Link 23 Perspectives in melanin chemistry

Fused with WORD Link 12 testo terzo.doc.Revised March 2005.

Melanin is instrumental for certain brain, ear, eyes and skin functions. It

would be rather difficult to distinguish blue or green colours of animal or

plant kingdom without melanin. No blue or green eyes to admire……

If you are skeptical about, imagine to remove the melanin from the

biological tissues and observe what happens.

Melanin chemistry produced poor results so far, due to the use of too raw

materials in chemical and physical analyses and to scarce knowledge of

solid state science by the researchers involved. The discovery of the

particle is expected, in our view, to change the strategy in future

structural and functional melanin studies, whereby mass spectrometry MALDI

( matrix-assisted laser desorption ionization ) or MALDI-TOF ( TOF = time

of flight ) are forecasted to be the most relevant fragmentation tool for

the particle and oligomers study. It is still unclear, which are, if existing at all, the

enzymes or factors regulating particle formation, how the particles operate

in vivo and if particle size, shape and internal organization are critical

for biological activity .

Remarks on melanin chemistry

The black matter is very common on our planet ( lithosphere, hydrosphere, atmosphere , biosphere ), as well as in the Cosmo if one considers the black matter found in interstellar spaces.

The black matter is composed by two main classes : The black cell matter ( ( BCM ) ; The black synthetic matter ( BSM )

The BCM are classified as :

Eumelanin particles generally derived from DOPA, and widely diffused in the animal kingdom ( skin,hair,eyes,brain, feathers etc. )

Pheomelanin particles generally derived from Cysdopa ( light brown, or reddish as protein complex alkali soluble animal pigments).

Pheochromes ( red , yellow crystallisable compounds derived from Cysdopa ) The only structure elucidated is that of red hair. ( Other names trichosiderins,tricochromes,pyrrotricholes ) .

Allomelanin derived from nitrogen-free precursors like catechol, 1,8-dihydroxynaphtalene etc.. ( plant, mould, bacteria,humic acid etc.)

WARNING :some melanin may be formed out of the cell .Copolymerization may occur between eumelanin’s and pheomelanin’s.Due to the binding effect analytical results may be erroneous.

Some well known BSM are :

Acetylene-black

Pyrrole-black

Aniline-black

Graphite

BCM and BSM are particles derived from polyphenols : DOPA= beta-(3,4-dihydroxy)-alpha-alanine , DHI or 5,6-dihydroxyindole , DHICA or 5,6,-dihydroxyindole-2-carboxylic acid , catechol, cyclodopa, aminocatechol, homocatechol, 1,8-dihydroxynaphtalene, 5,6-dihydroxytryptophan, opiod-peptide, tetrahydropapaveroline, xanturenic acid, cysteinildopa, dihydroxybenzoic acids, cinnabarinic acid ,

other aromatic and heterocyclic structures like benzene , pyrrole , acetylene , aniline , tryptophan , kynurenine.

Oxygen is found in polymer (particle) starting from an oxygen-free precursor.One atom of oxygen more in respect of the oxygenated precursor is found in polymer (particle )..The black particles ( melanins, BCM, BSM ) are amorphous insoluble and of variable shape and size, showing a tidy internal organisation ( AFM figures ) and are formed from radical-polarone oligomers ( see acetylene-black ). They show high binding power to organic and inorganic compounds,to liquids and gases.These properties may influence or distort the results of chemical and physical analysis.The black particles are good conductors expecially after doping showing the McGinness-Proctor effect ( melanin exhibits the unusual characteristics of an amorphous semiconductor threshold switch ).Melanin may act as innovative sensor and energy generator, with the capacity of growing and shrinking in length and volume,by electric stimulation .Melanins show typical broad EPR, IR,¹³C NMR ( some spectra show an aliphatic part ) spectra.The MALDI and MALDI-TOF procedures are not useful for molecular weight determination as in the case of proteins.LASER beams cause particle fragmentation The MALDI fragments might be useful in structural studies of the black particle.Melanin particles build up 15-70% of a melanosomes.They are sensitive to oxidants but resistant to reduction these properties being consistent with a non polyene structure Usually all oxygenated oligomers show one oxygen more than the precursor.

The ink in Sepia

.«Viscera autem nullum habet molluscum, verum id quod mytin vocant, supra quam atramentum, quod in sepiis et plurimum et maximum est;atque hoc emittunt,cum perterrefiuntpraesertim tamen sepia. ( Aristotelis Historia Animalium Lib IV, cap. I, 11 )

In the books of Aristotle, a number of passages are devoted to this pigment; Cicero also records that it was used for writing . Right until 1848 it was believed that indian ink was made from cuttlefish pigment , but during his zoological voyage to Japan in that year , Th. Von Seebold learned that Indian ink was in fact obtained by Buddhist priests collecting the deposits of black smoke from the walls of pagodas. Cuttlefish ink is still used today as basis of the colour known to painters as Sepia.

Sepia ink was used to paint this old disc with head. 4^th century BC . Lacco Ameno, Museo Archeologico di Pithecusae Isola d’Ischia

The ink of the Sepia gland is used to prepare the artist’s pigment called sepia.The pigment is prepared by boiling with lye the dried ink sack.The melanin solubilized ( part of all ) is precipitated with acid,washed , dried and ground with gum Arabic.

In addition to melanin the ink sack of cephalopods seems to contain active tyrosinase a dopachrome tautomerase catalysing the rearrangement of dopachrome with quantitative conversion into DHI and a dopadecarboxylase( gland) which produce dopamine with a biosynthetic pathway independent from tyrosinase and peroxidase (gland ) . Finally a tyrosine hydroxylase (gland) was undertaken. Both dopamine and dopa were identified in the ink.

An enzyme which convert dopachrome into DHI/DHICA mixture is quoted to be present.

The melanin present in the ink sack of the cuttlefish has been selected for the ease of collection.

Scientific research on the ink must be carried out with ( on ) fresh ejected ink.or mature particles.

The ink of the sepia is in fact constituted by a mixture of melanosomes, premelanosomes, (in which oxidative enzymatic or not activities are in act with the rearrangement, transferring and elimination or creation of atomic and radical groups) and granules of stabilised pigments. Enzymes like laccase, tyrosinase, peroxydase, tautomerase are present in the ink sack (48).

Among the various melanin-producing systems,the ink gland of the cuttlefish, has been regarded as a most covenient model for melanogenesis .The ink gland is a highly specialized organ with immature cells in the inner portion , from where the cells gradually mature, migrate towards the outer portion of the gland and become competent to produce melanin giving rise to particulate melanosomes (1),( 2 ). When cell maturation is complete, melanin is secreted into the lumen of the gland, accumulated into the ink sac and ejected on demand .

SEPIOMELANIN

The melanin present in the ink sack of the cuttlefish has been selected for the ease of collection.

Scientific research on the ink must be carried out with ( on ) fresh ejected ink.or mature particles.

Sepiomelanin occurs in the ink sack as Ca⁺⁺ Mg⁺⁺ salt. Sepiomelanin may be extracted from the cuttlefish in vivo ( fig.3 ) or from the ejected ink.

Sepiomelanin contains less carbon and nitrogen and more oxygen than calculated for a 5,6-indolequinone polymer.

Treatment with diazomethane of a suspension of sepiomelanin and DHI-melanin in ether gives an insoluble pigment lighter in colour. Methoxyl represents for sepiomelanin and DHI-melanin 18,8% , against a value of 24,4% calculated for a polymer of the type A indicated above , on the hypothesis that one methyl would be introduced into each indole unit by the diazomethane reaction. Naturally the formula A used above has none but a statistical value, and is not meant to indicate a regular distribution of the carboxyl group in the polymer.As will be brought out later, the structure of sepiomelanin under examination is considerably more complex and less orderly than would appear from the theoretical formula. The presence of dopachrome ( cyclodopaquinone ) units in the polymer ( particle ) is possible.

We knew from the literature that exstracts from the ink sack were capable of producing melanin in the presence of tyrosine. We have been able to confirm that tyrosine is the precursor of sepiomelanin by using C¹⁴ – labelled tyrosine both in position 2 and in the carboxyl (see figure 3 ) During preliminary experiments, in order to find out how long it takes for pigment to form in the ink sack ,we realized that the production of ink is slow ,and so decided to study only the actual melanin. We have shown that , 24 hours after tyrosine has been injected into the venous system through the peribuccal vein , (5uc/ml in an 0,2ml solution ), the ink contained in the gland is strongly radioactive. The fact that radioactive pigment is formed even when using tyrosine labelled in the carboxyl group, demonstrates that intermediates other then 5,6- indolequinone take part in the formation of the sepia ink.

The sepiomelanin found in the sack in the form of Ca⁺⁺and Mg⁺⁺ salts contains a protein which can be removed by heating with hydrochloric acid or more simply washing with water

After hydrolysis sepiomelanin contains about 0,2% of sulphur. This S is part of a cystine molecule, as can easily be shown by oxidizing the sepiomelanin with peracetic acid. Glycine and aspartic acid are also formed; they come, as will be seen, from the indole rings present in sepiomelanin. It is highly likely, therefore, that the protein is probably bound to the melanin by means of cystine .

H₂O₂ or KMnO₄ oxidation of melanins afford pyrrole polycarboxylic acids which were the first fragments of some significance obtained in the study of melanins. (1952). Degradation occurs with chlorine or bromine ( water or alkaline solutions ) with formation of products without relevant structural significance

A more complex pyrrole acid was isolated as barium salt C% 22.59 H% 2.1 N% 4.5 Ba% 43.6 , among the alkaline H₂O₂ oxidative mixture. It is interesting to note that a similar product (Table 1) appears in the Matrix-Assisted LASER desorption/ionisation ( MALDI ) spectra as peak 539. This means that melanin spectra are pictures of degradation products formed in the course of oxidative process which may be absorbed into the particle.

Mass spectroscopy of sepiomelanin, DHI-melanin, DHICA-melanin was studied.The spectra (peacks ) were different expecially those of DHI-melanin and DHICA-melanin as compared to sepiomelanin .

Sepiomelanin 273,313,335,349,363,369,373,391,450,526,552,685

DHI-melanin 457,516,542,572,579,612,722,744,778,874, 983,1047,1080,1136

DHICA-melanin 497,524,540,552,572,703,714,731,868.

Since spectra are not reproducible no conclusion about the molecular weight of oligomers which form the particle can be made.It is interesting that the MALDI study may show for the first time the formation of trioxygenated indole units during melanogenesis ( more oxygen in the polymer ).

Simple pyrrole polycarboxylic acid were found in the oxidative degradation of sepiomelanin with KMn O₄, H₂O₂ or alkaline hydrolysis like 2,3-pyrrole-dicarboxylic acid (PDA ), 2,3,5-pyrrole-tricarboxylic acid ( PTA ), pyrrole-2,3,4,5-tetracarboxylic acid ( PTECA) , 2-carboxyhydroxymethylpyrrole-3,5-dicarboxylic acid, 2-carboxymethylpyrrole-3,5-dicarboxylic acid, 2-hydroxymethylpyrrole-3,5-dicarboxylic acid. ( A.Napolitano, Tetrahedron 52, 8775-8780, 1996 ).

Pyrrole acids originate from carboxylated and non carboxylated indole units of the polymer.

Some of the breakdown products have been isolated in the crystalline state, others identified chromatographically. The major fraction of the oxidative degradation of the pigment is constituted of CO₂ , NH₃, COOH-COOH.

PTCA which forms generally with higher yields in respect to other pyrrolic acids is obtained from DHICA-black, DHI-black, Dopa-black, and natural melanins ( R.A.Nicolaus, et al., Tetrahedron , 20, 1163-1172, 1964)

The formation of 2-carbomethoxy-4,5-pyrroledicarboxylic acid through oxidation of the methylether of sepiomelanin can easily be understood with the presence of carboxylic group in position 2 is present in contrast with the hypotesis that melanin is a polymer of 5,6-indolequinone.It must be admitted that a carboxylated intermediate such as dopachrome may take part in the formation of the pigment.

It is not therefore without interest to note that a dopachrome unit has been found in the violet pigment of beetroot Beta vulgaris ; the oxidative breakdown of this substance leads,together with other products, to 2,3,5-pyrroletricarboxylic acid,while alkaline fusion gives 5,6-dihydroxyindole-2-carboxylic acid ( DHICA ) . In other words dopachrome or cyclodopa may be precursors of melanins.

