Word Link 12 testo terzo.doc

LINK 12 The melanin in Sepia

To M.Piattelli for his research on the melanin of Sepia officinalis and

to J. E. McGinness and P.A. Proctor who discovered the special electric conductivity of melanin

Melanin in Sepia

5,6-Dihydroxyindole (DHI) has been reported from biologist and chemist as the natural melanin precursor of Sepia arising from dopa--- dopachrome---- DHI reaction pathway. Oxidative polymerization of DHI yields the black pigment The black cell matter ( BCM, melanin ) is not a reducible substance but is sensible to oxidizing agents.Melanin binds a lot of organic and inorganic products, gas and ions, recalling the behaviour of charcoal. CO₂ and H₂O are lost by heating. Pulsed laser irradiation produces explosive fragmentation of melanosomes. Breakdown of melanin is observed in MALDI analysis.
Elemental analysis of natural and synthetic DHI-melanins with the corresponding methyl derivatives are reported . X-rays study may be related with fullerenic, graphitic or linear oligomers without distinction.

Natural and synthetic melanins ( 1-2 ) are still today considered polyindolequinones ( 6 ) , justifying the difference between the found and the calculated values of analysis by the presence of molecules of water. 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 ( 4 ),( 32 ). When cell maturation is complete, melanin is secreted into the lumen of the gland, accumulated into the ink sac and ejected on demand . 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. These enzymes are functionally interactive in close subcellular compartments of ink gland cells and appear to act in a concerted fashion during the process of melanogenesis in the mature portion of the gland.( 32 ) More recent studies have revealed that ink production and ejection are affected and modulated by the N-methyl-d-aspartate (NMDA)-nitric oxide (NO)- cyclic GMP(cGMP)signalling pathway . Glutamate NMDA receptor and NO synthase, the enzyme responsible for the synthesis of NO, have been detected by biochemical and immunohistochemical techniques in immature ink gland cells . Stimulation of NMDA receptors caused a marked elevation of cGMP levels, activation of tyrosinase and increased melanin synthesis in the mature portion of the gland, via the NO- guanylyl cyclase interaction ( 4 ), ( 32 ). This signalling is also present in different regions of the nervous system in Sepia and in certain neural pathways controlling contraction of the ink sac sphincters and wall muscle in the ejection mechanism. Overall, these and other findings allowed elaboration of an improved model of melanin formation in Sepia, which underscores the complex interplay of melanogenic enzymes and regulatory factors, highlighting both the similarities and the differences with melanogenesis in mammals.( 32 ).

The enzymatic equation can not be written being precursor and final product unknown.Dopachrome is only a red solution composed by phenols and quinones. Dopachrome would be yellow. Moreover synthetic and natural melanins ( BCM ) are chemically different, in enzymatic synthesis low yields are obtained , the natural pigment is in a degraded form ( 27 ), carboxylic group are present. Differences between DHI-melanin and the natural pigment are observed in IR, ¹³C NMR, MALDI spectra.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. Hydration reaction occurring before or after polymerization reaction stabilizes an oligomeric 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.
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 ). (10). A fragmentation which remember the LASER graphite fission.
. 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 : (organicsemicoductors.com ) ( 3 ).

IR, EPR spectra (1,2) and general chemical and physical behaviour are in agreement with a radical-polarone (radical-cation) conductive oligomers 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. (3).The broad IR spectra are typical of an amorphous material ( 10 ).The principal infrared absorption bands of the amorphous melanin 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 (16). As before mentioned 5,6-dihydroxyindole ( or dihydro derivative ) is the precursor in vivo of sepiomelanin but the chemical and physical ( ¹³C NMR) study always shows the presence of an aliphatic part in the polymer . Sepiomelanin is similar to the black pigment prepared in the laboratory from DHI in CO₂ evolution (from degraded units ) and methoxyl determination(5).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 (1 b, c, d), (2). It has been calculated that melanins contain one unpaired electron per 200-300 units. (14).Although the free radical represents only a minor part of the molecule (particle) it is of great importance for bio-physical studies (3), (15).Sepiomelanin and DHI-melanin methylated with diazomethane have similar percentages of -OCH₃ groups .

