Link 2-MELANIN 84

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Pigment cell research is rich in perspiration but poor in inspiration.

 

Recently the study of melanins has greatly intensified and has brought new results especially in the physical field. For this reason it appeared useful to me to publish a bibliographic adjournment of the book edited in the long ago 1968. The work, which the Accademia Pontaniana has accepted for its Quaderni, also wishes to illustrate the contribution of Neapolitan chemistry to the better understanding of the melanins and how, after the discovery of the Cystenyldopas, biological interest in melanocyte has grown.

 

BIOLOGY

 

The study of melanocyte, the cell which produces melanin has developed greatly in the last 25 years (1). The progress made covers various branches of science like physics, biology and medicine.

Melanocyte produces organelles called melanosomes which contain an enzyme called tyrosinase. The enzyme, a protein containing copper, catalyses the oxidative transformation of the DOPA (tyrosine) (2) into a pigment called melanin (melaV = black).

Pigments of red-brown colour can be produced by the cell. The pigments which are, among other things, responsible for the colouration of so-called red hair, take the name of the pheomelanin (jaioV = brown). A precursor of pheomelanin is the 5-S- cysteinyldopa which is formed by reaction between cysteine ( glutathione) with dopaquinone (3). For this reason the various colourations of hair and skin are due to two different chemical pigments: melanin and pheomelanin. (4). The physiological colour of skin is produced by a combination of different pigments, among which melanin, haemoglobin and carotinoids. Among these melanin is certainly the pigment which mainly determines the colour (5) of the skin and which is seen when the granules of the pigment transfer from the melanocytes to the keratinocytes. In the epidermis every melanocyte is associated to 36 keratinocytes in a characteristic biological unit (6). The colour of the skin, controlled genetically, may be modified by the action of physical agents (e.g. tan through the action of ultraviolet rays), or by the action of chemical agents (7). Besides, in pathological cases one can find features having different colours to the physiological ones. Addison’s syndrome produces a hyperpigmentation due, perhaps, to an alteration of the catecholic metabolism .Tyrosinase (8), the enzyme with a somewhat misleading name, should operate in the first phase with  transformation of  tyrosine into DOPA by consuming  oxygen;(For the 1978 Enzyme Nomenclature see the International Union of Biochemistry, Academic Press, Inc. New York 1979) .

 

As is noted the enzymes which oxidise the o. phenols to o. quinones, and which are very widespread both in the animal and vegetable kingdoms, are called polyphenoloxidases. Therefore the passage from dopa to dopaquinone is under the control of a polyphenoloxidase. Some authors limit the use of the name tyrosinases to the enzymes of animal origins but one also finds names like phenolases and dopaoxidases. The enzyme is proteic by nature and contains copper irrespective of the animal or vegetable source from which it is extracted. It should be noted that copper is necessary in the pigmentation of the mammals and that several complexes of copper oxidise the monophenols or the diphenols into o.quinones in the presence of oxygen. The enzyme does not seem to be very specific in its action (136): one cannot distinguish the stereoisomers of the DOPA, it is active on a great number of o. diphenols and oxidises with a velocity 1000 times greater than the catechol DOPA (enzyme extracted from the potato). On the other hand the enzyme extracted from human melanoma does not seem to have an appreciable action on the catechol (136): further investigations of the specificity of differing sources would be useful.

Many substances inhibit the activity of the enzyme in vitro. 2-mercaptoethylamine and N-(2-mercaptoethyl)dimethylamine cause depigmentation of the areas of the skin where they are applied (9) and in general the thiols are considered inhibitors of tyrosinase and the phenomenon of tanning is also explained by the oxidation of the thiols with the consequent activation of the enzyme. On this note one should observe that the thiols like cysteine or glutathione are biological metabolites of pheomelanogenesis (Link 14).

 

 

Fig. 1 - DOPA and tyrosine metabolism

 

According to several authors the enzyme is first activated by the reducing action of a catechol and then there is ortho oxhydrilation of the phenol (tyrosine) while according to others (10) the quinone produced by oxidation of the catechol (DOPA) is the agent which introduces the hydroxyl into the phenol (tyrosine).

More recently the classic role of the tyrosinase has been contested (11). The enzyme which, in mammals, converts tyrosine into DOPA and therefore into melanin would be the peroxidase, the enzyme of iron which separates the peroxides forming active oxygen. Confirming this there is the demonstration of the incapacity of the tyrosinase of the melanoma to transform the tyrosine into DOPA (11). The conversion of the tyrosine into DOPA is slow and requires an induction period which can be accelerated with the addition of small quantities of DOPA and for this reason the name which is given to the enzyme (12) seems little correct. Recently three factors which regulate melanogenesis have been identified (4, 24) .

According to Pawelek (4, 24) these factors could be in some way correlated to the action of melatonin. Pawelek has observed that cultures of non-pigmented melanoma cells possess an elevated expression of blocking factor while the pigmented cells have large quantities of indolic conversion factors.

The passage of the blocking factor to the conversion factor is stimulated by the exposure of the cells to MSH (melaoncyte stimulating hormone)(84), which also increases the activity of the tyrosinase. The dopachromic conversion factor is present in almost equal quantities both in the pigmented and in the non-pigmented cells and does not seem to be under the control of the MSH. Pawelek considers the new factors as being associated to the tyrosinase (4, 24) of the melanosome and therefore melanogenesis must be considered a more complex biological system, and different to how it has been considered up to now. It would be very interesting to isolate these factors from different biological sources and to know their chemical nature so as to be able to correctly insert them in the various schemes of melanogenesis which are fashionable.

Recently Ortonne (14) has given a very interesting picture of melanogenesis in the cephalopods Sepia officinalis, Loligo vulgaris and Octopus vulgaris. The sack containing the ink is composed of two distinct zones having different stages of maturation. The larger apical part of the gland is composed of pigmented tyrosinase-positive cells in different stages of maturation. In the caudal part of the gland the epithelial cells associated to the Golgi apparatus together with the small vesicles show tyrosinasic activity but do not produce melanin which suggests that the production of the tyrosinase is not necessarily linked to the production of melanin.

In fact the ink of the cephalopods contains notable amounts of free tyrosinases (14, 2), so that today it may be considered the most convenient biological source for the extraction of the enzyme. The presence of an active principle in the ink of the cephalopods is particularly interesting in relation to the defensive function of the secretion and it is probable that the tyrosinase shall be recognised as the composite which acts on the olefactive sense of the predator (15) with the production of quinones both in the sack (14, 2) and by oxidising phenolic composites of the predator. Besides, one should remember that insects can also produce quinones (p. benzoquinones) with a defensive purpose(4, 8).