The extent to which the different intermediates take part in the costruction of the particle probably varies with variation in the biological and chemical conditions leading to products which may be different even though they start from the same substrate. The final result is a particle built up from heterogeneous and easily tautomerizing units, which are linked together by bonds, probably of more of one type, that are not easily broken by hydrolysis.This situation makes it difficult to establish the sequence of the units and to obtain good yields of breakdown products of the oligomers.

Sepiomelanin appears from experimental data as a highly disordered polymer but particle show an ordered organization . The method to inquire into this structure requires an altogether new approach in organic chemistry and solid state.

The use of fresh ink particles (granules) is adviced.Granules are obtained by selective centrifugation at different speeds or are present in the naturally ejected ink of the animal

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Pheomelanins

Cysteinildopa is the precursor of pheomelanin and pheochromes.

Cysteinildopa was undertaken in Naples 1965 ( Nicolaus 1968 ).

Pheomelanins ( brown amorphous animal pigments ) and pheochromes

( other names : tricosiderins, tricochromes) yellow, red, cristallyzable animal pigments . Both are formed from Cysteinyldopa and have the same dibenzothiazine or dibenzothiazinone chromophores (man and chicken feathers ) . To the pigment of red hair was attributed the structure of cis-D ^2,2’-Bi[5-hydroxy-7-alanyl-2H-1,4-benzothiazine)-carboxy-3’-one (chromofore II).

Oxidative breackdown of eumelanin afford pyrrolic acid , whereas thiazolic acids are formed from pheomelanin and pheochromes.

Other pheomelanins are products of tryptophane metabolism in Kangaroo Allomelanins derive from nitrogen-free precursors ( catecholes, polyphenols, 1,8-dihydroxynaphtalene etc. ) Typical allomelanins are pigments from fungi and soil, the most studied being Aspergillin and humic acids.Generally they are aromatic polycondensed (graphitic) system..

Aspergillus niger appears to be the only microganism which is able to produce a graphite like pigment

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Melanins are particles with different shape and size .Melanins are particles built up from oligomers of unknown molecular weight. Oligomers, the fundamental stones of the particle, may be optically active intermediates . Isomers cis/trans have different conductivity ( pyrrole-black, acetylene-black etc. ) . Such oligomers contribute probably to the shape and size of the particles and other physical properties.The chemical and physical study of components of the fundamental structural units ( aggregates,filaments, smaller constituents, oligomeric molecules ) of the particles is of great interest for function melanins biology and materials science.

The presence of particles in light exposed tissues suggest that they are involved in photoreactions. Tetramers of the melanogen DHICA are found in the reflecting material of the tapetum lucidum of the sea catfish. Melanins might show various hydration and oxidation levels.The acetylene-black radical- polarone system is see in all natural (BCM) and synthetic melanins (BSM).

Biochemical studies carried out over the past two decades have shown that the ink gland contains a variety of melanogenetic enzymes, including tyrosinase, a peculiar dopachrome rearranging enzyme which catalyses the rearrangement of dopachrome to 5,6- dihydroxyindole and a peroxidase presumably involved in the later stages of melanin biosynthesis. The enzymatic equation can not be written being precursor and final product unknown. In other words dopachrome frequently used as substrate is only a red mixture of phenols and quinones. Dopachrome would be yellow.

Moreover synthetic ( BSM ) and natural melanins ( BCM ) are chemically different. Differences between DHI-melanin and the natural pigment are observed in IR, ¹³C NMR, MALDI spectra ( 8 ), ( 21 ), ( 28 ), ( 29 ), ( 38 ), AFM figures ( 42 ).Moreover it is to note that chemical composition of the natural sepiomelanin and the synthetic DHI-melanin suggest a formula in which every monomer contains three oxygen.
According to ketone compounds, quinone carbonyl groups of sepiomelanin can undergo hydration reactions yielding gem-diols through a reversible reaction.
As regards MALDI and MALDI-TOF spectra, all our attempts performed on very pure sepiomelanin samples to obtain high or medium mass fragment were unsuccessful ( see letter ). No high mass peaks have been detected, whereas a lot of small molecules, low fragments arising from melanin breakdown were recorded (mass range 50-600 ). (4). A fragmentation which remembers the LASER graphite fission.

Natural and synthetic melanins are still today considered polyindolequinones , justifying the difference between the found (three oxygen every indole unit ) and calculated values of analysis by the presence of molecules of water.
On the contrary, it is to note that chemical composition of the natural sepiomelanin and the synthetic DHI-melanin analysis are similar but not identical and that both compositions don't agree with the accepted polyindolequinone structure but suggest a composition in which every monomer contains three oxygen. According to ketone compounds, quinone carbonyl groups can undergo hydration reactions yielding gem-diols through a reversible reaction. The extent of hydration and stability of gem-diol depend on the structure of the carbonyl compound.
Thermogravimetric analysis performed on freshly prepared sepiomelanin samples recorded two transition temperatures of about 100° and 150° according to loss of two possibly different kind of water. ( 35 ) Sepiomelanin may be a linear polymer formed by units of hydrate indole-5,6-dione, coupled with a radical-polarone system like that occuring in acetylene-black.

Molecular modelling study on the 5,6-indole-dione monohydrate at 5 position showed that linear dimeric, trimeric, tetrameric, pentameric, etc. forms may assume two low energy conformation helixes and alternate sheets. The first conformation, sheet, shows two areas with different polarity, the second one is like a helix. ( 7f ,7 g )
Constrained by steric hindrance, the alternated, pleated sheet and the helix are held in their shapes by hydrogen bonds between a hydroxyl group and the heterocyclic nitrogen. The hydroxyl group over (or under) the indolic plane and the heterocyclic nitrogen generate helix, whereas a bond between the hydroxyl group, alternatively, over and under the indole plane and the heterocyclic nitrogen determines the alternated pleated sheet.

Whenever the helix behaved as an organic conductor, according to reported acquisitions on acetylene-black, it could constitute a very long solenoid able to generate a magnetic field at the far end of the molecule with an axis of the helix rolling around the backbone formed by double bond linking 4 and 7 positions of starting 5,6-indol-di-one monohydrate ( see also the letter ).
Even if the unsaturated carbon backbone is not completely planar as reported on the acetylene black, nevertheless partial orbital overlapping is possible as the bond length between the two junction carbons of the dienic unit demonstrates 1.49 Å. This distance is very close to the length of an aromatic conjugate C-C bond. Both proposed conformations can satisfy properties and different roles of melanin in the animal kingdom.
It is to note that reactions involving the radical or the cation site of the melanin radical-polarone produce a local carbon hybrid change and determinate variation of the strand course in the space. This response to chemical radical attack could determinate abrupt conductivity changes and indicate a possible chemical switching function of this pigments. On this topic, it was reported that melanins respond to a critical applied field by changing their conductivity and that the nature of response depends on hydration and temperature of sample and on external circuity..
Melanin exhibit two separate current-voltage characteristics, the on and off state. Experiments have demonstrated that their switching depends to hydration (gem-diol formation). Dried samples, kept 30 at 200°C minutes don’t switch. Re-hydration and drying at room temperature restore switching properties. (5 ), www.organicsemiconductors.com , IR, EPR spectra (6,7 ) and general chemical and physical behaviour are in agreement with a radical-polarone (radical-cation) conductive polymeric structure with peculiar electrical properties.

Experiments have shown that melanin exhibits rather exotic electrical characteristics similar to those found in the physics of the solid-state amorphous materials. The broad IR spectra are typical of an amorphous material.The principal infrared absorption bands of the amorphous sepiomelanin are at 2.9 microns, 5.9 microns and 6.1 microns. Absorptions at 2.9 microns and 6.1 microns are characteristic of -OH groups and of H-bonded quinones respectively . It has been referred that 5,6-dihydroxyindole (DHI) is the precursor in vivo of sepiomelanin but the chemical study always shows the presence of ciclodopa (leucodopacrome) or dopachrome units in the polymer (6, ) (7). On the other hand sepiomelanin has similar chemistry to the black pigment prepared in the laboratory from DHI .Sepiomelanin and DHI-melanin with HCl give chlorine derivatives according with their radical-cation nature. About one cationic center every 6-8 monomers may be calculated from chlorine values. It has been calculated that melanins contain one unpaired electron per 200-300 units. (10).Although the free radical represents only a minor part of the molecule (particle) it is of great importance for bio-physical studies ( 11 ).

Sepiomelanin and DHI-melanin methylated with diazomethane have similar percentages of -OCH₃ groups ( 6 )Formation of indolequinone hydrate was observed in the polymerization process of dopammine or DHI (14), (15) and in MALDI-TOF spectra of sepiomelanin samples . According to ketonic compounds,quinone carbonyl groups can undergo hydration reactions yielding gem-diols through a reversible reaction; the extent of hydration and stability of the gem-diol may depend on the structure of the carbonyl compound. In the case of dopachrome addition of water occurs via dopaquinone methide leading to an isomeric polymer . The methoxyl value, after methylation, is similar for sepiomelanin and DHI-melanin but different from the theoretical values.. A different values interpretation may be given assuming that about half of the hydroxyls present are masked and not methylable.

In all melanins, the unpaired electrons (one of two hundreds indole units calculated on the EPR signal) and the positive charged (one for eight units calculated on the amount of the counteranion; C1 in sepiomelanin) are distributed along the unsaturated, conjugated skeletons ( the red line ) and are responsible of their conductivity.

Biological electrical fields generated by the spine may change superficial properties of the pigment which could act as, easily removable (by H₂O₂), ideal equipment for cell assembly and movement. The results obtained by (14) show that the polymeric units are chiefly formed by DHI units with one oxygenated indole unit ( increments of 16 mass units in the MALDI-TOF analysis) for each DHI polymer. The percentage value of C, H, N, OCH₃ (after methylation of the polymer) for such a polymer don't agree with the values found for sepiomelanin or DHI-melanin. As you see in formulae presented the unpaired electrons and positive charges are distributed along the red line.

It is interesting to note that the system indicated with the red line corresponds to the structure of the acetylene-black (PA cis) and present in many BCM and BSM . Formation of indolequinone hydrate was observed in the polymerization process of dopamine or DHI and in MALDI-TOF spectra of sepiomelanin samples . In the case of dopachrome addition of water may occurs via dopachrome quinone methide. leading to an isomeric oligomer .. The indole units (4-16 monomer) may form a graphite-like stack with spacing of 3.4 Å (10), (13), a fullerene structure or according recent molecular mechanics (18) calculations they may have an alpha-helical structure.

Indole oligomers may form particles of different size and shape due to the preparation method.The size and shape, type of oligomers, may influence some physical properties like conductivity. Molecular modelling studies on the 5,6-indol-dione monohydrate at 5 position showed that linear dimer, trimer, tetramer, pentamer, etc. forms may assume two low energy conformations.(7 ) . From MALDI experiment we learn that :sepiomelanin is not DHI-melanin or DHICA-melanin. but may be a cyclodopa –melanin or dopa-melanin ( aliphatic moiety of IR and NMR spectra ).

Presumed oligomers for particle construction are showed.The degraded,colourless, pyrrole part of the structures are not showed. MALDI and MALDI-TOF spectra of purified sepiomelanin show no high mass peak but a lot of small molecules, low fragments arising from melanin breakdown, polluting compounds and melanogenesis intermediates (mass range 50-600 ).None of hydroxyindole found as intermediates of the melanogenesis were found in the MALDI spectra of purified samples of sepiomelanin mature granules

.Studies on chemical composition of the ink with labelled precursors were performed. Since animal ink formation requires a lot of time it was necessary to empty the sack. A higly radioactive pigment was synthetised in vivo with labelled tyrosine, phenol, catechol, 5-oxytryptophane.( 6 ) This means that no specific melanin-producing enzyme is contained in the ink sack. To obtain analytical melanin samples is a very difficult task. The pigment samples were found to differ in properties and chemical composition depending to the mode of extraction (for example with or without catalase) and storage time. Fresh ejected ink differs from the ink remaining into the sack. In some melanosomes melanogenesis is still active and H₂O₂ is present. Melanogenesis in immature melanosomes (IM) is an active process, and all the members participating in pigment formation (H₂O₂, enzymes, starting products and intermediates) are present. Melanogenesis components which come into contact with melanin during the pigment extraction can induce some transformations, like hydroxylation, further oxidation, ring opening and breakdown.

