair and epidermis by a process known as cytocrine transfer, in which portions of melanocyte cytoplasm, notably the ends of the dendritic processes, are endocytosed by epithelial cells (Wolff et al., 1974). Despite much speculation about possible processes of degradation, there is no evidence of breakdown of melanin, which appears to be resistant to enzymatic lysis.

P. A Riley

1238 BIOLOGICAL

FUNCTIONS

Structural Melanin has many putative biological functions (Riley, 1992). One of these is considered to be to strengthen structures by cross-linking of proteins. Melanins supply mechanical strength and may protect the protein from degradation. Melanization of seed pods in plants and insect cuticles confers increased rigidity, as does the browning of fruits (e.g. bananas) in response to surface injury. Antibiotic The generation of orthoquinones may have had evolutionary importance since their reactivity, especially towards nucleophilic groups such as thiols (-SH) and amino groups (-NH,), invests them with potential antibiotic properties. Examples of these are the insect immune system, the ink of cephalopods and the defensive sprays of insects. Photon shielding Light scattering and absorption by melanin is utilized in photoreceptor shielding and is thought to play a part in thermoregulation in reptiles by acting as a heat sink. Surface pigmentation is also widely functional in camouflage and display. In humans it is thought that melanin may play a role as a photoprotective pigment and a case has been made for it being genoprotective by acting as a photosensitizer of cells that have been exposed to radiation of sufficient energy to produce genetic damage. Chemoprotection Melanin may also function as a chemoprotective pigment by acting as a sink for free radicals (Sichel et al., 1987) or as a means of binding potentially toxic cations such as transition metals. It has also been suggested that melanin may have a function as a transcutaneous metal excretion pathway (Borovansky, 1994). MELANIN

IN DISEASE

Lack of melanin pigmentation occurs principally due to regional lack of melanocytes (e.g. piebaldism or vitiligo) or to the genetic transmission of mutations in pigment-related genes that give rise to hypopigmentation (e.g. albinism) when inherited in a homozygous form. There are several forms of oculocutaneous albinism (Summers et al., 1996). Failure

of pigmentation is associated with defects in neural development affecting the ocular pathway as well as visual impairment resulting from failure of light shielding in the eye. In vitiligo (also termed leucoderma) there is an acquired loss of melanocytes. The cause is not known, although there is a genetic component. Piebaldism is due to congenital lack of melanocytes caused by abnormalities in neural crest cell migration and is sometimes associated with related neural crest abnormalities, including deafness, as in the Waardenburg syndrome (Waardenburg, 1951). There is some evidence that melanization is increased in relation to oxidative stress, and hyper-pigmentation seems to be associated with inflammatory responses. Localized hyper-pigmentation is a sign of abnormal pigment cell function (e.g. malignant melanoma). The fact that most melanomas exhibit a high degree of pigmentation has encouraged the study of melanogenic pathway as a potential targeting strategy for anti-melanoma treatment and diagnosis. INDUSTRIAL

APPLICATIONS

The use of industrially-produced melanins as components of photo-protective creams is now well established. In view of the proposed mode of action of cephalopod inks melanins could possibly be investigated as a potential shark repellant. The properties of melanins as amorphous semiconductors have also received attention. REFERENCES

Berzelius J. J. (1840). Lehrbuch Chem. 9, 522. Borovansky J. (1994) Zinc in pigmented cells and structures, interactions and possible roles. Shorn lek 95, 309-320. Eisen T. G. (1996) The control of gene expression in melanocytes and melanomas. Melanoma Res. 6, 277-284.

Ito S. and Fujita K. (1985) Microanalysis of eumelanin and phaeomelanin in hair and melanomas by chemical degradation and liquid chromatography. Anal. Biochem. 144, 521-536.

Prota G. (1992) Melanins and Melanogenesis. Academic Press, San Diego. Riley P. A. (1992) Materia melanica: further dark thoughts. Pigment Cell Res. 5, 101-106. Sama T., Hyde J. S. and Swartz H. M. (1976) Ion exchange in melanin. Science 192, 1132-1134. Sama T. and Sealy R. C. (1984) Photoinduced oxygen consumption in melanin systems. Action spectra and quantum yield for eumelanin and synthetic melanin. Photochem.

Photobiol.

39. 69-14.

Melanin Scaly R. C., Felix C. C., Hyde J. S. and Swartz H. M. (1980) Structure and reactivity of melanins: influence of free radicals and metal ions. In Free Radicals in Biology (Edited by Pryor W. A.), Vol. 4, pp. 209-259. Academic Press, New York. Sichel G., Corsaro C., Scalia M., Scinto S. and Geremia E. (I 987) Relationship between melanin content and superoxide dismutase (SOD) activity. Cell Biochem. Funct. 5, 123-128. Summers C. G., Oetting W. S. and King R. A. (1996) Diagnosis of oculocutaneous albinism with molecular analysis. Am. J. Ophfhalmol. 121, 724726. Thathachari Y. T. (1976) Spatial structure of melanins. In

Pigment Ceil (Edited by Riley V.), Vol. 3. pp. 64 68. Karger, Basel. Waardenburg P. J. (i951) New syndrome combining developmental anomalies of eyelids, eyebrows, nose root with pigmentary defects of iris and head hair with congenital deafness. Am. J. Hum. Gen. 3, 195.-198. Winder A., Kobayashi T., Tsukamoto K., Urabe K.. Aroca P., Kameyama K. and Hearing V. J. (1994) The tyrosinase gene family-interactions of melanogenic proteins to regulate melanogenesis. Cell Mol. Biol. Rer. 40, 613-623. Wolff K.. Jimbow K. and Fitzpatrick T. B. (1974) Experimental pigment donation in rino. .I. (ilrraww~ Res. 47, 400--119.