Even though in contrast with experimental data it is still believed that melanins are high weight polymers formed by self-condensation of 5,6-indolequinone producing polyindolequinones (1) (6) (7). However, from a biological point of view, a quinone would be not a pleasent host for the cell. The difference between the laboratory findings and the calculated values of the elemental analysis of melanin has generally been justified by the presence of H₂O, -COOH groups arising from breakdown of benzenoid part of indole units (1) (6). 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 correspond to the structure of the acetylene-black (PA cis) present in many BCM and BSM . Formation of indolequinone hydrate was observed in the polymerization process of dopammine or DHI (8), (11) 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 depend probably on the structure of the carbonyl compound. 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 (12), (13), (2), (2d), (2e), (2f) or according recent molecular mechanics calculations they may have an alpha-helical structure.Indole units 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 (17) on the 5,6-indol-di-one monohydrate at 5 position showed that linear dimer, trimer, tetramer, pentamer, etc. forms may assume two low energy conformations.

From MALDI experiment we learn :

Sepiomelanin is not DHI-melanin

Sepiomelanin is not DHICA-melanin

Sepiomelanin is cyclodopa (DOPA)-melanin

Cyclodopa (DOPA)-melanin spectra are unknown.

But we learn also that the experiment is not reproducible.

Melanins as derivatives of acetylene-black

Presumed oligomers for particle construction.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.

.«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 perterrefiunt

praesertim tamen sepia. ( Aristotelis Historia Animalium Lib IV, cap. I, 11 )

How to take the ink from a living cuttlefish is see in Fig.6

The ink sack ( see the coloured picture ).From R.A.Nicolaus ‘’ Biogenesis of melanins ‘’ Idelson Napoli 1962

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. Fig 1. A higly radioactive pigment was synthetised in vivo injecting labelled tyrosine, phenol, catechol, 5-oxytryptophane. 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.

The black material contained in the ink sack of squid is a suspension of melanosome in various stages of maturation. 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 (1).

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 methyester obtained treating the pigment with diazomethane was in form of an infusible light brown powder (30) The change of ‘’ colour ‘’ indicate that a physical ‘’ color ‘’ contributes to the black ‘’ colouration ‘’ of the particle In 1987 a unified physical model of pigments ‘’colour ‘’ was presented ( 31 ) ( 31a ). 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 dependant 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 dependant 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 ( 31b ). 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.

The DHI- melanin and sepiomelanin are sensitive (expecially from a nanochemical point of view) to oxygen, oxidants, halogens, pressure, light and pH. Pulsed laser irradiation produces explosive fragmentation of melanosomes and granules. Breakdown of melanin is also frequently observed in MALDI spectra (2),(2d). Heating produces a loss of CO₂ (from degraded units ) and H₂O which arise very probably from degraded oligomers.

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 of aliphatic residues in all blacks ( 20 b ). Similar NMR spectra were obtained with sepiomelanic acid, an H₂O₂solubilized pigment.

The presence of an aliphatic part in NMR spectra (20 a- f ), the presence of VI and VII units in the oligomers showed by isotopic studies (21 a-n), allow us to assign the carboxylic group in position 2 of I (1b). On the contrary literature consider the acid IV (DHICA ) an important precursor of eumelanins (BCM). The importance of IV ( ? ) . is supported ( ? ) by the fact that acid IV was found in nature as polymer. The reflecting material of the tapetum lucidum of the sea catfish contains a mixture of polymers of IV among which the tetramer VIII predominate.Synthesis of VIII was recently reported ( 23 ) .

The dark brown eumelanin ( BCM ) according to ( 22 ) is built up from DHICA units. But carboxylate units were never found in the course of melanogenesis (35)

Oxidative polymerisation of DHICA produces four linear trimers in eight atropoisomeric forms ; evidence for chiral nature of the oligomeric species was given.

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 ) (1-2 ) occupy an unique position among the natural pigments.like the electrical conductivity in amorphous semiconductors and the display of threshold switching .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.