Fuller ink sacks ready for black ejection contain more mature melanosomes (MM). Consecutive ejections decrease the concentration of mature melanosomes and stimulate the synthetic processes. Sack content is, consequently, very variable from a chemical point of view and careful procedures are necessary to obtain reproducible results.

The pigment Na+ form occurs in the ink sac of Sepia officinalis as do Ca++ and Mg++ salts (6).The free acid obtained by acidification with HCl is called sepiomelanin ( sepiomelanic acid ) and is a black amorphous, insoluble, hygroscopic powder without a melting point.

The methylester obtained treating the pigment with diazomethane was in form of an infusible light brown powder The change of ‘’ colour ‘’ indicates that a physical ‘’ colour ‘’ contributes to the black ‘’ colouration ‘’ of the particle. In 1987 a unified physical model of pigments ‘’colour ‘’ was presented ( 20 ) The model is based on measurements of the optical constants of eumelanin and pheomelanin. Using the results of exact Mie calculations of the scattering and absorption cross- sections for individual pigment granules, it was showed that the colors produced by dispersions of eumelanin or pheomelanin granules are strongly dependent on the pigment granule size. Measurements of the granule size distribution in hair of differing ‘’colours ‘’ (blonde, brown, red, black ) are consistent with the predictions. The ‘’colour ‘’ is also found to be strongly dependent on the Mott- Davis optical energy gap parameter E_o. which controls the dispersion of the optical constant k (imaginary part of the complex index of refraction ) in an amorphous semiconductor ( 20 ).

Changes in E_o as small as 0. 2 eV (out of 1 .4 eV ) are sufficient to alter ‘’ colour ‘’ from one class to another , eg. brown to red. Conversely, sufficiently large granules (or clusters ) of both eumelanin and pheomelanin can produce dark brown and black dispersions.

Synthetic and natural melanins (dopa-melanin, melanoma melanin grown in culture, sepiomelanin ) were examined by solid state NMR using cross polarization,magic angle sample spinning , and high-power proton decoupling.Natural abundance ¹³C and ¹⁵N show resonances consistent with known pyrrole and indole structures within the heterogeneous biopolymer and indicate the presence aliphatic residues in all blacks . Similar NMR spectra were obtained with sepiomelanic acid, an H₂O₂solubilized pigment. The presence of an aliphatic part in NMR spectra (21 ), the presence of units in the oligomers showed by isotopic studies (22 ), allow us to assign the carboxylic group in position 2 . On the contrary, literature considers the acid (DHICA ) an important precursor of eumelanins (BCM). The reflecting material of the tapetum lucidum of the sea catfish contains a mixture of polymers of DHICA among which the tetramer predominates.Synthesis of the tetramer was recently reported ( 24 ) .Particles of BCM and BSM are different for shape and size.The difference is due to the molecular weight of the oligomers, the type of the precursors and method of samples preparation.

Melanins ( BCM ) (6-7 ) occupy an unique position among the natural pigments like the electrical conductivity in amorphous semiconductors and the display of threshold switching ( 5 ) .Peculiar are also the existence of planar stacks of monomer units , the absorption of ultrasound in the region of 1 MHz, the colour attributable more to the electronic transition of materials at bands than to orbital transitions, the capacity to form charge transfer complexes.

In the biological field it is be noted that the control of enzymatic melanogenesis is not well understood . The ionization technique (MALDI) , has extended to the study of synthetic and natural ( BCM and BSM ) pigments . A series of products of pyrolysis: toluene , ethylbenzene, phenol , cresol, methyl indole, methylprrole, indoline, are obtained from triptophane black oxidized by performic acid . With analogous methods , products not previously described , are obtained from sepiomelanin, hair- melanin, serotonin - melanin, tryptamine- melanin and tyrosine- melanin by enzymatic or chemical oxidation. Interesting results have been obtained with MALDI in the study of 5, 6- dihydroxyindole melanin (DHI) and 5, 6- dihydroxyindole – 2- carboxylic acid melanin (DHICA). The melanin particle would appear to be a mixture,not reproducible, of oligomers and pyrrolic polyacids ( 6a ) of low molecular weight (500-1500 Da ) arising from the breakdown of oligomers ( 25 ) (28 ) .It is not clear if the molecular breakdown occurs also in vivo

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Maldi Spectra

Sepiomelanin

273, 313, 335, 349, 363, 369, 373, 391, 450, 526, 552, 685.

DHI-melanin

497, 516, 542, 572, 579, 692, 722, 744, 778, 874, 930, 983, 1047, 1080, 1136.

DHI-melanin peroxidase

497, 524, 540, 552, 572, 703, 714, 731, 868

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The study of dopamine melanogenesis, performed by MALDI analysis on samples taken at time from the oxidation reaction mixture shows the melanin is formed by intact indole units. No pyrrolic polyacids were detected ( 14 ) .On the contrary extensive MALDI study of the natural and biosynthetic melanins leads to the conclusion that all are formed by groups of degraded oligomers with molecular weights between 500 and 1500 Da . It seems that melanogenesis, a typical radical process which produces a series of oligomers, is strongly influenced by the pH, by the concentration of the substrate, by light , by the time of the reaction , by the quantity of O₂ or other oxidizing agents present which furthemore produce degradation .In other words the study of melanin made until now has used synthetic material . Actually samples of sepiomelanin obtained from the same sourch but purified by different procedures were found to be different.The type of MALDI spectra obtained from sepiomelanin is depending on :

A ) centrifuge speed

B )time of storage

C )the presence of Ca and Mg ions

D )the acid treatment of the samples

E ) physical and chemical methods of purification adopted

F ) LASER energy and matrix adopted

It would be interesting to follow these modifications with the atomic force microscopy

The distribution of the oligomers is clearly different between the hot and the cold purified

sepiomelanin as well as a loss of CO₂from the degraded part of the molecule.Hot purification produces the disappearence of protein material . For the first time these methods have led to the identification of the initial states of melanogenesis : dopamine, dopaquinone, leucodopaminochrome , aminochrome, semiquinone. MALDI verifies the presence of oligomers.

At a later date the study of melanogenesis from dopamine with peroxidase /H₂O₂ in sodium phosphate buffer using MALDI-TOF- MS has revealed the presence of intact indole units up to a quantity of 11 units of 5, 6- dihydroxyindole (DHI) which appear also in sepiomelanin spectrum ( see Letter ). Oxidation of the indole with peroxidase / H₂O₂, tyrosinase from mushroom , sodium periodate, potassium ferricyanide produces high degraded melanin giving ionic mass spectra with weights between 500 and 1600 ba , and, none of these corresponds to intact oligomere of DHI. Analysis of the molecular weights and the difference in masses give a proof that significant quantities of polycarboxylic products explaining the CO₂ evolution by heating of the solid

Scheme I

Degradation products of sepiomelanin ( 7 ) The biggest fragment obtained.

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Table 1

Sepiomelanin and DHI-melanin

Sepiomelanin found %C 59.9 %H 3.6 %N 6.7 %Cl 3.2

DHI-melanin found %C 55.2 %H 3.3 %N 8.5

DHI polyindolequinone calc. %C 66.2 %H 2.0 %N 9.6

C₂₀ H₁₇ N₃ O₁₅ calc. %C 44.5 %H 3.1 %N 7.8

C₂₀H₁₁N₃ O₁₅ Ba₃ calc. %C 25.2 %H 4.4 %Ba 43.2

Barium salt of sepiomelanic acid (1), (1a), (10).

C%22.6 H%2.1 N%4.4 Ba% 43.6

C%22.4 H%1.8 N%3.0 Ba% 41.6

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The polyacids have already been obtained by mild oxidation of sepiomelaninin and were called sepiomelanic acids.These soluble acids can be purified under the form of barium salt.They react with 2,4-dinitrophenylidrazine ( 6a ) thereby showing the presence of carbonylic functions as depicted in formulae of the scheme..The molecular weight of C₂₀H₁₇N₃O₁₅ (539) corresponds to one of the peaks seen in the MALDI spectra (29) The melanins from Sepia, melanoma and human hair were methylated by suspending them in an ethereal solution of diazomethane and the percentages of methoxyl groups determined (sepia 18 %, melanoma 15 % human air 14% DHI-melanin 21.5 %, Dopa-melanin 19. 6 % ) Methylated melanins were oxidized with KMnO₄and analysis of the degradation products revealed the presence of 2- carbomethoxypyrrole –4, 5-dicarboxylic acid .

The pigment was extracted and purified.Oligomers of different type,including

pyrrole units, form round particles which are stable in structure .Sepiomelanin is solubilized by

alkali and H₂O₂ in mild conditions or degraded to a mixture of acids including oxalic and pyrrolic acids. To a more complex degradative compound obtained by us as barium salt the formula C₂₀H₁₁N₃ O₁₅Ba₃ was given ( 6,7 ) ( Scheme I ).A similar compound appears in MALDI experiments (29 ). Solubilization by alkaline H₂O₂of the sepia pigment occurs whithout important physical modification.Melanin behaves toward H₂O₂ like a catalase.

Breakdown of benzenoid part or indole units occurs by action of H₂O₂. Probably the reaction is a physiological one. Partially degraded oligomers,which contain pyrrole rings, were identified in the fragmentation ( MALDI) of sepiomelanin. The importance of pyrrole compounds in the formation of melanins was first pointed out by Angeli . He surmised that an oxidative fission of the benzenoid ring (30) (31) must be involved in the melanogenesis, perhaps through the formation of pyrrole acetic acids.

In conclusion, sepiomelanin is different from others melanins suggesting that it is a largely degraded DHI-melanin.The presence of an aliphatic part showed by NMR spectra remains an enigma. Pyrrole units are present.

Elemental analysis of sepiomelanin in Table reflects the degradation state of the samples used and the presence of more oxygen of the calculated. .It is not clear if these degradation processes occur in the cell.For chemical study of the ink it is necessary to use mature organelles ( melanosome full of melanin ) as material.

As radical scavengers, in scattering particles, as bio-organic conducting system and in numerous other instances melanins allow a rapid electron passage along their radical-polarone chain, and in behaving as an electric conductor in a crucial area of the brain could also induce magnetic fields useful for checking the heart's magnetic field, like hematite crystals in the turtle’s brain. Neuromelanin appears to be associated with the bio-electrical activity of neurons and with degeneration of substantia nigra and Parkinson’s disease. Not too much is known about the chemistry and biology of other melanins whose function can be interpreted taking into account their conductive oligomer property and particle structure (7). Contrary to what previously reported the particles found in the ink sack of the Sepia officinalis are formed from DHI, dopa, cyclodopa, decarboxycyclodopa, oligomers in agreement with biological studies .Some units are degraded ( fission of the benzenoid part ) with formation of carboxylic groups and pyrrole rings.It is probably that the degradation process occurs only in vitro. The melanogenesis is believed to be not an enzymatic process as the traditional meaning .

The melanin in Sepia, melanoma and human hair were methylated by suspending them in an ethereal solution of diazomethane and the percentages of methoxyl groups determined (sepia 18 %, melanoma 15 % human air 14% DHI-melanin 21.5 %, Dopa-melanin 19. 6 % , pyrrole-black 8.6 % ) Methylated melanins were oxidized with KMnO₄and analysis of the degradation products revealed the presence of 2- carbomethoxypyrrole –4, 5-dicarboxylic acid ( 6 )

It is possible that, in the mutated environmental conditions verified in the course of the extraction, the cellular process is modified with the formation of a melanin which is, effectively, artificial. The modification would seem to operate at the level of the dopachrome (2) with the formation of DHICA instead of DHI. That would lead to the description of a melanin formed in the prevalence of DHICA units instead of DHI contrary to that predicted in the scheme of the melanogenesis and by the activity of the tautomerase dopachrome . Another collateral process can be that of the opening of the benzenoid rings by hydrogen peroxide (a normal product of melanogenesis).
On the basis of the chemical and physical data available undoubtedly the study of sepiomelanin may contribute to the knowledge of the black matter.The studies furthermore suggest that melanins are polyindolquinones in a hydrate form.They are characterised by a radical-polaron system with stable unpaired electrons. Such a system is present in acetylene-black and in all the organic black materials examined. The oligomers (12-16 monomers) are settled in graphitic sandwiches (interspacing 3.4 A°) or in fullerene cages (interspacing 4.4 A°). The melanins are natural amorphous semiconductors with a model which corresponds to the band model of that of semiconductors and superconductors.(5) Amorphous semiconductor switching in melalnin was observed
On a biological level the black particles theoretically possess multi-functional properties yet to be discovered, like the capacity of molecular synthesis, of molecule and cell assembly, the function of communicating between tissue and the central nervous system, the storage of water, metals and gas. The particles of melanin explode under the action of the LASER and atomic bombardment continually transforming in cycles of synthesis and of break up (both on earth and in interstellar space)..