It is probable that the tyrosinases have a different function, besides that of producing pigment in biological situations differing from the cephalopods. Chen and Chavin (16) have used a precise radiometric method for measurement of enzymatic activity in the skin and have applied it to the study of tyrosinase in the vertebrates. The authors have often observed the presence of tyrosinases in soluble fractions, like in the guitarfish, in dogfish, in the eel of the Congo, in serpents, in frogs. In anurous (amphibian) the tyrosinase activity is concentrated in non-pigmented zones. In melanomas in general the enzymatic activity is very high but it is also high in non-pigmented tumours, as occurs for example in the crab Xiphophorus helleri.

The function of the tyrosinases could also be understood through the chemical mechanism with which it works and the properties of the melanin by studying the microorganisms and fungi.

In fungi tyrosinases, through the pigment, play a clear cytoprotective role. According to Liach and Ruban (4, 15) this role has two explanations:

a) protecting the cells from different types of electromagnetic radiation, so common in nature, and which in many cases are a severe ecological selection factor.

b) protecting the cells from the lythic action of the microbic enzymes, allowing the existence of a biologically highly active microflora like that on earth.

In general strongly pigmented fungi are found in zones rich in radiation, in the western Turkmenia (hyphomicetes of the families Dematiaceae, Stemphylium, Macrosporium, Alternaria, Cladosporium), in the desert terrain of Tadzikistan and in the uplands of central Asia (4, 15). Fungi of the uplands of eastern Pamir territory are principally represented by imperfect highly pigmented forms of the Dematiaceae family, most of the time one finds the species Cladosporium and Hormodendrum, and these forms are sometimes the only ones which are found in high mountain territory (4, 15). From the desert terrain of Nevada mainly fungi with brown coloured spores or mycelium have been isolated (Alternaria tenuis, Clodosporium herbrum, Phoma sp., Pullularia pullulans, Rhizopus nigricans, Stemphylium ilicis, Stachybotrys atra) while in the desert terrain of the Sahara the strongly pigmented species of Helminthosporium, Curvularia, Alternaria, and Stemphlium prevail (4, 15). Generalising the bibliographic data, Liach and Ruban conclude that the pigments present in the hyphomicytes allow life in the limiting conditions created by various ultraviolet coloured, thermic and ionized radiations, in that the spores with dark coverings are much more resistant than the non-coloured ones. Using glycosidase of basidiomycetes, the chitilinases of streptomycetes and proteases of Bacillus subtilis, Alexander (quoted in (4, 15)) found that while the coverings of the non-melanised fungi broke easily the enzymes did not act on the structures or parts containing melanin (121). On the other hand it should be remembered that the quinones produced by the enzyme are antibiotics with a wide range.

According to some authors tyrosinase participates in morphological and functional differentiation in some fungi.

In Neurospora crassa the enzyme is not absolutely necessary to life while for Stemphylium sar. it is indispensable, for which reason it has been suggested that it acts as a terminal oxidase (4, 15).

Parting from the fact that the appearance of the tyrosinasic activity in the mycelium of Glomerells cingulata coincides with the end of the growth period and with the period of autolysis of the mycelium, one presumes that the activity of the tyrosinase is induced by the accumulation of substances which appear as the result of autolysis (4, 15). Sometimes the activity of the enzyme is connected to the pathogenicity of the fungus, imagining, in the case of Fusarium vasinfectum, that it is employed for disintoxication of the phenolic composites of the host plant (4, 15).

Disintoxication from the phenols as a function of the tyrosinase is also suggested for the fungi Stemphtlium sarcinaeforme and Monilia (Sclerotina) fructicola. It is probable that also for other fungi the enzyme oxidises the phenols to quinones making them inoffensive by polymerisation. It is worth remembering that polymerisation is the essential part of disintoxication since mere oxidation of the phenols would bring about the formation of quinones which are highly toxic for the organism because of their reactivity. It is interesting to note that more recently these concepts have been transferred to melanogenesis in mammals (46) (4, 22) (49).

The enzyme is active towards a large number of different mono and diphenols (4, 1, 4, 8, 12, 14, 21, 24) (136) and it is so in a particular way in the fungi; perhaps it formed in evolution under the action of the large variety of toxic phenols, esogenes for the organism, with which it entered into contact (4, 15). According to Blois (4, 8) melanins, among which one prepared by photo-oxidation of phenylalanin, must be considered as matrices of chemical evolution (120) more than the mineral lattices to which the evolutionists always refer. According to this concept, in the course of evolution the melanin would have appeared before the proteins and DNA, and therefore with a different role to that sustained by evolutionist microbiologists. The study of Blois (5, 2-7) shows that the melaninic pigments possess several properties which are interesting for evolutionist molecular chemistry like, for example, the distribution of unpaired electrons in the melaninic particles which are stable and chemically highly reactive. Ascorbic acid and the oxygen do not manage to arrive in contact with the unpaired electrons while the copper ions, after saturation of the other binding sites of the "macromolecule", do manage, thereby profoundly influencing relaxation times. Therefore melanin has the property both of a molecular sieve and of an ionic exchange resin. It is still possible that the pigment exercises a catalytic activity or, as has been shown, functions as a capturer of O2- cells. (74) (108).

The participation of the tyrosinase in the respiratory chain seems to be excluded in the case of the fungi Glomerella cingulata while it seems accepted in the case of Aspergillus nidulans (Sussmann, Markert (1953) and Carter, Bull (1969) cited in (4, 15)). The authors also report a hypothetical scheme of the participation of tyrosinase in the joint transport of the electrons. The tyrosinase of the fungus introduces an hydroxyl in the ortho position to that already existing in the tyrosine (p. oxyphenilalanine), with the formation, that is, of DOPA which is oxidised to dopaquinone. The DOPA/dopaquinone redox potential is highly elevated and in relation to this the dopaquinone seems a very favourable vehicle for the electron in that, at least theoretically, because a contemporaneous variation of the free energy, the phosphorilation of 3 molecules of ATP can occur. One presumes that the DOPA system ¤ dopaquinone, in Aspergillus nidulans may function by regeneration of NADP or another oxidant (17).