It is well known the presence of BCM and melanin-concentrating hormome in animals brain (6 ), ( 25 ). In the biological field it is be noted that i mammals melanogenesis is the only non-enzymatic radical process occurring in vivo. A new ionization technique (MALDI) , recently made available , has extended the field of investigation for mass spectrometry (MS) 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, triptamine melanin and tyrosine melanin by enzymatic or chemical oxidation. Interesting results have been obtained with MALDI- MS : 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 ( 1a ) of low molecular weight (500-1500 Da ) arising from the breakdown of oligomers ( 22-24 ) .It is not clar if the molecular breakdown occurs also in vivo

SEPIOMELANIN

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

DHI

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

The study of dopamine melanogenesis, performed by MALDI-MS 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 .An extensive MALDI- MS study of the natural and biosynthetic melanins leads to the conclusion that all are formed by groups of oligomers with molecular weights between 500 and 30,000 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 futhemore produce degradation .In other words the study of melanin made until now has used synthetic samples. 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 :

:

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

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MALDI of sepiomelanin. Different type of samples from different treatments used. Adapted from A. Pezzella et al., Rapid Comm. Mass Spectrometry 11, 368-372, 1997

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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- MS verifies the presence of oligomers. ( For MALDI studies made at Padua see LINK 4 references ( 56-57 ), ( 60-61 ), ( 65-69 ) .

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 a MALDI which follows ).. 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 1500 ba , and, none of these corresponds to intact oligomere of DHI. Analysis of the molecular weights and the difference in masses gives a proof that significant quantities of polycarboxylic products which explain the CO₂ evolution by heating of the solid Analytical data compared with those obtained from natural sepiomelanin are presented in Table I.

Scheme I

Sepiomelanin oligomers degradation products ( 1a )

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Table 1

Sepiomelanin and DHI-melanin

Sepiomelanin (1a) 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 polymer 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 had 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 ( 1a ) thereby showing the presence of carbonylic functions as depicted in formulae.

The molecular weight of C₂₀H₁₇N₃O₁₅ (539) correspond to one of the peaks seen in the MALDI spectra (19)

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 II.

Trimethylamine and ammonia were found among the oxidation products of the methylated pigments. Trimethylamine was not found in oxidation of sepiomelanin (R.A. Nicolaus at al. Tetrahedron 20, 1163 –1172, 1964 ). Since trimethylamine forms during oxidation of compounds, having alfa-amino acid chains previously methylated with diazomethane, it is to be supposed that , different from sepiomelanin, --CH (NH₂ ) –COOH groups, are present in melanin from rat- tumor and human hair ( presence of proteic material is possible ) . This would appear to be borne out in Swan’ s work (G.A.Swan, Ann. New York Ac .of Sciences 100, 1005, 1963 ) on the autoxidation of VI which has been deuterated in alpha- position

The pigment was extracted and purified as before reported ( LINK 22 pag.8 ) Oligomers of different type ( 8 ), including pyrrole units, form round particles which are stable in structure ( LINK 22 references 42-47 ), ( 10 ), ( 11 ).

The pigment has the property of binding organic products, drugs, ions and gases.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 ( 1a ), ( 2g ) ( Scheme I ).A similar compound appears in MALDI experiments (19 ) 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.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 (28), (29) 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. Pyrrole units are present.

Elemental analysis of sepiomelanin in TableII reflect the degradation state of the samples used .It is not clear if these degradation process occur in the cell.

Literature oft report or conclude that sepiomelanin is a DHI-melanin and melanoma-melanin is a DHICA-melanin

To a more complex degradative compound obtained as barium salt the formula C₂₀ H₁₁ N₃O₁₅ Ba₃was given (1a) (2g). A similar compound is obtained in MALDI experiments (19).

.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 (18). Electrically conductivity may be also related to shape and size of the particle and method of synthesis of BCM and BSM.

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 (2), (2e), (2f).

Contrary to what previously reported the pigment particles found in the ink sack of the Sepia officinalis are formed from DHI units in agreement with biological studies (4), (32 ) (35).

Some units are degraded ( fission of the benzenoid part ) with formation of carboxylic groups and pyrrole rings.CO₂ evolution by heating of the pigment derive from the fission of the benzenoid part of the molecule.

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 .

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Lettera con I risultati sperimentali ottenuti con la tecnica di spetroscopia MALDI

To be translate

See Link 22 reference (139)

I receive and I report

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 (1) 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 (2), (3). In 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 (5), (6), (7), 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 (8) (3) (4) (32) 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 (9) oppure ( 139 ) del Link 22.. Lo spettro non mostra picchi molecolari relativi a prodotti di degradazione o picchi a peso molecolare elevato (10). La mancanza di ioni molecolari della melanina e’ in accordo con la nota sensibilità della particella alla radiazione LASER(11).

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 ( 35 )(12), (13)

A.Bolognese

Dipartimento di Chimica Organica e Biologica

Via Mezzocannone 16, 1-80134 Napoli.