Breakdown of benzenoid part or indole units occurs by action of H₂O₂. Probably the reaction is a physiological one.A partially degraded oligomers,which contain pyrrole rings, were identified in the fragmentation ( MALDI) of sepiomelanin. The importance of pyrrole compounds in the formation of melanins was first pointed out by Angeli . He surmised that an oxidative fission of the benzenoid ring must be involved in the melanogenesis, perhaps through the formation of pyrrole acetic acids.In conclusion sepiomelanin is different from others melanins suggesting that it is a largely degraded DHI-melanin.or ciclodopa-melanin Pyrrole units are present.The pigment may form without intervention of enzymes ( W.Brackman, E.Havinga, Rec.Trav.Chim.des Pays-Bas 74,1107, (1955).

Samples preparation of melanin from biological sources.

The preparation of samples of melanin regards, for the moment, only melanin from the sepia. In a similar way would be possible to obtain samples of melanin from different biological sources.
The sepia ink is a complex mixture of organelles, premelanosomes, melanosomes, granules, proteic material (enzymes), glucosamine, and phospholipids in suspension or solution liquid. At the moment of extraction the mixture is still active and contains some hydrogen peroxide. An artificial melanin, that is a chemical product different to the physiological one with a possible formation of a system built on units of DHICA rather than DHI or brocken units may be formed.
The composition of the mixture is very variable according to whether one is dealing with the ink of a live animal or a dead one, and on the time spent between one emission and another of the black of the animal. For this reason the goal of obtaining a reproducible sample is difficult and laborious to reach. The main problem is to use samples almost formed of granules or particles and material not contaminated of hydrogen peroxide.
Samples obtained from naturally fresh ejected ink are recommended or to proceed in the following manner :

Sepia was killed with urethane.The sepia ink pouch is opened and the liquid gently squeezed ( a preferable method is to use the black naturally ejected ) out to the black suspension catalase ( amount to be defined ) and water (20% distilled, deionizated and deoxygenated water) is added and centrifuged at 2000-3000 cycles. The black solid is washed with H₂O x 3, CH₃COCH₃ x 3, H₂O x 3, and dried on KOH pellets. All the operations are conducted at room temperature and away the contact of light and as much as possible away from atmospheric oxygen.
The black solid ( A ) thus obtained is rich in ashes (Na, K, Ca, Mg up to 20-25% expressed in sulphates) and contains about one oxygen atom for every IQ unit ( meaning addition of water to quinone group, storage of O₂, water, presence of carboxylic groups )
This sample (A) called sepiomelanin is a salt, the Mg and Ca salt, and can be used in the same way either in the form of a free acid treating it with HCl 2N x 3, H₂O x 3, in centrifuge, or briefly boiling with HCl 6N ( 15 min.) obtaining (B), the sepiomelanic acid.
The sepiomelanic acid (B) can also be obtained by the following method:
The solid (A) is suspended in 80 cc of H2O and taken to pH 10 by adding NaOH N, passed through ultrasound (15 min 80W) and eventually filtered or centrifuged. The filtrate is taken to pH 1 with concentrated HCl and the solid centrifuged and washed with HCl N x3, H₂0 x3, acetone x3, H₂0 x3, dried on KOH drops at room temperature and away from light.
Both sepiomelanin and sepiomelaninic acid can be further purified using various methods .
Samples of different composition can be obtained varying the speed of the centrifuge. Using MALDI mass spectrometry and MALDI-TOF on these samples it is possile to carry out an in depth examination of the process of melanogenesis which happens in the ink sack of the sepia Sepia officinalis .
Summing up: successful work needs a homogeneous preparation of the melanin granules, working when possible in the absence of hydrogen peroxide ( peroxide is present in the cell or formed by action of atmospheric oxygen on o.diphenols ) light, oxygen, at a physiological pH. The samples for centesimal analysis must be dried at room temperature.
The samples undergoing analysis must give values of C, H and N taken from the ashes, in agreement with the theoretical values calculable for a polyindolequinone hydrate (various quinonic structure).Conductivity, gases , liquids and drugs absorptions are determined . Typical broad IR, ¹³C NMR, EPR, spectra MALDI and MALDI-TOF fragmentation, AFM pictures are to be compared with DOPA-melanin, dopamine-melanin, DHI-melanin and DHICA-melanin. All the analysis must be carried out for a correct identification and classification of the melanin

Oxidative degradation of natural pigments : identification of 2,3,5-pyrroletricarboxylic acid ( PTA ) ( 6f )

Samples of natural pigments (25mg) were dissolved or suspended in 2n potassium carbonate (2ml) and oxidized at room temperature by the gradual addition of saturated potassium permanganate solution.When the colour of permanganate persisted for about 10 minutes excess of the oxidant was destroyed by addition of a little sodium sulphite.The solution was briefly boiled and freed from manganese dioxide by filtration or centrifugation.The manganese dioxide was washed with hot distilled water (3ml)the washing being added to the main filtrate . The combined filtrate and washing,acidified to congo red and if necessary filtered was adjusted to ph 4-4,5 by addition of 2N NaOH . 50% Calcium chloride solution (1-2 drops ) was added and a precipitate forming during 1 hour was removed by filtration or centrifugation. After ensuring that a fportion of the solution afforded a precipitate with ammonium oxalate solution it was made strongly acidic to congo red by addition of conc.hydrochloric acid, pyrrolic acids were now extracted with peroxide-free ether ((4 x 2.5ml). The ethereal solution was washed with distilled water (0.5ml) dried over magnesium sulphate and evaporated to 2-4ml in vacuo.Finally evaporation to dryness was effected in a small text tube at 60-70°.Appropriate water solution were chromatografated on Whatman 1 ( butanol-acetic acid-water ) spray with diazotised sulphanilic acid for red spots.

TABLE of elemental analysis

Centesimal composition of sepiomelanin.Sulphur always present in little amounts ( % S 0.2-0.8 ).Aging or speed of the centrifuge used may alter morphology and composition of the samples ( 24).Values are affected by purification and extraction methods which cause breakdown of benzenoid part and decarboxylation. Therefore sepiomelanin samples are chiefly formed of partially degraded oligomers of DHI. Values are meaningful only for experienced people.

(1)Nencki and Sieber, Arch.Path. 24,17,1888 ; (2) Neuberg, Zeitschr. fur Krebsforschung, 8, 1909 ; (3) Piettre, Co mpt.Rend.153, 1037, 1911 ; (4) Panizzi e Nicolaus, Gazz.Chim.Ital. 82,435,1952 ; (5) Piattelli et al.Tetrahedron Letters 21, 14, 1959 ; (6) Nicolaus et al., Rend.Acc.Sci.Fis.Mat XXVII,13,1960 ; (7) Piattelli et al.Tetrahedron 15,66,1961 : (8) Nicolaus, Rassegna di Medicina Sperimentale,IX,1-30,1962 ; Ed.Idelson Napoli 1962 ; (9) Nicolaus et al. Rend.Acc.Sci.Fis.Mat. XXXII, 83,1965 (10) J.P.Ortonne et al. Pigment Cell 1981, Ed. Seiji,University of Tokyo Press 1981 (11) M.Benathan,These Facultè des Sciences,Universitè de Lausanne 1980. Dir.de These H.Wyler.(12) Ito, BBA,883, 155, 1986.

% C 56. 3 %H 3.6 % N 12.3 (1)

% C 57. 0 %H 3.4 % N 11.3 (2)

% C 58. 0 %H 3.3 % N 11 .3 (3)

% C 57. 5 %H 2.9 % N 10.5 (4)

% C 54. 9 % H 3.0 % N 10.1 (4)

% C 55. 0 % H 4.5 % N 8.8 (4)

% C 64. 1% H 3.0 % N 8.5 (5)

% C 58. 6 %H 3.2 % N 9.3 (6)

% C 59. 8 % H 3.2 % N 9.5 (6)

% C 63. 1 % H 2.4 % N 8.9 (6)

% C 58. 3 % H 3.0 % N 9.8 (6)

% C 58. 5 % H 3.0 % N 9.6 (6)

% C 64. 2 % H 2.8 % N 8.6 (6)

% C 63. 9 % H 2.9 % N 8.9 (6)

% C 63. 3 % H 2.4 % N 8.8 (7)

% C 59. 9 % H 3.4 % N 8.2 (7)

% C 44. 2 % H 5.3 % N 9.9 (8)

% C 54. 4 % H 3.0 % N 8.1 (8)

% C 53. 6 % H 4.0 % N 8.9 (8)

% C 55. 0 % H 4.5 % N 8.8 (8)

% C 60. 8 % H 3.4 % N 8.5 (9)

% C 54. 3 % H 2.9 % N 8.8 (10)

% C 54. 3 % H 2.9 % N 8.7 (10)

% C 54. 3 % H 2.9 % N 8.5 (10)

% C 57. 7 % H 2.6 % N 6.2 (11)

% C 52.2 % H 3.4 % N 7. (12)

Fig. 1 Fig. 2

Melanin particles ( 40-42 ) are also contained in branched cell called chromatophores (melanophores) of the sepia skin. In melanophores the particles can become dispersed into the branches or aggregated in the middle of the cell thus rapidly causing the animal to appear darker or lighter.

It is not yet known whether melanin particle move to form the zebra patters of the cuttlefish ( from fig 1 to fig 2).

The bright colored pigment cells of fishes and amphibians include xantophores,erithrophores and iridiphores.The role of the hypophysis is discussed.The nervous system may be involved in physiological responses of the iridophores.Intermedin causes iridophorestocontract as doseveral drugs the effect of intermedin can be reversed by still other agents.Melatonin has no effecy on iridophores.Xantophores of some fishes and amphibians are induced to expand by intermedin.Thr morphological effects of intermedin at the organellar level are presented in terms of ultrastructure and pigmentary composition

J.T.Bagnara, Mac E.Hadley, Am.Zoologist, 9, 465-478 ( 1969 )

E.Florey,American Zoologist, 9, 429, 1969.

From the symposium organized by R.R.Noveles for American Association Zoologists, for Advancement of Science, for Division of Comparative Endocrinology

Cephalopod particles ( chromatophores ) are deformed by radially attached muscle fibers.The change in colour of a squid is controlled by a motor innervation that activates the chromatophore muscle.Thus the colouration of a cephalopod obeys the laws that govern the coordination of muscular movement : there are coloured twitches, red, yellow ( see Link 9 ) , brown tetani,and there is paling relaxaction.

Cephalopod chromatophores are deformed by radially attached muscle fibers.The change in colour of a squid is controlled by a motor innervation that activates the chromatophore muscle.Thus the colouration of a cephalopod obeys the laws that govern the coordination of muscular movement : there are coloured twitches, red, yellow ( see Link 9 ) , brown tetani,and there is paling relaxaction.Photomicrograph integumental chromatophores of Rana pipiens. . Dermal preparation showing the tipical morphology of chromatophores as they are disposed in Ringer solution Melanophores are punctuate whereas the iridophores are highly irregular in shape . Pure epidermal sheet showing the punctuate state of the epidermal melanophores as they appear in Ringer solutioThese melanin-containing cell may in some skins,however, remain dendritic in Ringer solution. Dermal preparation showing the response of chromatophores to MSH.Melanin particles ( granules ) become dispersed out into the arms of the melanophores (arrows ) The reflecting platelets within the iridophores become aggregated and these cells now appear round in shape, Epidermal sheet showing melanin granule dispersion within melanocytes in response to MSH(Mac E.Hadley,J.T.Bagnara,Endocrinology, 84, 69-82 ( 1969 )

The particle may assume the fullerene or the graphite form.