Also in man, through the products of melanogenesis, tyrosine seems to exercise a disintoxicating activity via melanocyte-keratinocyte. It has been found, in fact, that a large number of chemical substances (Tab. 1) and drugs accumulate in the pigmented tissues. This accumulation can also provoke chronic skin lesions, lesions to the eyes, internal parts of the ear and in certain cerebral nervous cells. Such manifestations may be, perhaps, explained by the denaturation of some properties of the melanins like that of amorphic semiconductors and "electron transfer" molecules. Besides, the pigment possesses a notable complexing capacity which makes it similar to an ionic exchange resin.

Many of the chemical and physical properties, common to all the melanins, from fungus to man, could justify the very different functions. More rigorous proofs and a differentiation between melanins with differing chemical structures, however, are still lacking.

In man the melanin is generally considered as an agent protecting from solar radiation even if it does not produce complete protection. The cytotoxicity (49) of the intermediaries and as a consequence the hypothesis of disintoxication should be considered: a function which has been attributed to the melanin by researchers of prebiological systems and of their molecular matrices. The various functions attributed to the melanins would therefore be seen as a memory of the functions of the preenzymatic era.

Chemically the accumulation of so many different substances ( Table 1 ) in melanins  can be interpreted in two ways with the high reactivity of quinones, by linking effect or clatratisation .Unfortunately the process which occurs, both in vivo and in vitro  has not yet been studied in depth, neither for its chemistry nor for its biology.

The melanins are among the most widespread common pigments in nature. They are responsible for the brown and black colour of skin, hair and eyes in mammals, in feathers, the eggs and skin of birds, the scales and the eggs of reptiles, the amphibians, of the cuticle of insects, the ink of the cephalopods, for several microorganisms and fungi (4, 19). In man the pigment is also found located in the Substantia nigra, in the Locus coeruleus, in the internal parts of the ear, in the hepatocytes, in the muscular cells of the heart, in the leucocytes, in the pineal tissue and in the surrenal gland. Yellow, red and brown colourings are due to a particular class of melanins the so-called pheomelanins (hair human, feathers of birds, etc).

The melanins are also responsible for blue and green coloured characteristics (schemochromes), very common in nature, and in man appear in the eye: these colours are due to a physical effect called the Tyndall effect (4, 5, 19).

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These pigments are also found in several pathological forms (melanoma, vitiligo, Acanthosis nigricans, Schizophrenia, plant parasites etc).

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Tab. 1. Substances which are copolymerized or linked to melanins both in vitro and in vivo.

 

Tryptophan (4, 7, 14) (151), Tyramine (4, 7, 14), Phenylalanine (4, 7, 14), Histhidine (4, 7, 14), Phenol (4, 7, 14), Valine (18), Chloropromazine (19-28), Phenothiazine (40), Aminoacids (16) (4, 7, 14) (18), Thiouracil (36) (46) (47), Iodoquine, Chloroquine (37-39), Inorganic Ions (4, 14) (41-42) (83), Nucleides (41), Dyes (43), Amphetamine (44), Epinephrine, Ephredine, Octopamine, Norepinephrine, Cocaine (44), Atropine (44) (147), Porphyrin (45), Nicotine (46) (128), Aniline (43) (46), Paraquat (46), Dopamine (48), Diethylamine (81), Ametryn (125), Amanitin (126), Vitamin C (127).

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The melanins give the terrain its characteristic colour and contribute to its fertility with their property of being resins with ionic exchange and gas agent transport. These melanins, often of microbic origin, taking the name of humic acids, while many pigments of artificial origin are given the generic name of melanoidins.

An attempt to describe the pigments correlated to them on a prevalently chemical basis is presented.

The natural melanins have been attributed different properties and functions like filters of radiation in the skin and the eye, mimetic function, antimicrobic function, disintoxicantifiers, protection from noise in the ear, fixers of free radicals, eliminators of oxygenated water, ionic exchange resins with a particular importance in the skin and in the coroide, in hair, in soils, and finally the function of redox and of semiconductors with a charge transfer effect at physiological electrical potentials.

The only generally accepted function is that of filter (partial) of light and especially of the dangerous ultraviolet rays. For the other functions, certainly worthy of the utmost attention, a deeper examination is necessary extending to philogenetically different species. For example is the antimicrobic action of the melanins (or intermediaries) of the fungi also carried out in the skin of man? Different research is currently under way to give an adequate and not hypothetical explanation to the biological functions of the melanins and of melanogenesis.

The "colour" of the melanins has always been a puzzle for chemistry. In fact, for a black substance which absorbs all the wavelengths, it is necessary to imagine having an electrical conductor (metallic dust, amorphous carbon) or a complex mixture of different chromophores. A radical polymerisation of the precursors of melanin would seem an ideal condition for the formation of such a "chromophore" complex.

Effectively one should also think that the spacial positioning of the molecules or the atoms plays an important role for colour: its enough to think of amorphous carbon and diamond. Important research of Blois in California has shown, with X-rays, (see) that a special packaging of the melaninic macromolecules can contribute to the characteristic "colour". In other words if the distance between the various molecular strata of the melanins themselves could be increased they would no longer be black.(4,19) (5)

 

CHEMISTRY

 

The study of the melaninic pigments, as almost always happens with substances not easily reconductible to crystalline and homogeneous products, has given scant information about their chemical structure (4, 14, 18) (50).

Essentially research has turned in two directions: a) the study of the nature and reactivity of the precursors of the pigments; b) the direct structural study of the natural melanins according to the classical lines of organic chemistry.

 

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Table 2. - Melanin classification.

Eumelanins

Pigments derived from DOPA (3,4-dihydroxyphenylalanine): like Sepiomelanin (50, 3). Pigments derived from Dopamine like Neuromelanin (140).

Pheomelanins  ( Link 14 )

Brown, red, yellow pigments derived from 5-S-Cysteinyldopa from hair and chicken feathers -1 (50, 33, 36, 43, 45- 47, 50- 52) (4, 21) (www.tightrope.it/nicolaus/pheomelanin.htm). Brown, red, yellow, pigments derived from tryptophan (58) (95) (113) (151) like those extrated from Australian kangaroo furs (65).

Allomelanins

Brown pigments from catechol and others polyphenols like Alternaria-melanin (61), Ustilago-melanin (4, 14), Daldinia-melanina (4, 14) (149), microrganism-melanin (4, 15), humic acids (141) (137) (4, 14) (64) (142), fungi (63), Aspergillin (4, 1, 14).

 

 

Melanogens

Black pigments are formed in different conditions from indole (82) and pyrrole (4, 1). The structure of these polymers was not yet elucidated but a trimer was isolated by Pieroni (143). Brown pigments form in oxidation of tryptophan and derivatives (113) (151) (144), hystmine, phenol, phenylalanine (black material is also formed from aromatic amine and heterocyclic systems), tyramine (132). The existence in Nature of such melanogens is not known.