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

Bibliografia

1).H.S. Raper Biochem J. 21,89, 1927

2). A. Palumbo et al., Biochem J. 299,839,1994.

3). A.Palumbo et al., Biochem.J.,323,749,1997

4). M.Piattelli et al., Tetrahedron, 15, 66,1961

5). A.Napolitano et al. Rapid Comm.,Mass Spectrometry, 10, 468, 1996

6). A.Pezzella et al. Tetrahedron,53,8281, 1997.

7). A.Pezzelle et al. Rapid Comm.Mass Spectrometry, 11, 368, 1997

8). M.Seiji et al. Nature 197,1082, 1963

9). C.Kroesche et al. Tetrahedron, 52, 3947, 1996.

10). L.Seraglia et al., Biol.Mass Spectrometry, 22, 687, 1993

11). http://www.tightrope.it/nicolaus/index.htm

12). H.S.Mason The Pigmentary System : Advances in Biology of Skin pag.293-312. Eds. W.Montagna,F.Hu, Pergamon Press,Oxford 1967

13). H.S.Mason,Pigment Cell Biology , pag. 563-582, Ed. M.Gordon, AP New York 1959

35 ) see 35 of general Bibliography

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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 II.

B The case of sepiomelanin is relevant.The most studied natural black substance, for the ease of access to it at the biological source, is the melanin which can be extracted from the ink sack of the sepia and is called sepiomelanin. The extraction of sepiomelanin presents great difficulties not only because of its insolubility.
The ink of the sepia is in fact constituted by a mixture of melanosomes, premelanosomes, (in which oxidative enzymatic activities are in act with the rearrangement, transferring and elimination or creation of atomic and radical groups) and granules of stabilised pigments. One thinks of diffusion and therefore of the presence of enzymes like laccase, tyrosinase, peroxydase, tautomerase (48). And for this reason 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 principle modification would seem to operate at the level of the dopachrome (2e) (8) 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 (8a). 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 to date it is possible to conclude and summarise:
The melanins are polyquinones in a hydrate form.They are characterised by a radical-polaron system with stable unpaired electrons. Such a system is present in DHI-melanin 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.
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).(12), (14), (15).

In conclusion sepiomelanin is different from others melanins suggesting that it is a largely degraded DHI-melanin.or ciclodopa Pyrrole units are present.

-------------------------------------

O.Diphenol formation from phenol without enzyme.

From W.Brackman, E.Havinga, Rec.Trav.Chim.des Pays-Bas 74,1107, (1955).
It was found that the primary reaction consists in the introduction of a hydroxyl group into phenol.This reaction occurs within a complex of copper with morpholine, phenol and hydrogen peroxide. The catechol formed is oxidized further either by oxygen or by a cupric-morpholine complex to give ortho-benzoquinone. This takes up morholine to give the morpholinocatecol, which is subject to a rapid autoxidation to a morpholino-ortho-benzoquinone. The addition of a second morpholino molecule followed by another autoxidation gives rise the main reaction product: dimorpholino-ortho-benzoquinone.The hydrogen peroxide necessary for the primary attack upon the phenol originatfrom the autoxidation of the catechol formed. The primary reaction occuring in the hypothetical copper-morpholino-phenol-hydrogen peroxide complex explains the specific ortho-oxidation as well as the exclusive activity of copper in these catalytic oxidations.

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Sample preparation of melanin from biological sources based on laboratory experience with Sepiomelanin

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, 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 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 x3, CH₃COCH₃ x3, H₂O x3, 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 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 ( 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 x3, H₂O x3, in centrifuge, obtaining (B), the sepiomelanic acid.
The sepiomelaininc 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 (43).
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 (5).
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). Typical broad IR, ¹³C NMR, MALDI spectra are to be compared with DOPA-melanin and DHICA-melanin.

Extraction of melanin from biological sources

The dried fresh material was treated with 5% ammonia solution and few mg of sodium bisulphite.Sometimes sonication ( see sepiomelanin) is necessary.The cooled yellow-brown solution was acidified with conc. hydrochloric acid to Congo red , centrifuged and dried at room temperature.

IR, MALDI, ¹³C NMR spectra, analysis for C, H, N, S, Fe, Cu, Ni, Cd, C are compared with those of DOPA-melanin and DHICA-melanin.The storage of gases and the bindig of foreign substances are determined .

Oxidative degradation of natural pigments : identification of 2,3,5-pyrroletricarboxylic acid

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 portion 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 ((4x2.5ml). 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°.