MALDI and COMPUTER STUDIES

Lettera con risultati MALDI e commenti ai modelli del computer

With english text

Caro Professore Nicolaus,

Le comunichiamo in breve i risultati ottenuti recentemente nello studio della melanogenesi dello inchiostro del sacco della seppia Sepia officinalis. E’generalmente ritenuto che la melanogenesi negli animali sia il processo enzimatico che trasforma la tirosina in melanina. Gli studi del fisiologo inglese H.S. Raper portarono all’isolamento di alcuni intermedi della melanogenesi fra cui il 5,6- diidrossindolo (DHI) e il 5, 6- diidrossindolo-2-carbossilico (DHICA) ; il DHICA, meno reattivo, non fu ritenuto del Raper un intermedio di rilievo della melanogenesi. Lo schema della melanogenesi qui riportato fu il risultato di studi effettuati con enzimi vegetali estratti dal Tenebrio molitor sulla tirosina:

TIROSINA---DOPA----DOPACROMO----DHI------MELANINA

Piu recentemente lo schema e’stato studiato con 1’uso di enzimi animali e precisamente con quelli contenuti nel sacco della Sepia officinalis. Nella borsa dell’ inchiostro e’ stato trovato un enzima che opera la trasformazione del dopacromo in DHI il che costituisce una importante conferma della validità dello schema di Raper anche per gli animali superiori Come conseguenza di cio si deve ritenere che il nero di seppia e il nero di DHI, ottenibile in laboratorio, siano molto simili se non la stessa cosa. Lo studio chimico della sepiomelanina sembra apparentemente portare invece a conclusioni che sono in contrasto con quelle ricavabili dallo studio enzimatico. L’ analisi chimica (4)dei prodotti di degradazione indicano che il nero di seppia e un copolimero di DHI e Dopacromo mentre gli spettri di massa dei campioni di sepiomelanina indicano che la melanina e’ una miscela di oligomeri, a relativamente basso peso molecolare, derivati per il 75% dal DHICA e per il 20% dal DHI. I risultati da noi ottenuti sembrano chiarire i dati contrastanti con lo schema di Raper. L’ inchiostro della seppia e’ formato di granuli di melanina mescolati a premelanosomi e melanosomi a diverso grado di maturazione e densita, essendo i granuli piu densi dei melanosomi. La separazione fra granuli ed altri organelli componenti del sacco si realizza abbastanza facilmente centrifugando (a 1000—3500 giri) 1’inchiostro fresco proveniente possibilmente da una seppia viva. Le proprietà chimiche e fisiche del campione variano col variare della velocità di centrifugazione I1 campione si prepara a pH fisiologici, fuori del contatto della luce e dell’ ossigeno ed a temperatura ambiente. L’ analisi centesimale C, H, N eseguita su campioni di melanina che non abbiano subito drastici trattamenti da valori che, se corretti per le ceneri e molecole di acqua o frazioni di essa, sono in buona approssimazione vicini a quelli calcolabili per un poliindolchinone nella forma idrata oppure con la presenza di carbossili. I dati analitici sono confermati dallo studio da noi effettuato sui campioni con 1’ausilio della spettrometria di massa.

Lo spettro MALDI ottenuto su uno Spettrometro Voyanger DE, PerSpective Biosystem, Boston MAUSA, Nitrogen LASER=337,1 nm e’ stato effettuato su campioni di melanina ottenuti per centrifugazione a 3500 giri. La sospensione e’ stata preparata mescolando 1mg di pigmento in 1mldi acqua La sospensione e’ stata sonicata per 15 minuti in un bagnetto ad ultrasuoni. 1ml di questa miscela e’ stato caricato sulla piastrina autocampionatrice e lasciata seccare. I campioni sono stati esaminati o tal quali o con l’ aggiunta sul pozzetto di una matrice DHB (1 ml di una soluzione di 10ml in 1ml di una soluzione 70% CH3CN-0.1%TFA contenente 250 fentomoli di insulina) mostra picchi identificabili con oligomeri del DHI in buono accordo con quanto osservato nella preparazione della melanina da dopammina o da DHI . Lo spettro non mostra picchi molecolari relativi a prodotti di degradazione o picchi a peso molecolare elevato . La mancanza di ioni molecolari della melanina e’ in accordo con la nota sensibilità della particella alla radiazione LASER.In conclusione tutti i dati sperimentali fin qui raccolti confermano lo schema di Raper cosi come ere stato originariamente concepito per via chimica e pongono in evidenza il ruolo giocato dal DHI nella melanogenesi animale

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The first conformation in alternate sheets shows two areas with different polarity, the second one like an right-handed helix constitutes a very long solenoid able to generate a magnetic field at the far end of the molecule with the axis of the helix rolling around a backbone formed by a double bond linking 4 and 7 of starting 5,6-indol-di-one monohydrate. The pleated sheet and helix are held in their shapes by hydrogen between a hydroxyl group and the heterocyclic nitrogen. The hydroxyl group over (or under) the indolic plane and the hydrogen on the heterocyclic nitrogen generate a helix, whereas a bond between the hydroxyl group alternatively over and under the indole plane and the hydrogen on the heterocyclic nitrogen determines the pleated sheet. In this system only one indole hydroxyl group is available to methylation.Both conformations can satisfy properties and different roles of melanin in the animal kingdom.It should be noted that reactions involving the radical or the cation site of the melanin radical-polarone produce a local carbon hybrid change and determine variations of the strand course in space. This point of view explains the high reactivity to oxidation showed by melanins and resistance to reduction reactions.As expected, no information about MW of sepiomelanin could be obtained by Mass Spectrometry. MALDI and MALDI-TOF spectra of purified sepiomelanin show no high mass peak but a lot of small molecules, low fragments arising from melanin breakdown, polluting compounds and melanogenesis intermediates (mass range 50-600 ).None of hydroxyindole found as intermediates of the melanogenesis were found in the MALDI spectra of purified samples of sepiomelanin.Sepiomelanin was studied as a molecular model of melanins arising from different sources (5,6-dihydroxindole, adrenalin, serotonin, tryptamine, dopamine and cysteinyl-DOPA) all yielding unknown melanins. Nevertheless, the shapes into which sepiomelanin arranges its backbone, allows the hypothesis that the black pigments can play different roles in different districts according to their features. Folding of the helix backbone upon itself can form spheres; stretched-out zig-zag chains, which alleviate steric hindrance and permit hydrogen bond interactions, and can build strata.Sepiomelanin like an electrically conducting polymer may reversibly change, with an applied potential, their surfaces properties.

It was experimentally found that conducting polymers represent a type of culture substrate which could provide a noninvasive means of controlling the shape and function of adherent cells indipendent of any medium alteration (Ingber) . As radical scavengers, in scattering particles, as bio-organic conducting system and in numerous other instances melanins allow a rapid electron passage along their radical-polarone chain, and in behaving as an electric conductor in a crucial area of the brain could also induce magnetic fields useful for checking the heart's magnetic field, like hematite crystals in the turtle’s brain. Neuromelanin appears to be associated with the bio-electrical activity of neurons and with degeneration of substantia nigra and Parkinson’s disease. Not too much is known about the chemistry and biology of other melanins whose function can be interpreted taking into account their conductive polymer property and structure . 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 region (for example, the midbrain). A singular 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 determing the nature of these states is a meausurement 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 electron-phonon 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.The pigment found in the ink sack of the squid is probably formed from 5,6-dihydroxyindole, or cyclodopa a reactive eumelanin precursor. The pigment like a polyindolequinone hydrate (5-gem-diol of polyindole-5,6-dione) radical-polarone system, may represent a new structure for organic chemistry The units may form a graphite- like stack with spacing of 3.4 Å or, as molecular computer calculations show a helical or an alternate sheet structure. The structures proposed explain many of the experimental data accumulated over the past half century of study on melanins. Although for such a peculiar structure further investigations are required, the chemical enigma of melanins is being solved. New perspectives in Biology have opened up but a fundamental question is still unsolved. What was the role played by the black conductive material in the past and what is its role in Nature today ? What is the function of conductive material in the brain?

Elemental analysis was performed by REDOX (snc) I-20052 Monza.

Physical and Chemical data are available.

A.Bolognese , Orazio Mazzoni, Dipartimento di Chimica Farmaceutica e Tossicologica, via E.Montesano 46, 1-80131 Napoli . A.Malorni , F .Talamo,A.M.Salzano Centro Internazionale di Servizi di Spettrometria di Massa del CNR di Napoli, Istituto per la Chimica di Molecole di Interesse Biologico del CNR,Arco Felice, Napoli . B.Nicolaus, I.Romano ICMB del CNR, 80072 Arco Felice , Napoli.

Pheomelanin,Pheochromes,Chromatophores

Melanin

Pheomelanin

Marsupial pigments are probably triptophane derivatives

Marsupials arrived in Australia from South America via Antartica some time prior to 43 million years ago when the three continents were believed to costitute one land mass (E.M.Nicholls, K.G.Rienits ‘’ Marsupials Pigments ‘’ in Pigment Cell , vol. 1 ,pp. 142-150, Karger, Basel 1973 .

The red hairs

This nice neapolitan boy provided us with the material for pheomelanin studies

The pheochrome of red hairs The chromophores

Pheomelanins are alkali soluble pigments of feathers, hair and furs. Pheomelanins are formed starting from cysteinyldopa (1) (2). Pheomelanins are divided into two groups:
Pheochromes, yellow, reddish crystallizable, dilute HCl soluble pigments
Pheomelanins, red-brown, brown, amorphous, dilute HCl insoluble pigments.
Pheochrome cis-D^2,2'-Bi(5-hydroxy-7-alanyl-2H-1,4-benzothiazine)-3-carboxy-3'-one (I) together with gallopheomelanin-1, have been isolated from New Hampshire chicken feathers and characterized. Cysteinyldopa is the biological precursor of (I), a yellow-orange compound recovered as crystals. Its chromophore system is responsable of the carrot-red hair of the young Neapolitan boy see in the photography. The structure of pheomelanins is not well understood but the structure (I) may be part of gallopheomelanin-1
The pheochromes (other names like trichosiderins, trichochromes, are reported in litterature) obtained during acid extracion of feathers and hair are artifacts. The chromophores may be (II) (III) (IV) type . Melanin, Pheomelanin, Pheochromes may coexist in Nature.
Pheomelanins and pheochromes as radical-polaron structure are biological electric conductors. The EPR spectra is similar to that of melanin. Actually, DOPA a versatile aminoacid, precursor of melanins and alkaloids, provides benzopyrrole, benzothiazine and isoquinoline skeleton to eumelanin, pheomelanin and neuromelanin, respectively. The red, orange, brown pigments of some rodents and kangoroo furs are probably tryptophane derivatives.

Allomelanins

Nitrogen-free polyphenols are the precursors of many allomelanins ( plant pigments,mould-pigment, bacteria-pigment,humic acids etc. )

Melanogenesis is given by the co-occurrence of tyrosine and dopa in a number of melanin-producing plants and by the observation that the blackening of plant tissue sometimes proceeds via a red phase, as, for example, when potato slices are ground up or exposed to chloroform vapor. Very interesting is the mould Aspergillus niger which produces Aspergillin, a graphite- like material .

The photo was a gift of Professor A.Quilico (Milan 1960). Aspergillus niger an organism that generates a rare graphite-like pigment. (A.Quilico ‘’ I pigmenti neri animali e vegetali ‘’ Tip.Fusi,Pavia 1937 ; R.A.Nicolaus ‘’ Melanins ‘’ Hermann, Paris 1968 ).

Black pigmentation is often highly localized in individual plants in contrast to the more general distribution of the enzyme ''tyrosinase''. Polyphenols as catechol, 1, 8-dihydroxynaphtalene are known as precursors of allomelanins

Warning : Various papers still quote descriptions of BCM and BSM which are not up- to- date and do not fully comply with the chemical and physical experimental data.

The particle

The concept of particle was used for sepiomelanin in 1992. A deeper analysis of sepiomelanin granules was made by use of atomic force microscopy AFM.

The radical-polarone oligomers, which can form a particle, are characterized by electronic lacunae and by positive charge on an extensively conjugated carbonium skeleton.The system show a typical EPR signal.The most simple example is acetylene-black .

‘’ Colour ’’ depends not only on the gap (expressed in eV ) of the semiconductor model but also by particle structure ( morphology) and the doping state which could explain why the methyl ether is not black anymore. Blue or green pigments don’t exist in animal or plant kingdom ( Link 9 )

.Melanin is responsible for blue , green or yellow colours in animal and plant kingdoms.