The first type of approach is described in the  monograph of Swan (4, 18). The identification of some intermediaries of melanogenesis from the work of Raper brought about the construction of the following scheme of biogenesis:

Tyrosine-----> DOPA-----> Dopaquinone-------> Dopachrome--------> Leucodopachrome -------> 5,6-dihydroxyindole (DHI)------> 5,6-Indolequinone--------> melanochromes (oligomers of the preceding) ---------> melanin.(51) (compare to Link 20 ).

This scheme, constructed in an arbitrary way both from a biological and a chemical point of view, was given the name Raper. According to such a scheme the passage from tyrosine to dopaquinone is under the control of the tyrosinase while the successive phases are represented by an ordered series of spontaneous oxidative transformations which lead to a regular polymer of the 5,6-indolequinone.Even though reference to this scheme is still made today melanogenesis must be thought of as a much more complex semi-enzymatic biological process (4, 7) not yet describable in exact terms because any intermediates may contribute to melanin formation.Furthermore no eumelanin has ever been found to correspond to DHI-melanin or its corresponding indolequinone.

The various products described in the scheme of melanogenesis have been isolated in the pure state or in forms of derivatives, or characterised by spectroscopy (52) and each of them converts into melanin either by oxidation or in the presence of inorganic catalysts (57) (104) (135). The 5,6-dioxyindol easily oxidises even in the presence of atmospheric O2 (50, 28) (59) and the pigment which forms, with yields which do not correspond with the theoretical values, shows a centesimal composition not corresponding to the theoretically calculable value for a polymer of 5,6-indolquinone; such a discrepancy is generally explained by the supporters of Mason’s biological scheme by the presence of molecules of water strongly bound to the melanin (59).

Also the fact that the oxidation of the tyrosine, dopa, dopamine, 5,6-dioxyindol brought about physically and chemically different melanins, while according to Mason’s scheme of melanogenesis they should be identical, has not yet found an explanation. From the start, these contrasting data were the object of a scientific dispute (50, 30), based especially on the theory of H. S.Mason , that is, whether the pigment were or were not formed of only units of 5,6-indolequinone, briefly whether it was a poikilopolymer or a homopolymer. Interesting studies with models made in Great Britain by Bu'lock, Kirby, Harley-Mason, and Swan (4, 18) showed that the melanin was composed of different units and that given the negative chemical results obtained with the melanins it was not possible to write a formula of structure (4, 7).

In 1926 Raper, pioneer of melanogenesis, isolated DOPA, 5,6-dihydroxyindole and its acid 5,6-dihydroxyindole-2-carboxylic acid (DHICA), under the form of ethers/methylic esters, after enzymatic oxidation of the tyrosine. Later Piattelli isolated 5,6-dihydroxyindole, in very low yield, by simple extraction with ether from the DOPA solution, oxidised enzymatically at pH 6.8 (50, 14).

The possibility that, in the course of melanogenesis, there is the formation of different composites to those foreseen in Mason’s scheme (53) was discussed by Graham in the case of the composite II (54) (97) (92). The composites III, obtained by synthesis, are interesting products for melanogenesis (55) and composite IV is isolated from the tapetum lucidum of the catfish (56): these two composites, very interesting in relation to the biogenesis of the melanins, were not studied any further. The 5-hydroxy-tryptophan V, which can supply the 5,6-dihydroxyindole by oxidation, has been suggested as a precursor of the melanins (58) (151). The recent discovery of melanogenes and pheomelanogenes biogenetically correlated with tryptophane suggests that the role this aminoacid plays in melanogenesis is less hypothetical than it has been considered up to now.

Recently the differences between the natural melanins (melanosomes) which had already been demonstrated (4, 14, 18) were quantified, by Wyler at Lausanne, with the nomination of the acid functional groups present in the "macromolecule" (4, 23) (60). One finds, within the limits deriving from working in heterogene phases, that the proportions among the moderately acidic (carboxyls) groups and weakly acidic (like phenols) groups is the following:

natural melanin 1.1:1

dopa-melanin (self-oxidative) 1.7:1

dopa-melanin (enzymatic) 5.5:1

which means that melanins obtained by self-oxidation have a higher number of carboxyls, while the number of carboxyls of the natural melanins is different to that of the enzymatic melanins. This difference may, in fact, give rise to different hypotheses like: the presence of a pheomelanic fraction in the natural melanins , the nature of the enzyme, size of the particles (granules),oxidative processes etc.

 In summarising the study of the nature and the properties of the precursors of the pigments (the melanins) it is usual to indicate that they possess a complex structure, from a point of view of the monomers, even if characterised by a certain order in which the heterogeneous "macromolecules" are aligned and packaged. The enzyme, often little specific, transforms the orthodiphenols in the pigment and only operates in the first phase, even if later the final result is the synthesis of melanin. In our opinion, this would deserve the denomination of: Polyphenoloxidases poikilopolymerases. (with sub-enzyme factors)

Direct chemical study of the brown and black pigments aimed at obtaining structuralistic information of some relevance from the degradation products was completely negative up to 1952 (4, 1, 2, 5). Dealing with substances of great molecular complexity which are insoluble, infusible, incrystalisible and non-hydrolisible made their mixing and isolation in states of chemically defined comosites practically impossible. Also the terms molecule and macromolecule which are used in organic chemistry to indicate a definite and unitary structure cannot be used in this field.

The melaninic pigments are more realistically definable in terms of particles or granules instead of molecules or macromolecules, even though this is more than a little disturbing for chemists. A different meaning is almost always possible after the obligatory step in the structuralistic study of every natural substance, that of purification, as it is possible to follow two paths, either the drastic treatment with acids and bases to remove the soluble or solubilisable products, or the bland treatment which is limited to simply washing the pigment with solvent.

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Table 3- Elemental Analysis

C H N S

Bakunin (50, 1) melanoma C% 52.5 H% 3.6 N% 7.9

Panizzi (50, 3) sepia C% 57.5 H% 2.9 N% 10.5

Piattelli (50, 11) sepia C% 64.7 H% 2.5 N% 9.7

Ortonne (14) sepia C% 54.3 H% 2.9 N% 8.7

Benathan (4, 23) sepia C% 57.7 H% 2.6 N% 6.2

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In the drastic treatment, which can lead to not easily observable structural modifications, every result obtained must be controlled on native product which has not undergone chemical treatment. The differences which are seen in the elementary analysis (Tab. 3) are probably to be put down to the different conditions used in the purification of the pigments. The differences seen, at that time, in the melanins extractable from different sources could be explained today by the different type and grade of copolymerisation of the intermediates . These and other unfavourable properties and the scarce success obtained in the structure investigation with physical methods make it very difficult if not impossible to reach the ultimate goal of organic chemistry: the formula of the structure of the molecule.

