Word  Link 12 testo terzo.doc

LINK 12     The melanin in Sepia

 www.tightrope.it/nicolaus/index.htm

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  Eo. which  controls the dispersion  of the optical constant  k (imaginary part of the complex  index of refraction ) in an amorphous semiconductor ( 31b ).  Changes in Eo  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 H2O2  solubilized 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 CO2 from 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 /H2 O2  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

C20 H11 N3 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 KMnO4 and 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 H2O2      of 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 Ba3 was 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 KMnO4 and 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 H2O2      of the sepia pigment occurs whithout important physical modification.Melanin behaves toward H2O2 like a catalase.

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).

 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.or ciclodopa Pyrrole units are present.

 

 

 

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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. ASwan "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. 4th  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.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

.
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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 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.

 

 

 

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.