In nature the black particles ( BCM = black cell matter, melanin , natural melanins , eumelanins etc.) may form from many different precursors like decarboxycyclodopa , DHI, serotonine, catecholamines, dihydroxytryptamines, dopamine, adrenaline, dihydroxyquinolines that is from polyphenols . The heterogeneous black material is generally formed by oxidation of o.diphenols with CO₂ evolution and H₂O₂ formation.The carboxylic group present in many polymers of the particle are formed from the breakdown of the benzenoid part of the indole units. The elemental analysis of the black material show in general the presence of one oxygen more than the precursor. This may be explained with storage of oxygen, water, addition of water to the o.quinone or the quinone methide structure, presence of carboxylic groups, or with a trihydroxylated-phenylalanine precursor . Pyrrole rings are often present in indole melanins .

Melanins show typical broad EPR, IR (K.Jimbow et al., Cancer Research, 44, 1128-1134, 1984 ; S.N.Desiderio et al.,cfograeff@ffelrp.usp.br ; melanin IR spectra (Google) http://www.accelrys.com/cases/melanin.html ; B.Bilinska ,Spectrochimica Acta Part A : Molecular Spectroscopy 52, 1157-1162, 1996 ) , (¹³C- NMR B.Bhavin, Magn.Reson.Chem., 41, 466-474, 2003 ) spectra of amorphous state but an ordered state appear under the AFM figures. .The spectra may be used for eumelanin identification comparing it with a know material like dopa-melanin, cyclodopa-melanin, DHI-melanin, DHICA-melanin.

As long-awaited the MALDI procedure is not useful for molecular weight determination, as occurs for proteins. LASER operates a fragmentation (explosion, depletion ) of the particle. The MALDI fragments are useful in structural study of melanin .

The material obtained from the ink sac (sepiomelanin) is a mixture of oligomers, brocken oligomers, premelanosome, melanosome, particles at different maturation grade and activity. Some chemical and physical data obtained from such a heterogeneous material are probably to be revisited like binding effect and storage of gases and linquids, electrical behaviour. The recent results obtained by J.D.Simon (2003) with at, omic force microscopy tecnique will contribute to elucidate the structure and biological functions of the melanin particle ( melanins,eumelanins, BCM,BSM,allomelanins,pheomelanins)

Atomic force microscopy is used to investigate the structural organization of the melanin ( not purified ) of the ink sack of Sepia (40-53 ) .The structure is an aggregate comprised of particles with diameters of 100-200nm.Manipulation of these structure using the AFM tip show that these particle are not the fundamental structure unit but are aggregate of smaller constituents.Images of the bulk pigment also reveal the presence filaments that have an average height and width of circa 5nm and tens of nanometers,respectively.The particle is formed of small oligomers as showed before, and the structural morphology reflects aggregation of these oligomers.Chemical and physical agent may influence the morphology of the particle (42 ). AFM microscopy reveals eumelanosomes and pheomelanosomes have ellipsoidal and spherical shape respectively ( 49 ).

Pheomelanins (brown,red-brown proteic amorphous pigments ) and pheochromes yellow red cristallyzable animal pigments.Both are formed from Cysdopa and have the same dibenzothiazine or dibenzothiazinone chromophores. (7g ), ( Link 14 of the WEB )The structure of the red hair pigment was reported. (7g ), ( Link14 of the WEB ) , ( 54 - 91 )

Melanin ( BCM or BSM ) is a mixture of particles : properties and biological function may be linked to size, form,exterior and internal structure of the particle. Melanin exhibit two separate current-voltage characteristics, the on and off state. Experiments have demonstrated that their switching depends to hydration . Dried samples, don’t switch. Re-hydration restore switching properties ( 5 ) . Electrically conducting polymers can non invasively control the shape and the growth of mammalian cells ( 32 ). The particle has the property of binding organic products, drugs, ions, liquids and gases ( 33 ) .

Warning : School, journals, books, encyclopedias, dictionary, internet, forum, scientific meeting, pigment conference, etc. still quote descriptions of black matter ( BCM and BSM ) and melanogenesis which are not updated and are not in agreement with the most recent findings.

Relevant Papers with comment ( 1-81 )

1) ....melanin a bioelectronic mechanism in brain function....M.E.Lacy, Physiol.Chem.Phys.,13, 319,(1981).

melanin pigmentation in the skin has a protective value against the harmful effects of ultraviolet UV radiation....W.C.Quevedo et al., '' Light and skin color in Sunlight and Man , T.B.Fitzpatrick, M.A.Pathak, I.C.Herber, M.Seiji, A.Kukota, eds. pag.165-194, University of Tokyo Press,Tokyo (1974).

2) ……. Chemistry of sepiomelanin .Melanin from Sepia officinalis is present in the ink sac as the Mg and Ca salt….. M.Piattelli, et al., Tetrahedron, 15, 66-75, 1961; M.Piattelli et al., Tetrahedron, 18,941-949, 1962 ; 19, 2061-2072, 1963 ; R.A.Nicolaus et al. Tetrahedron, 20, 1163-1172, 1964.

3). H.S.Mason et al., Archives of Biochemistry and Biophysics (ABB)86, 225-230, 1960 ; T.Sarna et al., Science, 192, 1132-1134, 1976 ; K.Stratton et al., ABB, 123, 477-483, 1968 ; M.A.Pathak, ABB, 123, 468-476, 1968.

Sepiomelanin show a typical EPR ( about g = 2.003 ) signal which is similar to other BCM and BSM.

4) The ink secretion of Octopus contains compounds capable of paralyzing the olfactory sense of its enemies.

G.E.MacGinitie, N.MacGinitie, ''Natural History of Marine Animals'' MacGraw Hill,New York (1968).

5) .....pigmentary disorders are well known as in albinism, vitiligo, mongolian spot, melanoma .... P.A.Riley in '' The Physiology and Pathophysiology of the skin '' pag.1149-1167,A.Jarret ed.,AP New York (1974).

6) Melanins increase the optical efficiency of the eye, are responsible for production of color patterns, usually of adaptive significance, in the hair and or the superficial epidermis.

G.Prota ''Melanins and Melanogenesis'' pag.1, AP San Diego (1992).

7) Melanins are able to bind a number of foreign substances and to keep potentionally harmful substances which are released after in low,non toxic form.

N.G.Lindquist, Acta Radiol.,325,1-92, (1973) ; Uppsala J. Med. Sci., 91,238-288 (1986) ; Pigment Cell Research, 1, 133, (1987).

8) Melanins as a matrix for chemical evolution .

M.S.Blois in the ''Origins of Prebiological Systems'' pag 19. S.W.Fox ed. AP,New York (1965).

9) Melanin particle acts as a coloured amorphous semiconductor ,the colour depending to the amplitude of the prohibited Fermi's band.

B.J.R.Nicolaus et al., Atti Accademia Pontaniana, Vol.XLV, 365, (1997) ; Rend.Acc.Sci.Fis.Mat.,Vol. LXIV, 325, (1998) ; www.tightrope.it/nicolaus/index.htm

10) Neuromelanin as serving a biocybernetic function.Depletion causes the substantia nigra to revert from its neuroendocrine role to its original motor role .

F.M.Forrest, Adv.Biochem.Psychopharmacol., 9, 255, (1974).

11) Degeneration of substantia nigra is associated with Parkinson's disease .

E.Hirsch et al., 334, 345, (1988).

12) The binding of drugs aside neuromelanin suggest certain forms of chemically induced parkinsonism .

R.M.J.Ings, Drug,Metabol., Rev., 15, 1183, (1994)

13) Melanins from tetrahydroisoquinolines found in the brain of post mortem of Parkinson's patients are similar in dopamelanin regarding NADH oxidizing properties , oxy-radical scavenging activity, and ability to form soluble mixed polymers with melanins from opioid peptides .

L.Mosca, C.Blarzino, R.Coccia, C.Foppoli, M.A.Rosei, Free Radical Biology and Medicine, 24, 161, (1997).

14) Antimitotic and antibiotic activity is showed by certain allomelanin of bacteria and fungi.

S.P.Ljach et al., Akad., Nauk SSSR, Institut Mikrobiologii, Izd., Nsuka Mosckva, 1-86, (1972).

15) Melanin particle was discussed as a dark screen , lying beneath certain physical light-reflecting structures whose purely schemochromic colours it emphasizes merely by absorption of the longer, more deeply penetrating rays .

D.L.Fox, ''Animal Biochromes'' pag.222, Cambridge University Press, (1953).

(Link 5,6,9,12,19,21,22,)

16) Chordate melanins particle have a mainly protective function, e.g, in shielding underlying tissue from shortwave irradiation , in heat control, adaptive coloration. The significance in plants and the more primitive animals is still obscure .

R.H.Thomson in Comparative Biochemistry pag 727, N.Florkin, H.S.Mason eds., Vol.III, Part A, AP (1962).

17) Studies on the opticalspecificity of tyrosinases from different sources reveal the preference for D-isomer of tyrosine

Y.M.Chen, W.Chavin, Experientia, 23, 997, (1967).

18) Melanin particle forms 18-72% of the melanosomes.

J.Duchon, J.Borovansky, P.Hach, Pigment Cell, 1, 165, Karger,Basel, (1973).

19) Melatonin administered to the fish induces the change from day to night colouration .

A.B.Lerner et al., W.Mori, J.Am.Chem.Soc., 80, 2587, (1958).

20) Amorphous melanin is capable of absorbing ultrasound in the I-MHz range and converting this energy into a form which kills the tumor cells.

J.Mc Ginness, P.M.Corry, E.Armour, Pigment Cell, Vol.3, P.Riley ed. ,Karger Basel (1976). ; U.Mizutani et al., Nature, 259, 505, (1976).

21) Melanin exhibits the unusual characteristics of an amorphous semiconductor treshold switch.

J McGinness et al., Science, 183, 853, (1974). www.organicsemiconductors.com

22) All black materials are electroactive .

B.J.R.Nicolaus, et al., Atti della Accademia Pontaniana, Vol.XLV, 365, (1997); Rend.Acc:Sci.Fis.Mat., Vol.LXIV, 325, (1998) ; www.tightrope.it/nicolaus/index.htm ( Link 19 )

23) Explosive vaporization during pulsed laser irradiation of melanosome.

S.L.Jacques, D.J.Mc Auliffe, Photochemistry and Photobiology,53,769, (1991).

24) Melanin is found to be not volatile in mass spectrometry. www.tightrope/nicolaus/index.htm

25) Melanins natural or prepared by synthesis are amorphous semicoductors.

A.Pullman,B.Pullman, BBA,54,384,(1961); T.Strzelecka, Physiol. Chem.Phys. 14, 219-233, (1982)

26) It is shown further that the superconducting state of these polymers should be distinguished by certain unique chemical properties which could have considerable biological significance.

W.A.Little, Physical Review. 134, A1417, (1964).

27) Electrically conducting polymers could provide a noninvasive means to control the shape and function of adherent cells.

J.Y.Wong, Proc.Natl.Acad.Sci.USA, 91,3201, (1994)

28) The influence of UV radiation on collagen from rat tail in the presence of melanin was investigated .

A.Sionkowska, J.Photochem.Photobiol., 124, 91, (1999)

29) Effects of L-Cysteine on the oxidation chemistry of Dopamine.

F.Zhang, G.Dryhurst, J.Med:Chem. 37, 1084, (1994).

30) ........the doping effect on melanins particle is not known.........DHI-black, Adrenalin-black, Catechol-black are radical-polarone of hydrate polyquinones ......the counterion of melanin is not known........the chemistry of interstellar black matter is similar to the terrestrial black matter chemistry.......

www.tightrope.it/nicolaus/index.htm

31) ......dopachrome which is transformed by Sepia dopachrome-rearranging enzyme to DHI .......

A.Palumbo et al., Biochem.J. 299, 839, (1994) ; A.Palumbo et al. Biochem J., 323, 749, (1997)

Apparently sepiomelanin is similar to DHI- melanin.

32) Many dopachrome units are present in sepiomelanin.M. Piattelli et al., Tetrahedron, 19, 2061-2072, (1963)

33 ) R.A.Nicolaus et al., Atti Accademia Pontaniana , Vol. XLIX, 197-233, 2000 ; R.A.Nicolaus et al., Atti accademia Pontaniana, Vol. L, 225-243, 2001 ; A .Bolognese et al., Atti Accademia Pontaniana, Vol. LIII in press

The radical polarone system which form the melanin particle.Structure proposed for melanin oligomers taking in account the electric semiconductor property.The acetylene-black system is shown.