The most studied melanin to date is that obtainable from the ink sack of the squid Sepia officinalis (50, 3) (4, 14) .

In 1952 Panizzi isolated the acid 2,3,5-pyrroltricarboxylic XI (50, 3) from the mixture of oxidation of the pigment with H2O2 (KMnO4) which at that time represented the first fragment of a certain structural interest obtained by degradation. Successively, with a very low yield, different products of degradation among which the acids VII, VIII, IX, X, XII (4, 14, 23, 75) (62) were obtained by different techniques. The acid XI is also formed by alkaline treatment of the sepiomelanin (50, 23, 30).

For some of these acids it is difficult to explain the genesis from a polyindolequinone polymer via 3-7 as foreseen in the Raper-Mason scheme and from the study of models (4, 14, 18). The tetracarboxylic acid XII, for example, seems to form from preexisting polycyclic structures which can originate from the intermediate heterocyclic enamines, which form by cyclisation of the dopaquinone according to a chemical process already clarified in the case of the alkaloids (111). It is worth pointing out how the formation of the polycarboxylic acids is a characteristic of the melanins.

An attack with H2O2  conducted at room temperature transforms the sepiomelanin (50, 3) into sepiomelanic acid soluble in the carbonated alkalis and insoluble in the acids. The soluble fraction both in the acids and in the alkalis already contains the acid XI. It is interesting to note that Wolfram (138) managed, with a bland attack with oxygenated water, to solubilise the pigment and to show the proteic matrix to which it is linked. According to other experiments of Wolfram the pigment (hair) should have a molecular weight of 11,400 (osmometry) and of 15,000 (gel-filtration). The proteic matrix which makes up 8-10% of the weight of the melanin has a composition in similar aminoacids, even if one operates with melanins from different biological sources, which suggests a specific type of protein.

The study of degradation products has also provided evidence for the presence of dopachrome units in the polymer ( 50,11,18,21 ). The participation of dopachrome in melanogenesis does not occur in vitro to any extent, probably owing to the fact that conditions of pigment formation in vivo are not reproducible (50,18 pag 946)

The oxidation of the sepiomelanin always shows a fraction more resistant than the others to degradation but if this is to be attributed to the residue proteic fraction or to a polycyclic "heart" as has been shown for the melanins of soil by Haworth (64) (137) it has not been studied.

The acid XI which is formed with the total yield (successive oxidations) of about 3% may originate either by rupture of an "indolic" unit of the macromolecule or from a unit already open, of the type XIII or of the type VI, found by Wyler among the products of selfoxidation of the DOPA and which most probably comes from a sequence of oxidative transformations of the dopachrome (60). Further structuralistic information can be found from the study of the esters and the ethers of the sepiomelanin and of their degradation products: the carboxyl is also present in the pigment of phenolic groups (weakly acidic groups). Among the degradation products it is possible to show mere traces of indolic substances, for this reason it is not possible to have direct chemical proof of a polyindolquinonic structure for the melanin.

The direct chemical study was then extended to a large number of natural melanins (50, 8, 16, 20, 22, 25 - 27, 49) and biosynthetic melanins (50, 18, 24) which allowed identification, in some cases, of the precursor chemical of the pigment and showed the complex heterogenity common to all the melanins.

As is well known the first objective in the study of the structure of natural substances is the isolation in the crystalline state of the substance which one wants to study. The appearance of crystals is, on the psychological plane, a source of great pleasure for the researcher but this joy was for a long time denied to people who worked in Naples on the melanins, until studies on the pheomelanins, classes of pigments little known chemically,were started (4, 5).

The study of the pheomelanins started with the extraction of the pigments of the feathers of the New Hampshire or Rhode Island hen, (130) (Link 14). Even with repeated extraction it is not possible to decolour the feathers. The yields which are generally reported are therefore relative. The pigment is collected in oval or globular granules somewhat smaller than the black ones (0.5-1.3 mu). The yellow pigments are contained in even smaller granules.

Unlike for the melanins the contribution of the pheomelanins to the so-called structural colours or schemochromes which are very common in birds is not known (4 ,2, 19). Other modifications of colour are due to the co-called dust which covers the barbules of the feathers (4, 5) and which seem due to a process of keratinization or disintegration of the cells.It seems that this dust contributes to creating the soft colours (viola, green-yellow etc). The chemical nature of these pigments  or of the schemochromes is, for most of the cases, still unknown.

The pigments of the feathers were given the name gallopheomelanins (50, 33, 36, 43, 45, 46, 52). In the course of extraction of the gallopheomelanins, (mixtures of pigments mixed with proteins with a continuity of molecular weights (from 1800 upwards)), a compound called C1 was isolated and was assigned the structure XIV. This and other correlated compounds or isomers were given, in chronological order, the names of tricosiderine by Flesch (85) (4, 17), of pyrrotricole

by Boldt (86) (4, 17), and of tricochromes (pheochromes Link 14 ) by Prota (4, 17). (www.tightrope.it/nicolaus/pheomelanin.htm ).

The synthesis of the pigments strictly correlated to the trichromes (tricosiderines) was realised in 1974: Kaul prepared (115) the cis-D2,2’-Bis(2H-1,4-benzothiazine)-3(4H)-one showing its characteristic photochromism.(Link 14)

A derivative of this was found to be the colouring pigment XIV of human red hair. It was also the only crystallizable pigment obtained from red hair and New Hampshire chicken feathers.

In precedence a series of model composites, which were very useful for understanding the chemical and physical behaviour of the pigments, were realised by Sica (50, 37, 39) and by Santacroce (50, 38, 41,58) (146). A notable contribution to the understanding of the pheomelanogenetic process was also given by Crescenzi (40, 59) in the synthesis of the D2,2’-Bis(5-oxy-7-methyl-2H-1,4-benzothiazine).

Unfortunately the biological significance of the tricochromes (pheochromes) (87) is still uncertain because it has not yet been demonstrated that they preexist in exactly the same way in the various biological sources from which they, or their artefacts, are extracted (133) (93).