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.

Board of Consultants

yorick@inwind.it ( computer model )

Dr. A. Bolognese (Organic Chemistry).

bologne@cds.unina.it

Dr. B. Nicolaus (Preparative Biochemistry).

bnicolaus@icmib.na.cnr.it

Dr. B. J. R. Nicolaus (Neuropsycho Pharmacology).

Dr. R. Nicolaus (Melanin Chemistry).

rnicolaus@tightrope.it

gparisi@cds.unina.it (biochemistry)

BIBLIOGRAPHY

1) a.L.Panizzi,R.Nicolaus,Gazz.Chim.Ital. 82,435, (1952); b.M.Piattelli,R.A.Nicolaus,Tetrahedron 15,66-75,(1961); c. 18,941,(1962); d.19,2061, (1963) ; e. R.A.Nicolaus, Rassegna di Medicina sperimentale,Anno IX, Supplemento 1,Ed.V.Idelson, Napoli 1962

2) a.R.A.Nicolaus,Rend.Acc.Sci.Fis.Mat.,Vol.LXIX,325,(1997);b.B.J.R.Nicolaus,R.A.Nicolaus,Atti della Accademia Pontaniana, Vol. XLV, 365,(1997); c. G.Nicolaus,R.A.Nicolaus, Rend.Acc.Sci. Fis. Mat. Vol. LXVI, 131, (1999); d. M.Olivieri, R.A.Nicolaus, Rend.Acc. Sci. Fis. Mat., Vol. LXVI, 85, (1999); e. R.A.Nicolaus, G.Parisi, Atti della Accademia Pontaniana, Vol.XLIX, (2000); f.A.Bolognese, R.A.Nicolaus,Atti della Accademia Pontaniana,Vol XLIX (2000); www.tightrope.it/nicolaus g. R.A.Nicolaus, Rend. Acc. Sci. Fis. Mat., Vol. LXIV 315-324 (1997).

3) J.E.McGinness, P.H.Proctor, J.theor.Biol.,39,677,(1973); J.McGinness, J.Corry, P.Proctor, Science, 183, 853, (1974); P.H.Proctor, J.E.McGinness, P.M.Corry,48, 19, (1974); U.Mizutani, T.B.Massalski, J.E.McGinness, P.M.Corry, Nature, 259, 505, (1976). http://www.organicsemiconductors.com/

4) A.Palumbo, M.d'Ischia, G.Misuraca, L.De Martino, G.Prota, Biochem.J. 299,839, (1994)

5) R.J.S.Beer,T.Broadhurst, A.Robertson, ‘’ The chemistry of melanins. Part V . The autoxidation of 5,6-dihydroxyindoles ‘’ J.Chem.Soc, 1947-1953, (1954)

6)K.Hempel ‘’ Investigation in the structure of Melanin in malignant melanoma with 3H-and 14C-DOPA labelled at different positios ‘’ Symposium on structure and control of the melanocyte , Sixth International Pigment Cell Conference, SV Heidelberg, 1966 ; G.Prota, Melanins and Melanogenesis, Academic Press, San Diego (1992).

7) D.S.Galvao, M.J.Caldas, J.Chem.Phys., 88,4088,(1990).

8) C.Kroesche, M.G.Peter, Tetrahedron, 52,3947, (1996).

9) The percentages of C H N is to the method of samples preparation depending.

10) Private communication of Professor S.Califano ( Fi ).

11) A.Bertazzo,C.Costa,G.Allegri,R.Seraglia, P.Traldi,Rapid Comm.,Mass Spectr.,9,634,(1995).

12) J.Cheng, S.C.Moss, M.Eisner, Pigment Cell Research,7, 263,(1994) ; G.W.Zajac, J.M.Caldas, J. Cheng, M.Eisner, S.C.Moss, A.E.Alvarado-Swaisgood, BBA, 1199, 271, (1994).

13) M.G.Bridelli , Biophys.Chem., 73, 227, (1998).

14) M.S.Blois, A.B.Zahlan, J.E.Maling, Biophysics J., 4,471, (1964).

15) T.Sarna, I.A.Menon, R.C.Sealy, J.Photochem.Photobiol., 39, 805, (1984); T.Sarna, 12, 215, (1991).

16) G.Tollin, G.Steelink, BBA,112,377 (1966).