An o-quinone hydrate may be the monomer repetitive unit of DHI-melanin

- Three units of o-quinone hydrate three-dimensional arrangement is represented (Chemdraw software ver. 5.0))

- This unit satisfies the elemental analysis requirements for the DHI-melanin with polyindolquinone structure (Calculated for (C₈ H₂ N O₂): C%, 65.3; H%, 3.4; N %, 9.5. Found: C%, 56.3; H%, 3.0; N%, 8.2 (Beer)) taking in account the presence of larger amount of oxygen found and giving a rationale interpretation of the strongly linked water molecules invoked to justify the non-coherent analytical results.

-This hydrated quinone participates to stability and to planarize the unsaturated backbone of melanin and account of the radical-polarone structure related to the conductive properties of melanin.

Destruction of this hydrated structure pushes the indole rings about at 90° each from other and, determining a structural conformation changing, strongly affects the electric properties

34) ...... no melanins (BCM) derived from tyrosine are 5,6-polydolequinones......

www.tightrope.it/nicolaus/index.htm

35) Cyclodopa as precusor of eumelanin (BCM).

A. Bolognese et al., Atti Accademia Pontaniana Vol. L, 2001. Pontaniana.unina.it accponta@tin.it

Melanins obtained either enzymatically or by autoxidation of DOPA or Dopamine largely retain the hydrogen atoms of the methylene groups of the original side chains.

G.A.Swan, Ann.New York, Acad.Sciences, 100, 1005-1016, 1963

NMR spectra show always the presence of an aliphatic part. In BCM or BSM melanins.

(Link 5,6,9,12,19,21,22)

36) BCM ( Black Cell Matter ) and BSM ( Black Synthetic Matter ) have one oxygen more than their precursors.

www.tightrope.it/nicolaus/index.htm March 2003

37) ...... tyrosinase is the only enzyme of melanogenesis...... www.tightrope.it/nicolaus/index.htm March 2003

38 ) During the extractive process melanins (BCM) are oxidized by H₂O₂ contained in melanosomes or formed from atmospheric O₂

www.tightrope.it/nicolaus/index.htm March 2003

39) The red solution is called dopachrome. The solution is a mixture of quinones and phenols. Any substance or material which decolorizes the red solution is believed to be an enzyme.

www.tightrope.it/nicolaus/index.htm March 2003

40)………melanogenesis universal model for earth and space……… The WEB. Link 7

41) …optically active tetrameric melanin intermediates are discovered…… A.Pezzella et al. Tetrahedron Asymmetry 14,1133-1140, (2003)

42) Melanin particle ( L.Zeise, B.L.Murr, M.R.Chedekek, Pigment Cell Research, 5, 132-142, 1992 )

42 bis) Sepiomelanin is formed by mixture of oligomers species with molecular weight in the range of 500-1500 Da A.Napolitano et al., Rapid Comm. Mass Spectrom., 10,204-208, 1996 ; 10,468-472, 1996 ; A.Pezzella et al. Tetrahedron, 53, 8281-8286, 1997.

43 ) The black pigment from Ustilago maydis spores has been studied. Analytical data and degradation experiments show it to be a catechol-melanin.

M.Piattelli et al., Tetrahedron, 21, 1229-1236, 1965.

44) Melanin is not a polymer. Melanin is a particle ( L.Zeise et al., 5, 132-142, 1992 ) formed by radical-polarones of polyene systems the most simple one being acetylene-black.

Melanin is not a biological garbage as commonly believed.Melanin is one of the most important substance of living matter.

Some peculiar properties known until now are:

a. Sound and electrical conductivity ( BCM and BSM )

b. Storage of gas, water, liquids ( Earth and Interstellar spaces ).

c. Binding of ions and organic molecules

d. Conducting melanins ( BCM, BSM ) and control of adherent cells………

e. Form, size and structure of the particle, may change the chemical and physical data.

45 ) Melanin standard method : particle description.

L.Zeisel et al. Pigment Cell Research, 5, 132-142, 1992.

46 ) ….. the effect of preparation procedures on particle morphology of sepiomelanin……

Y.Liu et al., Pigment Cell Research, 16, 72-80,2003

47 ) ….¹³C and ¹⁵N NMR of sepiomelanin and sepiomelanic acid------------ B.Bhavin et al., Magn.Reson.Chem., 41, 466-474, 2003

48 ) Electrical conductivity of DOPA-melanin.

M.Jastrzebska et al. J.Biomater.Sci.Polym., 7, 577-586, 1995 ; G.M.R.Robinson at al., Electrochimica Acta,43,3489-3496,1998

Melanins conductivity and related papers.Research of pigment conductivity in the area of Biology is strongly recommended.Tecnique of conductivity determination in tissues are to be developed.

B.T.Allen, et al., in ‘’Free Radicals in biological systems ‘’ ,211,AP,NewYork,1961.

M.H.Brodsky, ‘’ Amorphous semiconductors ‘’ Springer,Berlin,1979 ; R.Bibang,et al.,Pigment Cell Res.,2,395-400,1989 ; 387-394 ; A.Berlin et al. Mat. Res. Soc. Symp. Proc. Vol.413 (Materials Research Society) ,1996 ; A.Berlin,et al.,Synthetic Metals , 84, 451,1997 ; A.Berlin et al . ,Tetrahedron, 52,7947, 1996 ; B.Commoner et al., Nature, 174, 689, 1954 ; F.W.Cope et al., Arch. Biochem. Biophys. , 100, 71, 1963 ; K.K.Darrow, Endeavour XIII N. 50 pp. 101- 106 ,1954 ; C.C.Felix, J. Am. Chem. Soc. 100, 3922-3926 ,1978 ; J.Filatovs et al . , Biopolymers 15, 2309, 1976 ; D.S.Galvao et al., J.Chem.Phys. 93, 2848- 2853,1990 ; E.P.Goodings, Endeavour XXXIV, 123- 130, 1975 ; S.K.Kurtz et al., Pigment Cell Res. 1, 261- 262,1987 ; Scientific Seminar of the Society of Cosmetic Chemists, Orlando,Florida USA ; Little et al. ‘’ Organic Superconductivity ‘’ pp.1-386,1990,PP,New York ; Scientific American 212, 21-27, 1965 ; H.C.Longuet-Higgins, Arch.Biochem.Biophys., 86, 231, 1960 ; McGinness et al., Pigment Cell Res.,2, 316, 1960 ; J.Theor.Biol., 39, 677-678, 1973 ; A.Menon et al., Can.J.Biochem., 55, 783-787, 1977 ; M.Okasaki, Arch.Biochem.Biophys.123, 197-205, 1985 ; A.Rabenau, Endeavour, XXV, 158-165, 1966 ; G.A.Pagani, Heterocycles, Vol.37, n° 3, 1994 ; D.Slawinska et al., Physiol.Chem.Phys., 14, 363-374, 1982 ; J.E.Simmons, Endeavour, XXVII, 138-143, 1968 ; L.J.Wolfram et al., J.Invest. Dermatol., 87s, 396, 1986 ; G.W.Zajac et al., J.Vac.Sci.Technol., 12, 1512, 1994.

49) Semiconductor properties of natural melanins.

T.Strzelecka, Physiol.Chem.Phys,14, 223-231, 1982.

50 ) Melanogenesis in the ink gland of Sepia.

A:Palumbo, Pigment Cell Research, 16, 517-522, 2003

51)…the color loci of mice……

D.C.Bennett et al., Pigment Cell Research, 16, 333-344, 2003

52) Melanins,melanocytes, melanogenesis : an illustrated history of the past half century

G.Prota, Pigment Cell Research, 13, 283-293, 2000 .

53 ) For the first time the sepiomelanin particle was described . Chemical Duke University , physical, biological properties of units and subunits would be of interest .Simon 2003, Duke University see text.

54 ) Electric and acoustic conductivity, electric switch, photoelectric conductivity, conductivity depending to preparation methods and doping .Melanins are biological material with striking large conductivity.

The semiconductor band model ( Link 9,17,21,22 ) parameters are :

EG = Optical band gap ( colours band , condudctor or insulating state) The same model has been qualitatively suggested for amorphous semiconductors.

BW= band width of valence band.

EA = electron affinità.High value indicated easy reducible materials.

IP = ionization potential . Low value indicated easy oxidable

Melanins have a little gap.

Melanins can be made from a poorly conducting to a highly conducting state at fairly low electric state i.e., from 10 K ohm ^–cm to 100 ohm ar a field of 300 V cm^-1 . ( McGinness et al., Science, 183, 853-856, 1974 ). The gap and conductivity of dopa-melanin and natural melanins was measured ( T. Strzelecka, Physiol.,Chem.,Physi., 14,219-222, 1982 ; 14, 223-231, 1982 ; 14, 233-237, 1982 ; M. Jastrzebska et al., Stud. Biophys., 122, 39-43, 1987 ; M.Jastrzebska et al., J. Biomater. Sci.Polym.Ed. 7, 781, 1996 ; 7, 577,1995 ; 33, 4023-4028, 1998 ; M.Jastrzebska et al., Gen.Physiol.Biophys., 9, 373-383, 1990 ; L.Zeise 1992 ; L.Hanyz et al., Cryst.Res.Technol., 38, 325-330, 2003 ; K.B.Stark et al., J.Phys.Chem.B., 107, 11558-11562 , 2003 ; V.Horak et al., Biorganic Chemistry, 21, 24-33, 1993.

Threshold switching in hydrated melanin was first reported by McGinness et al. in 1984.The American researchers opened a new area for switching studies by showing that low electric field switching occurs in organic semiconductors and in living system.This discovery was not taken in the right consideration by ‘’expert biologists ‘’. See the recent paper R.J.R.Nicolaus “ Neuromelanin “ Atti della Accademia Pontaniana, Vol. LIII,2004. and www.brunonic.org

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 mechanism.Double-pulse measurements add to the evidence that thermal effects dominate electronic effects in melanin.A pseudomemory was found in melanin ( C.H.Culp et al. J.Appl.Phys. 46,3658-3660 ,1975 )

55 ) Particle binding power for organic and inorganic materials, ions and molecules

( B. Larsson et al.,Biochem.Pharmacol., 28, 1181-1187, 1978 ; Pigment Cell Research, 6, 127-135, 1993 ; U. Mars, PHD Thesis, University of Uppsala 1998. ) a typical behaviour of the particle. Cosmochemistry.

56 ) Ability of store liquids and gases ( meteorites, humic acids, cellular oxygen, active charcoal ) . Cosmochemistry.

57 ) Active culture substratum controlling cell growth and form. (J.Y.Wong et al., Proc.Natl.Acad.Sci.USA, 91, 3201, 1994 ; D.E. Ingber, J.Cell Sci., 116, 1397-1408, 2003 )

58 ) explosive fragmentation by exposure to LASER ( S.L.Jacques et al.,Photochem. Photobiol. 53, 769, 1991.) occurs with melanosome.

MALDI spectra show complex pyrrole acids of obscure origin.

59 ) Behaviour as innovative sensor and energy generator with the capacity to grow and shrink significantly in length and volume when subjected to electric stimulation .Biosensors..( A. Berlin et al.,Chem.Mater.6, 1742-1748, 1994 ; A.Berlin et al., Tetrahedron 23, 7947-7960 , 1996 )

60 )

61 ) Sepiomelanin composition change with time in vivo and in vitro ( Link 5,6,9,12,19,21,22 )

62 ) Communication between tissues and between tissues and brain. ( Link ,2122, )

63 ) Presence of organic black matter in interstellar spaces ( Link 7,8 )

64 ) Primordial soupe ( S.W. Fox ‘’ The origins of Prebiological Systems and their molecular matrices ‘’ AP , New York, 1965 )

65 ) Melanin contribute to the blue,green,jellow physical colours formation in Nature ( Link 9 )

66 ) Some kinds of biological tissues generate fast electrical responses to intense light and both stable and unstable responses have been detected.The unstable response is known to be associated with visual pigments.The possibility that melanin may be responsible for the photostable electrical response of the eye ( no large photostable response in albino ) is supported by a study of electrical photoresponse from various tissues.

T.G.Ebrey et al. Nature, January 28, 360-362, 1967 .