The chemical study of the pigments of feathers was carried out mainly on the gallopheomelanin - 1, fraction without proteins. For action of the acids or of the different oxidants characteristic products of degradation were obtained. Link 14 .(50, 35, 36),

In parallel with the direct chemical studies, synthetic and biogenetic research were also carried out in the laboratory. Fattorusso (50, 46) found that the enzymatic oxidation of DOPA in the presence of cystein gave a pigment (134) very similar in chemical and physical properties to gallopheomelanin - 1. Besides in 1961 it was found that the 5-S-Cysteinyldopa synthesised (50, 35) and its isomers coming from the cysteinic residue in 2 and in 6, synthesised by Fattorusso (50,46), also yielded a product chemically and physically similar to gallopheomelanin - 1, for which reason it was clear that the natural pigment was a product of a complex oxidative reaction (polymerisation) which involved these aminoacids (50, 46) (88). Successively experiments carried out in vivo by Misuraca (50, 48) demonstrated that the 14C cystein and the 2-14C tyrosine (precursors of DOPA) were incorporated in the papillae of the feathers of the embryo of the New Hampshire hen. Another experiment conducted at Naples (50, 54) gave information about the first stages of pheomelanogenesis with the isolation of the cyclocysteinildopa (Fig. 3) from the mixture of enzymatic oxidation of the 5-S-Cysteinildopa (89) (107).

The interpretation of the successive stages of pheomelanogenesis or the structure of the pigments is more difficult, seeing the creation, that is, of situations similar to that of the eumelanogenesis where some intermediaries (see Fig. 3) are known and others supposed present, but how polymerisation occurs and what the structure of the final product is, are not proved.

According to Minale (50, 45, 50) the analytic data available allow hypothesising both a partial structure of the pigment of type XXVII in which there are present "benzothiazolic" units forming through a Mannich and a XXVI structure with "benzothiazinic" rings forming by 2-8 polymerisation. Chioccara, instead (50, 65), following the biogenesis of the alkaloids (111) and on the basis of experiments carried out on 2H-1,4-benzothiazine, thinks that the pheomelanic pigments can form through a process of aldolization which would lead to cyclic trimer intermediaries as represented in the hypothetical compound XXVIII. The work, though, does not state how the uncoloured composite XXVIII becomes the pigment. It is interesting to note that the model polymers (50, 65) easily convert into the artifact tricochromes which would lead to the conclusion that, effectively, the natural tricochromes are the artefacts or products of degradation of the pheomelanin. Another hypothesis on the first stages of pheomelanogenesis is that the composite I

 forms: the closure of the "thiazolic" ring is made between N1 and C2, that of the isoquinolinic ring between C3 and C4 and finally with N1 and C3 and C4 the closure of the "thiazinic" and "isoquinolinic " rings occurs with the eventual formation of a spiranic system. The hypothetical structure I  can also be considered a potential source of tricochromes. The action of the acids on I could, in the presence of O2 , bring about a series of transformations like hydrolysis of the base of Schiff, N1-C3 cyclization and dimerization to tricochromes. The closure of the "isoquinolinic" rings by the action of acids on the bases of Schiff has been known for time in the laboratory (reaction of Pictet-Spengler) and was used by Minale (50, 51) in the study of pheomelanic models. Perhaps it is worth recalling that the isoquinolinic compounds XXII and XXIII are obtained by acid degradation of the gallopheomelanin. Even these degradation products leave doubts about the preexistence of isoquinolinic structures in the pigment.

The degradation products of gallopheomelanin - 1 have been used by Fattorusso (50, 49) to characterise the pheomelanin in various biological sources. These studies, given that the structures of the pigments are not known, also represent a chemical way even if approximative of differentiating the melanins among themselves, and of identifying the precursor or the precursors. The observation that the melanin which can be extracted from the hair of deer supplies both the degradation product of the sepiomelanin and that of the gallopheomelanin is interesting and suggests that one is dealing with a mixed melanin  (150).

More recent work by Iso (50, 70) (90) and Rorsman (91) (92) has demonstrated that the phenomenon of copolymerisation between the intermediates of melanogenesis of the DOPA and that of the 5-S-cisdopa and 2-S-cisdopa is perhaps more vast than previously believed. These fundamental works assign a new role, still to be clarified, to the cystein (glutathione) in the area of melanogenesis and in the cellular metabolism (4, 22) (50, 68, 69). From a chemical point of view the sulphur present in many eumelanins, for example in the melanoma melanin, and attributed to a cysteinilproteic link (50, 17) (13) is probably included in "thiazolic" or "thiazinic" systems.

From 1968, the year in which Prota synthesised 5-S-Cysteinildopa and Fattorusso 2-S-Cysteinildopa, interest in these aminoacids has been slowly growing thanks especially to the research of Rorsman and Lund (91) (4, 14) (92). The 5-S-Cysteinildopa (5-cisdopa) which had been seen spectroscopicly by Bouchilloux (98) in 1960 has been found again today in the feathers of the red hen (91), in the skin, in the bull’s eye (114) (101), in some sea anemones, in crustaceans (99), in hair of mice (129), in the siero and urine of man . A derivative of 5-S-CISDOPA, the aminoacid 3-(2,5-S,S-dicysteinyl-3,4-oxyphenyl)alanine has been isolated from tapetum lucidum of the catfish (lepidosteidi) by Ito (100).See recent papers.

The 5-S-cisdopa and its methylic ethers are found in the urine of patients with melanoma next to 6-S-cysteinildopa and to 2-S-cysteinildopa (50, 62), to 2,5-S,S-dicysteininildopa (50, 62), and to the B and C tricochromes (50, 61) (102). One should stress that the measure of the 5-S- cisdopa and its derivatives, realised with refined analytic methods by Rorsman (91), by Hansson (102), by Crescenzi (50, 67), by Ruffo (131), in the urine and siero of carriers of melanoma is of a diagnostic value in the initial and therapeutic phases of the tumour. The 5-S-cisdopa, which is found in physiological conditions in the urine and siero of mammals increases not only in pathological cases but also after the exposure to ultraviolet radiation (106).

Recently Hagström (103) has shown the effect of 5-cisdopa both on the fertilisation and in the successive stages of embryo development in the eggs of the sea urchin Paracentrotus lividus. The property of this aminoacid, demonstrated by Palumbo (104), of forming reversible complexes with metals and their role in the metabolism in vivo is interesting.

All these studies show that perhaps this new catecholic aminoacid has a role and a biological meaning which goes beyond a purely pheomelaninic interpretation. The discovery of dopa (105) and of glutathiondopa (91) in malign tumours poses, then, a series of different problems relative to the role of the glutathione in the cells (6 ,6) (4, 22) (50, 68) (109).