17) Private Communication of A. Bolognese

18) J.Y.Wong, R.Langer, D.E.Ingber, Proc. Natl. Acad. Sci. USA, 91, 3201, (1994).

19) A. Napolitano, A. Pezzella, G. Prota, R. Seraglia, P. Traldi, Rapid Comm. Mass Spectrom. 10, 204-208, 1996 ; 10, 468 (1996).

20) C.Lambert et al. ,BBA 993, 12-20 1989 ; G.A.Duff et al., Biochemistry, 27,7112-7116, 1988 ; M.G. Peter et al., Angew.Chem.,Int., 28, 741-743 ,1989 ; S.Aime et al., Gazz.Chim.Ital. 120, 663-664, 1990 ; S.Aime et al., Pigment Cell Research, 4, 216-221, 1991 ; M.Hervè et al. BBA, 1204, 19-27, 1994.

21) a. G. A. Swan "Chemical Structure of Melanins" Annals of the New York Academy of Sciences Vol. 100, 1005 (1963)

b. F. Binns, G. A. Swan "Oxidation of some Synthetic Melanins" Chemistry and Industry 396 (1957)

c. G. A. Swan "Some studies on the formation and structure of melanins" Rendiconti Accademia Scienze Fisiche Matematiche, Vol. XXXI (1964)

Studies are described of the formation of melanins in vitro enzymically and by oxidation from dopa and from dopammine. When these precursors were labelled with deuterium in the alfa and beta positions of the side chain, and then converted into melanins large retention of deuterium in melanin was observed. This means that melanins are not DHI-melanins.

d. S. N. Mishra, G. A. Swan "Studies related to the chemistry of Melanins. -Part II- Synthesis of 5,6-Dihydroxyindoline" Soc. 1424 (1967).

e. S. N. Mishra, G. A. Swan "Studies related to the chemistry of Melanins -Part V- Investigations on the Specific Deuteriation of 5,6-Dihydroxyindoline and 5,6-Dihydroxyindole" Soc. 1431 (1967).

f. F. Binns, J. A. King, A. Percival, N. C. Robson, G. A. Swan "Studies related to the Chemistry of Melanins -Part IX- Syntheses of Specifically Deuteriated 3,4-Dihydroxyphenethylamines and (simbolo 177 \f "Symbol" \s 12) 3,4-Dihydroxyphenylalanines" Soc. 1134 (1970).

g. F. Binns, J. A. King, S. N. Mishra, A. Percival, N. C. Robson, G. A. Swan, A. Waggott,"Studies related to the Chemistry of Melanins -Part. XIII- Studies on the structure of dopamine-melanin" Soc. 2062 (1970).

h. G. A. Swan "Structure Chemistry and Biosynthesis of Melanins in Fort. Chem. Org. Natur. Vol. 31, 522, (1974) Springer-Verlag, Wien 1974

The experiments show that 20% of the polymer units are formed by DOPA and cyclodopa units either in the autoxidative or enzymatic process. This value was cofirmed by (58) by NMR.

i. G. A. Swan '' Current knowledge of Melanin Structure '' In Pigment Cell,Vol. 1, Eds. V. J McGovern,P. Russel. S. Karger,Sydney, (1973), pag. 151.

If the Raper Scheme were correct it would be expected that the same melanin would be obtained from tyrosine, DOPA, dopammine,DHI. . Although these melanins have the same radical- polarone system, they are,in part, chemically different.

l. J. A. King, A. Percival, N. C. Robson, G. A. Swan "Studies related to the Chemistry of Melanins -XI- The distribution of polymeric linkages in dopamelanin" Soc. 1418 (1970).

m. G. W. Kirby, L. Ogunkoya "Structure of melanin derived from -3,4 -dihydroxy- 1 - (14C, 3H) -phenylalanine by oxidation with tyrosinase" Soc. Chem. Comm. 21 546 (1965).

n. V. J. Hearing, T. M. Ekol, P. M. Montague, J. M. Nicholson "Mammalian Tyrosinase Stoichiometry and measurement of reaction product" BBA 611, 251 (1980).

The authors reported that there is no incorporation of DOPA, dopachrome, leucodopachrome (cyclodopa), DHICA into dopamelanin. . In other words in melanins the carboxylic group is not present and melanins are pure DHI-melanins.

22). A.Pezzella et al., Tetrahedron, 58, 3681-3687, 2002.