67 ) The ink gland of the cuttlefish Sepia officinalis has been shown to contain a variety of melanogenic enzymes as tyrosinase, dopachcrome tautomerase, peroxidase.

A.Palumbo et al. Biochem.J., 323, 749-756, 1997

68 ) Sepiomelanin radical-polarone oligomers are formed from cyclodopa.

Generally melanins ( BCM and BSM ) are hydrate-quinones..That is melanins have one oxygen more in respect of the precursor.

The theory of quinone hydrate is published also on ATTI ACCADEMIA PONTANIANA

69 ) Quantitative determination of PDCA, PTCA, specific degradation products, carboxylic content, rate of ferricyanid comsumption,absorption spectra, MALDI experiments revealed that sepiomelanin consist of a mixture of oligomers incorporating over 75% of DHICA and 20% of DHI units occurring for the most part in the degraded pyrrole form.A.Pezzella et al., Tetrahedron 53, 8281-8286,1997

Since such a pigment would be colourless we are induced to suppose that new and strange oligomers bluilt up the black particle and a new melanogenesis must be suggest.

70 ) Polymerization of monomers into melanin indicate that uncyclised and carboxylated derivatives are not incorporated into the polymer in vitro.The paper clash with the above mentioned results.

V. J. Hearing et al., BBA 611, 251-268, 1980.

Since there are often relation between laboratory findings and biological data we conclude that no carboxylated units are present in BCM

71 ) It was found that about 96% of the CO₂evolved in the reaction DOPA-----Melanin arise from the breakdown of the benzene nucleous.G.A.Swan et al., J.Chem.Soc.381-384, 1954.

It would interesting to know if such a degradation process which remember that of Parkinson disease occurs also in the cell.

72 ) Composition of melanin samples depends on centrifuge speed

73 ) Binding property ( solid, liquid and gases ) of the black material is of interest for humic acids

74 ) MALDI spectra of DHI-melanin, DHICA-melanin, sepiomelanin are reported.

In contrast to the commonly held notion of melanins as high molecular weight polymers all the pigment analysed are mixtures of oligomers species with molecular weight in the range of 400-1500.A.Napolitano et al., Rapid Comm. Mass Spectrom., 10, 204-208, 1996 ; A.Napolitano et al., Rapid Comm. Mass Spectrom., 10, 465-472, 1996 ; A.Pezzella et al., Rapid Comm. Mass Spectrom., 11, 368-372, 1977

The MALDI spectra interpretations were a great surprise to the pigment cell researchers.

Although the peaks of the spectra do not correspond to oligomers but to degradation products of obscure origin it seems possible that the oligomers of low molecular weight are necessary to built up the particle.

75 )

The conclusions about the BCM (eumelanin ) structure in 1992

‘’ While dopachrome and leucodopachrome can be ruled out merely on the basis of their reactivety there is now fresh evidence that DHI and DHICA when allowed to co-oxidize under suitable biomimatic conditions give mainly mixtures of the corresponding homopolymers,in keeping with their markedly different redox pontential.Thus, eumelanins would show now more as mixtures of oligomers of DHI and/or DHICA, partially oxidized,rather than as intimate copolymers of various precursors. In conclusion , though many structural details of eumelanins are uncertain, new evidence is rapidly accumulating that is expected to lead to a fairly realistic picture of such unfathomable materials.Verification of this preview is expected in the very near future. ‘’.

G.Prota’’ Melanins and melanogenesis ‘’ , pag 118, AP, San Diego 1992

To be noted that dopachrome and leucodopachrome are very reactive substances and no evidence was given about the oligomers structure.

76. ) Pheomelanins ( hair, chicken feathers ) and pheochromes ( other names tricosiderins, thricochromes are derived from the new aminoacid 5-S-Cysteinil dopa ( CYSDOPA) : Chromophores are dibenzothiazines and dibenzothiazinones . The structure of the red hair pigment was established.

R.A.Nicolaus ‘’ Melanins ‘’ pag.190-199, in Methodicum Chimicum, Vol. 11, Part 3, Eds. F.Korte, M.Goto, AP,Tokyo 1978

Link 14 del WEB www.tightrope.it/nicolaus/index.htm

77. ) The pigmented life of a redhead

As a redhead I have had a personal interest in red hair, freckles and sunburns since childhood. An observation of a formaldehyde-induced fluorescence in human epidermal melanocytes initiated my scientific interest in these cells. Prota and Nicolaus demonstrated that oxidation products of cysteinyldopas are the main components of pheomelanin. Our identification of 5-S-cysteinyldopa as the source of formaldehyde-induced fluorescence of normal and pathological melanocytes started a series of investigations into this amino acid, enzymatic and non-enzymatic oxidation of catecholic compounds and the metabolism of thiols. All melanocytes with functioning tyrosinase produce cysteinyldopas and the levels of 5-S-cysteinyldopa in serum and urine are related to the size and pigment forming activity of the melanocyte population. The determination of 5-S-cysteinyldopa in serum or urine is a sensitive diagnostic method in the detection of melanoma metastasis. Some non-specific formation of cysteinyldopa is present in the body, as demonstrated by 5-S-cysteinyldopa in individuals with tyrosinase-negative albinism.

Rorsman H.

Department of Dermatology, University of Lund, Lund, Sweden.

hans.rorsman@derm.lu.se

(.April 2004 Pigment Cell Research )

1. Rorsman H. Pheomelanin, freckles, and cutaneous aging. In:Kligman AM, Takase Y, eds. Cutaneous Aging. Tokyo: Universityof Tokyo Press; 1988. pp. 193–200

2. Falck B, Jacobsson S, Olivecrona H, Rorsman H. Pigmented neviand malignant melanomas as studied with a speci.c .uorescence

method. Science 1965;149:439–440

3. Prota G, Nicolaus RA. On the biogenesis of pheomelanins. In:Motagna W, Hu F, eds. Advances in Biology of Skin, Vol. 8. New

York: Pergamon Press; 1967. pp. 323–328

4. Nicolaus RA. Melanins. Paris: Hermann; 1968

5. Bjorklund A, Falck B, Jacobsson S, Rorsman H, Rosengren AM,Rosengren E. Cysteinyl dopa in human malignant melanoma. Acta Derm Venereol 1972;52:357–360

6. Rorsman H, Rosengren AM, Rosengren E. Fluorimery of a dopa peptide and dopa thiethers. In:McGovern VJ, Russell P, eds. Pigment

Cell, Vol. 1. Basel: Karger; 1973. pp. 171–179.

6. Rorsman H, Rosengren AM, Rosengren E. A sensitive method fordetermination of 5-S-cysteinyldopa. Acta Derm Venereol

1973;53:248–250

7. Agrup G, Falck B, Jacobsson S, Rorsman H, Rosengren AM,Rosengren E. 5-S-cysteinyldopa in melanomas of Caucasians. ActaDerm Venereol 1974;54:21–22

8. Vogel CL, Dhru D, Rorsman H, Rosengren AM, Rosengren E.Dopa and 5-S-cysteinyldopa in malignant melanoma in UgandanAfricans. Acta Derm Venereol 1974;54:19–20

9. Agrup G, Falck B, Kennedy BM, Rorsman H, Rosengren AM,Rosengren E. Dopa and 5-S-cysteinyldopa in the urine in healthyhumans. Acta Derm Venereol 1973;53:453–454

10. Agrup G, Agrup P, Andersson T, Falck B, Hansson J-A, Jacobsson S,Rorsman H, Rosengrem A-M, Rosengren E. Urinary excretion of

5-S-cysteinyldopa in patients with primary melanoma or melanoma

metastasis. Acta Derm Venereol 1975;55:337–341

11. Rorsman H, Rosengren AM, Rosengren E. Determination of5-S-cysteinyldopa in melanomas with a .uorimetric method. Yale JBiol Med 1973;46:516–522

12. Aubert C, Rosengren E, Rorsman H, Rouge F, Foa C, Lipcey C.5-S-cysteinyldopa in diagnosis and treatment of human malignantmelanomas and ultrastructural observations. Eur J Cancer1977;13:1299–1308

13. Aubert C, Janiaud P, Rouge F,Hansson C,RorsmanH, Rosengren E.Melanogenesis in cultured human neuroblastomas. Ann Clin Res1980;12:288–294

14. Rorsman H. The melanocyte illuminated. Trans St Johns HospDermatol Soc 1974;60:135–141

15. Prota G, Rorsman H, Rosengren AM, Rosengren E. Phaeomelanic pigments from a human melanoma. Experientia 1976;32:970–971

16. Prota G, Rorsman H, Rosengren AM, Rosengren E. Occurrence of trichochromes in the urine of a melanoma patient. Experientia

1976;32:1122–1124

17. Prota G, Rorsman H, Rosengren AM, Rosengren E. Isolation of2-S-cysteinyldopa and 2,5-S,S-dicysteinyldopa from the urine ofpatients with melanoma. Experientia 1977;33:720–721;

Pigment Cell Res. 17, 2004.

18. Agrup G, Falck B, Hansson C, Rorsman H, Rosengren AM,Rosengren E. Metabolism of 5-S-cysteinyldopa by O-methylation.Acta Derm Venereol 1977;57:309–312

19. Hansson C, Agrup G, Rorsman H, Rosengren AM, Rosengren E,Edholm LE. Analysis of cysteinyldopas, dopa, dopamine, noradrenaline

and adrenaline in serum and urine usinghig h-performanceliquid chromatography and electrochemical detection. J Chromatogr1979;162:7–22

20. Hansson C, Edholm LE, Agrup G, Rorsman H, Rosengren AM,Rosengren E. The quantitative determination of 5-S-cysteinyldopa

and dopa in normal serum and in serum from patients withmalignant melanoma by means of high-pressure liquid chromatography.

Clin Chim Acta 1978;88:419–427

21. Hansson C. 6-Hydroxy-5-methoxyindole-2-carboxylic acid in normalhuman urine. Acta Derm Venereol 1984;64:185–190

22. Rorsman H, Agrup G, Hansson C, Rosengren E. Biochemical recorders of malignant melanoma. In: MacKie RM, ed. PigmentCell, Vol. 6. Basel: Karger; 1983. pp. 93–115

23. Agrup G, Edholm L-E, Rorsman H, Rosengren E. Diastereomers of5-S-cysteinyldopa. Acta Derm Venereol 1983;63:59–61

Pheomelanin,Pheochromes (tricochromes )

78) 1,4-benzothiazines as key intermediates in the biosynthesis of pheomelanins.

Napolitano A.
Pigment Cell.Res., 16, 575, 2003
University of Naples "Federico II", Complesso Universitario Monte S. Angelo, Department of Organic Chemistry and Biochemistry, Naples, Italy.

79 . ) Ultrafast absorption and photothermal studies of decarboxytrichochrome C in solution.

Ye T, Lamb LE, Wakamatsu K, Ito S, Simon JD.

Photochem.Photobiol.Sci.,2, 821-823, Department of Chemistry, Duke University, Durham, NC 27708, USA.

The trichrochromes are natural constituents of pheomelanins. Herein, time-resolved spectroscopic techniques are used to quantify the energetics and dynamics of the primary photoprocesses of trichochromes following excitation into the lowest excited singlet state of the molecule. The absorption spectrum of decarboxytrichochrome C (dTC) reveals multiple electronic states are accessible upon visible and UV excitation. Emission is not observed upon excitation into the lowest energy absorption band. Photothermal measurements reveal 91% of the photon energy is promptly released as heat. Femtosecond time-resolved absorption studies reveal a ground-state recovery time of approximately 2.4 ps. Complete recovery of the ground state is not observed; 15% of the initially excited molecules do not recover on the approximately 150 ps timescale. The combination of ultrafast absorption and photoacoustic data suggest photoexcitation produces a long-lived intermediate and the energy of this species is at least 133 kJ mol(-1) above the ground state of the dTC molecule.

80. ) 1,4-benzothiazines as key intermediates in the biosynthesis of red hair pigment pheomelanins.

Di Donato P, Napolitano A.

Pigment Cell Research,16, 532-539, 2003 , Laboratory of Biochemistry and Molecular Biology, Zoological Station Anton Dohrn, Naples, Italy.

For chromophores and the name pheochromes ( trichochromes are attributed to hair.Curious thing is that the pigment of red hair was extracted from the chicken feathers ) see Link 14 of the WEB, www.tightrope.it/nicolaus/index.htm

Link 14, reference 32