In relation to the cytotoxicity of the catechols and to the genesis of the neuromelanin, Ito (108) has studied the oxidative process of DOPA in the presence of cystein with different systems of biological interest: tyrosinase, peroxidases, hypoxantin-xantin, O2 + Fe/EDTA, H2O2 + Fe/EDTA. One reaches the result that the Cisdopas can also form without tyrosinasic activity and that their synthesis can be mediated in vitro by peroxydases, by superoxide radicals, by oxygen in the presence of kelated Fe, and by oxyhydeinic radicals. Ito (110) has also found that considerable quantities of DOPA and 5-S-cisdopa are found in the hair of white mice which completely lack follicular melanocytes and which give a negative tyrosinase test.

From the proceeding it is also possible, in a rapid summary of the literature like this, to note that after the discovery of 5-S-cisdopa and 2-S-cisdopa, chemical research has served to develop biological studies inherent to pigmentation, with very interesting results from all points of view.

Chemical knowledge on the melanins which results from work carried out in the recent years can be summarised as follows.

The chemical precursors of the natural melanins identified to date are DOPA, 5-S-CISDOPA, 2-S-CISDOPA and most probably catechol and 1,8-dioxynapthalin; probable precursors of the pheomelanin of the kangaroo and the melanins of some invertebrates (sponges, leeches etc) are derived from tryptophane. In general the diphenols suffer different known transformations, among which the fundamental one into quinonic systems, but the mechanism and the way in which the various units link among themselves, and the type of units which concur in forming a particle granule are still uncertain.

A large number of chemical substances can be involved in polymerisation or link to the melanin and it is difficult to say if, in the world, there are two equal melanins. The mechanism of copolymerisation which leads to the mixed melanins is not known.

Synthetic melanins can be obtained also from the non-phenolic precursors (Tab. 2) like indole and pyrrole. Also for these pigments the chemical structure is unknown but the physical properties of all the melanins, either of biological or of synthetic origin, appear very similar to each other (Raggi X, IR, etc). On the basis of the deluding chemical results to date obtained two structuralistic hypotheses seem to be the possible for the natural melanins:

 a) melanins with polycondensate cyclic systems.

 b) linear polyindolquinone melanins.

 The formation of polycarboxylic acids among the oxidative degradation products of different natural melanins speaks in favour of the presence of condensated aromatic nuclei in the pigments. The 2,3,4,5-pyrroltetracarboxylic acid of the sepiomelanin, the 2,4,5,6-pyridintetracarboxylic and 2,4,5-thiazoltricarboxylic acids of the gallopheomelanin, the mellitic acid of the Aspergillus niger melanin are indicative of the polycondensated polycyclic structure. The benzenpolycarboxylic acids which are obtained from Aspergillin or from humic acids . The interpretation of data is to be made with great caution and great care because it is now knownthat melanin samples are always altered.

Physics

 Solubility

 Little soluble in water even as salts, insoluble in organic solvents. The solubility varies with the treatment undergone. In some cases they can be solubilised with "Solvene 100" (116) or liberate proteic material with Triton X-100 + SDS (117).Sonication may be useful.The pheomelanins or the mixed melanins are more soluble especially in the alkalis. In the melanoma melanin there is also a part that is soluble in piperidine (50, 1). When the solubility is sufficient it is possible to purify the melanins for chromatography (4, 23) (50, 49) (60) (132). The use of spectroscopic methods is often limited by scarce solubility. In the case of the pheomelanins it is possible to make them more soluble in organic solvents under the form of salts of TEA (triethylamine) or after treatment with BSA (bis-sylilacetamide) or with acetoacetic esters (94). The acetoacetic esters, already used as N-group protectors of the aminoacids (E. Dane et al, Angew. Chemie Vol. 74, 873, 1962), can be removed by simple acidification. The same DOPA becomes soluble in alcohol and other solvents which makes more controlled study of the oxidation products possible.According to Das (145) the solubility of the melanins in water increases notably after reduction with sodiumborohydride

 

UV Spectrophotometry

Spectra without definite bands with high absorption which grows from the visible to low UB (4, 14, 18) (132)

 NMR Spectroscopy

Badly defined spectra (94) (119) (142). The scarce solubility, the presence of free radicals, the molecular complexity are the causes which limit the structuristic information to some function.

 IR Spectrophotometry

The spectrum shows wide bands with a difficult interpretation (4, 14, 18) (5, 7) (132) (139) (145).

 ESR (electron spin resonance or EPR) Spectroscopy

The signals which are obtained seem due to free radicals trapped in the "macromolecule" (66). All the melanins, from the humic acids to those of the skin show a signal (122) (4, 14, 18) (70) (71) without hyperfine structure which is present instead for the precursors (68).According to Mason (53) (67) the melanins contain one radical for about 1000 units indolquinonic. Blois (5, 2, 6, 7) makes a higher estimate of one radical per about 200 units. Considering that the particle of melanin has a volume of about 10-3 m3, with about 106 monomeric units there would be about 104 spin in the particle (5, 7).According to Mason (67) there are differences in the quantity of free radicals of indolesemiquinone type, in the various melanins. The sepiomelanin loses many of its radicals after reduction with ascorbic acid which is not confirmed for other melanins. The paramagnetism of the melanins is explained by Longuet-Higgins (Arch. Biochem. Biophysics Vol. 86, 225, 1960) with the property of a monodimensional conductor with the protons which act as a trap for the electrons.Zanotti has obtained simple ESR spectra from both white and black hair and pelts (bovine) while albinos present a very weak spectrum or it is completely absent (69). Compared to black ones the ESR spectra of red hair and red animal hair are different and more structured (69) and besides all the red hairs (different bovine races) possess a structured spectrum. Recently Sealy (72) (73) found a new, pH dependent free radical, of o. semiquinonic structure, studying the synthetic pheomelanin obtained from 5-S-Cysteinildopa. The ESR study of the natural eumelanins and pheomelanins (72) (73) shows that copolymers are in question . Sarna has applied ESR united to Mossbauer spectroscopy to study the ion exchange properties of the melanins (83).Constant dielectric, conductivity, ESR, of the water-melanin mixtures are sensible to pH, to the ionic force, to the grade and time of hydration (75).

 X-Rays

Allow establishing that in all the melanins the plane structures tend to align in a parallel way with the distance between the different strata of 3.4 Å (5). The total lack of crystallinity, even if in a certain "order", is necessary to package the various strata. for the study with synchrotron radiation see (5, 10).