23). A.Pezzella et al. Tetrahedron : Asymmetry, 14, 1133-1140, 2003 ; S.I t o et al., Biochem.J.,, 143,207-217, 1974.

24 ) . A.Pezzella et al., Rapid Comm. Mass Spectrometry, 11, 368-372, 1997.

25 ) . D.J.Bird et al., Gen.Comp.Endocrinol., 121, 232-241, 2001

26). A.Pezzella et al., Rapid Comm., Mass Spectrom., 11, 368-372, 1997.

27). A.Pezzella et al., Tetrahedron,53, 8281-8286, 1997.

28). Z.E.Jolles, Chemistry and Industry, 845-846, 1953.

29). A.Quilico ‘’ I pigmenti neri animali e vegetali, 1-178, Ed.FUSI, Pavia 1937

30). M.Piattelli et al., Tetrahedron, 15, 66-75, 1961

31). S.K.Kurtz, L.Albrect, T.Schultz, L.Wolfram ‘’ The physical origin of colour in melanin pigment dispersion ‘’ Communication presented at the first meeting of The European Society for Pigment Cell Research held in Sorrento October 11-14, 1987.

a). S.K.Kurtz, S.Kosikowski, L.J.Wolfram, J.Invest., Derm., 87, 401A, 1986

b). K.Nassau ‘’ L’origine dei colori ‘’ in Le Scienze quaderno n° 21,1985, pag.59-72

32 ) A.Palumbo ‘’ Melanogenesis in the ink gland of Sepia officinalis‘’ Pigment Cell Research, 16, 517-522, 2003.

33 ) Unpublished results

34 ) J.Borovansky et al., Cell Biology International Reports, 1 n° 6, 1997 ; L.Zeise et al. ,Pigment Cell Research, 5, 132-142,1992 ; Suppl.2 , 48-53, 1992 ; C.M.R.Clancy et al., J.Phys. Chem B, 104, 7871-7873, 2000 ; Biochemistry 40, 13353-13360, 2001

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

TABLE II

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, Compt.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)

Under construction

The Story of Melanin in Sepia

Corrections and suggestions wellcome

Melanins were considered polyindolequinones derived by enzymatic oxidation of tyrosine ( Raper 1928 ).

Pyrrole-2,3,5-tricarboxylic acid was isolated from the oxidative mixture of sepiomelanin ( Panizzi 1950 )

Many dopachrome units were found to be present in sepiomelanin polymer

( Piattelli 1960 ).

Electrical properties of sepiomelanins , a bistable switch melanin ( Proctor 1974,1976 ), ( Crippa 1978,2001 ), ( Strzelecka 1982 ), ( Jastrzebska 2002).

The sepiomelanin particle morphology ( Crippa 1977, 1986 1990, 1998 , 1999 ), ( Aime 1991 ) ( Bridelli, 1998 ), ( Simon 2000, 2003 )

Trihydroxyindoles and melanochromes appear in the MALDi spectra Graham ( 1977 ), ( Allegri 1993,1995 ) ( Peter 1996 ).

The melanogenesis of the ink gland ( Ortonne 1981 ).

Sepiomelanin is a particle and not a polymer (Chedekel 1992 )

Sepiomelanin particle is formed by oligomers of low molecular weight

( Prota 1996,1997 ).

Sepiomelanin samples contain many localised free radicals ( g= 2,007 ) ( Bowers 2003 )

Sepiomelanin news : http://tightrope.it/nicolaus/indrx.htm

Naples Feb 2004.

Octopus, squid, cuttlefish ink is ejerted to decrive attacking fish. The Ink is a black suspension of particles

Fig. 1 Fig. 2

Melanin particles are contained in branched cell called chromotophores (melanophores) of the 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 (fig 1 and 2).

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 (the spine) 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.

Sepiomelanin particle are built up from DHI and DHHCA as showed or formed by cyclodopa and decarboxy cyclodopa units.

The particle may assume the fullerene or a graphitie form.

Sepia particles

Old but the best book for students interested in natural pigments. An original feature of the book is the inclusion of instruction for practical clam work. This book gives a concise but comprehensive account of animal colors physical, chemical and biological standpoints.

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

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.

.
.

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

The authors wish to thank Miss R. Liotto and Miss C. Di Somma ( Library of the Zoological Station of Naples) for assistance in the bibliography research , Dr. A. Alberti (CNR-ICOCEA, Bologna ) for EPR spectra and finally Dr.A.Palumbo for interesting biological talk.