 XPS (X-ray photoelectron spectroscopy)

This relatively new technique allows having, among other things, information about the state of oxidation of the nitrogen and of the sulphur in the melanins and in other biological material in the solid state. Williams-Smith (76) have examined different natural and synthetic melanins. Typical spectra found N1s and S2p for the melanoma-melanin (horse) and for the melanins of the human skin. This technique can be used with great advantage for differentiating eumelanin and pheomelanin and their grade of copolymerisation, besides establishing the grade of oxidation of N and S.

 Electrical characteristics

 All black material are electroactive. Conductivity is strongly increased by doping. Electrical and sound conductivity show by melanins are of biological interest. Superconductivity may be present.Both the natural and the synthetic melanins behave as amorphous semi-conductors with threshold switching. (77-81), (152), (50,81) ( www.organicsemiconductors.com ).The method of isolation may produce artifacts which affect the electrical behaviour of the melanin.

 ...The melanosome has long been considered a passive cellular organelle. Its considered role as a photoprotective agent in the skin and other illuminated areas, could not explain its presence and function in the non-illuminated areas (for example, the midbrain). A single hypothesis was developed, on the basis of a quantum mechanical model of disordered materials (amorphous semiconductivity) to explain the functional role of the melanosome in both illuminated and non-illuminated areas. This hypothesis was based on electron-phonon interactions, which seems to be particularly strong in melanins, and on the large density of available energy states. A particularly useful probe for determining the nature of these states is a measurement of low temperature specific heat. The measurements presented here include two anomalies, a transition and an unusually high linear contribution. The observed anomalies probably arise as a result of the electronphonon coupling and high density of unpaired spins, which until now were difficult to correlate. further experimental measurements at near the transition temperature may yield a detailed quantum mechanical description of the states, which will then afford a more precise understanding of the biological functions of melanosomes than has been possible to date....

U. Mizutani, T. B. Massalski, J. E. McGinness, P. M. Corry, Nature 259, 505-507 (1976).

 

...Threshold switching in hydrated melanin was first reported by McGinness at al. Their discovery opened a new area for switching studies by showing that low electric field switching occurs in organic semiconductors. They, and others, have discussed some of the ramifications of this discovery to living systems. No attempt was made to explain the mechanism of the melanin threshold switch. Switching has been studied in amorphous semiconductors for several years. It has been shown that in some bulk amorphous semiconductors the switching action can be explained on the basis of a thermal model. Two types of switching experiments were done on bulk melanin samples to distinguish between electronic and thermal effects. Measurement of the sample current vs voltage as a function of time provides information on the delay time and on the time to traverse the negative-differential-resistance (NDR) segment of the current-vs-voltage trace. The delay time is the time between the application of a voltage greater than the threshold voltage and the attainment of low resistance in the sample. Thermal models imply a much slower time to traverse the NRD segment than electronic models. A more definitive experiment to distinguish between electronic and thermal thermal effects is a double-pulse experiment, which reverses the polarity of the applied voltage before the sample switches. Voltage reversal cannot alter the Joule heating which is responsible for the thermal switching effect but would be expected to reverse electronic effects. Threshold-switching measurements of synthetic melanin confirm that this organic semiconductor switches to a low-resistance state in low electric fields as reported by McGinness at al. Time-dependent current-vs-voltage curves show that the time to traverse the negative-differential-resistance (NDR) segment is much slower than would be expected from electronic-switching mechanisms. Double-pulse measurements add to the evidence that thermal effects dominate electronic effects in melanin. A pseudomemory effect was found in melanin....

C. H. Culp, D. E. Eckela, P. H. Sidles, J. Appl. Phys. 46, 3658-3660 (1975).

 

...Basic semiconductor characteristics of natural melanins isolated from bovine eye, human dark hair, and banana peel were obtained by means of the dc dark conductivity experiments and optical absorption measurements. The results were compared with results obtained for synthetic melanin. Specific conductivity in natural melanins is of the order 10-11 W -1 cm-1 and in synthetic melanin 10-8 W -1 cm-1 . Thermal activation energies in the range 298-333° K are eye melanin, 0.93 eV; hair melanin, 1,01 eV; banana melanin, 1,04 eV; whereas synthetic melanin has two values of activation energy: up to 311° K, 0,1 eV; above 313° K, 0,78 eV. Optical gaps are: in eye melanin, 1,73 eV:in hair melanin, 1,35 eV; in banana melanin, 1, 55 eV; and in synthetic melanin, 1,40 eV. The observed differences between natural melanins and the synthetic one could be explained by either the presence of protein residues in natural melanins or the influence of the isolation method on their electrical properties....

In amorphous semiconductor the optical absorption coefficient follows the relation (ahv)1/n  ~ (hv-Eo), n=1,2, or 3 where a=optical absorption coefficient, hv=photon energy of incident light, Eo=optical gap Optical absorption were performed with melanin disolued in NaOH in the region of 250 to 840 nm. Dependance of (ahv)1/4 on hv plotted yields a straight line which gives an optical value Eo= 1,4 ± 0,1 eV.

T. Strzelecka, Physiol. Chem. Phys. 14, 219-236 (1982).

 

TSDC (Thermally stimulated depolarisation current)

 

Both the natural and the synthetic melanins are able to conserve electrical charge and/or polarisation. This effect (electret effect) depends on the content in water (80).The pigment cell research on the behaviour of melanins with respect to the electromagnetic spectrum were not much considered until now. All black materials are or may be electric or sound wave conductors. Superconductivity in melanins is to be taken in account.The concept of the melanosome as an inert biopolymer is still largely accepted..Now at least one biological material has been shown to have a strikingly large conductivity when correctly excited. Melanins can be made to switch from a poorly conducting to a higly conducting state at fairly low electric fields ( from 10K ohm-cm to 100 ohm-cm with a field of 300 V cm-1 ). This remarkable phenomenon occurs both in melanin made synthetically from tyrosine and in that extracted from human melanoma ( Nature Vol. 248, April 5, 1974, pag. 475. ''News and Views").Many results are obtained from artifacts and are to be revisited. Conductivity, like other physical parameters, must be measured in physiological conditions, that is, in the presence of water, and on samples which have not been altered by heating. Some difficulties also arise in the study of pigment conductivity due to the lack of elemental analysis of the material under study. It is surprising that many scientifical journals continue to accept papers without the analytical data.(elemental composition).

bruno.nicolaus@virgilio.it