www.tightrope.it/nicolaus/index.htm 

In the second half of the last century despite being there intense the study(1-7) of synthetic and natural melanins this did not lead to good results.  Structurally, physically and functionally our knowledge of the melanins developed in a misleading way.  There are several  reasons for this and many considerations may be made.   Here, we recall a few.

Chemical and biological research is useful for the study of crystalline substances, of ordered polymers, of natural organic substances and not for an amorphous material.  The belief that the melanins were polmers created a series of false problems like that of the positions involved in polymerisation or the problem of the homogenous and heterogenous polymers.  It was said: the question is whether melanin is a regular, polymer of 5,6-indolequinone (poikilopolymer), or whether  it is a random polymer consisting of several different monomers which are coupled by means of various  bond types. 

In  itself this question contains a number of rather special queries, some of which seem of fundamental  importance. 

Under the principle of biochemical unitarianism which had proven so fruitful, it was  tempting to assume from the start that melanin, which is clearly an unusual biological''  polymer '', is synthesized in a manner similar to all biological polymers, That is, its  synthesis is controlled  by enzymes which are  specific for  substrate and/or bond type.  Despite the complexity of the nucleic acids, proteins, and polysaccharides, they obviously all have a high degree of order or of non- randomness.  These polymers are each characterized  by a single type of linkage,  the phosphate  ester bond, the peptide bond and the  glycoside  bond respectively.  If  we follow the unitary  principle then,  melanin  too  should have one  characteristic type of linkage which couples the monomers together.  As to the nature of the monomer, the Raper-Mason scheme, as presented in the past,  suggested that it is indole 5,6-quinone.  Perhaps an extreme statement of the  homopolymer view would then  be that  melanin  consists of 5,6-indolequinone linked through  a single bond type(1).  This concept requires no special assumption other than that  the premelanosome  contains  the necessary enzymes to carry tyrosine through  the Raper-Mason scheme, and to direct the polymerization  of the indole 5,6-quinone.

The concept of the polyindolequinone structure for BCM dominated the scientific scene of the past half century

The centesimal values for a polyindolequinone structure  (C8H3O2N) as formed following the  Raper-Mason scheme (an oxidative product of DHI  formed from tyrosine) are:

C%   66. 2   H%   2. 1   N%   9. 6

The DHI-melanin analysis (R. J. S. Beer, T. Broadhurst,A. Robertson, '' The chemistry of melanins-Part V-The autooxidation of 5,6-Dihydroxyindoles '' Soc.  1947 (1954) gave  

C%   56. 6   H%   3. 1   N%   8. 2

which approximates to the formula  (C8 H5 O3 N)

Surprising the values of BCM  were  different as for the ink of cephalopds

C% 54. 3  H%  2. 9  N%  8. 8

(J. P. Ortonne, C. Voulot, C. Khatchadourian, A. Palumbo, G. Prota '' A reexamination of melanogeneis in the ink gland of cephalopods '' in Pigment Cell 1981 page 49-57, Ed. M. Seiji, University of Tokyo Press, 1981 but may approximate the centesimal value for C8H3O2N (depending of physical methods of purification adopted) (62a), (62b).

From the start it was clear that :

a)     The  black material obtained from tyrosine  was different to the black material obtained by oxidation of 5,6-dihydroxyindole (DHI)

b)     Natural melanins  (BCM) were not polyindolequinone (C8H3O2N).

c)     In the oxidation of 5,6-Dihydroxyindole (DHI) the polymer itself must be much more complex. In the oxidative process CO2 is evolved and H2O2 is formed so that there may be  some very complex reactions which are not showed in the Raper-Mason scheme.  In addition the black material cannot be reduced by aluminum hydride or Zn+HCl.  If it were a quinone it could be reduced.

.  Yet general methods for the solubilization of natural melanins under physiological conditions have not yet been found, excepting mild action of H₂O₂ in alkaline medium with transformation of the original structure (opening of the benzenoid part).  Once these other biopolymers  have been solubilized, they may be hydrolyzed under physiological conditions with the use of specific enzymes, or under quite mild conditions  of acidic or basic hydrolysis.   Although melanin -in common with any organic  substances may be degraded in mild conditions by H₂O₂, or photooxidatively  degraded by ultra violet light, in both cases the degradation products are formed with low yields   It is interesting that the colour changes  (black, reddish, gold yellow) during the H₂O₂ oxidation corresponds to those of some inorganic sulphides (8).  The colour depends on the gap amplitude of the semiconductor model (8), (27d).   (Fig.  1).

It is now amply confirmed, by EPR, that natural and also synthetic melanins are paramagnetic  and that they are paramagnetic because they contain unpaired electrons within their molecules (see acetylene-black) and are amorphous semiconductors : (www. organicsemiconductors. com ;  www. tightrope. it/nicolaus/index. htm ), (27 a-d), (27h), (27l) (27m).  The EPR spectra of natural melanins (BCM) and synthetic material  (BSM) are quite similar. The EPR spectra  cannot distinguish between these pigments which we know are in some ways  chemically different (catechol-melanin, dopa-melanin,cyclodopa-melanin, DHI-melanin), that is the EPR spectra are of no relevance to the melanin problem. 

It is our view that the EPR results raise significant questions : first the EPR signals (electrical conductivity) which one invariably observes  in BCM and BSM, are not seen in any other biochemical compounds, or in tissues or organisms not containing melanin. 

Thus melanins (BCM) seem to be unique among the costituents of living organisms  in that they exhibit  intrinsic semiconductivity and have charateristic band gaps 1,2-1,7 eV (black of the semiconductor model), gas-storage properties and particle structure.  All this  eventually requires an explanation  in terms of  molecular  structure. 

Some theoretical studies reported in the past (63) are interesting for understanding the electronic structure and statistical properties of quasi-particles in polymeric materials that is solitons, antisolitons, polarons, bipolarons,. Polyacetilene black was used to study the distortion associated with quasi-particles in oligomers and infinite polymers.

The radical-polarone (cationic) system seems to be present in all the BCM and BSM until now examined. The models explain a number of properties like optical, magnetic, electrical, chemical assembling of oligomers.  (27 b-d).  The description of neutral and charged defects in conjugate polymers is based on Huckel calculations which have quantitative limitations.  More sophisticated treatment of neutral and charged defects in polymers differ from Huckel description in quantitative but not in qualitative conclusions. Whereas Huckel treatment  of a radical defect on polyacetylene yields a distortion of the lattice extending about seven carbon atoms on either side from the defect, detailed calculations yield an entity localised on three carbon atoms. The difference has no important consequence in the application of the theory to the description of defect formation, trasport in acetylene-black and limits of the applicability of the soliton model.

Secondly the EPR absorption and other properties which melanin alone appears to have in living organisms are found abundantly among products of non-living  systems (BSM) including probably black interstellar matter (27f), (27g).  

It is partly because  of these findings that we have seriously entertained the concept that melanin  itself  may be formed through a free radical method and without the intervention of enzymes (except the first step), leading to the formation of an amorphous material formed by black particles. 

Fig  1.  The yellow cadmium sulphide has a gap 2,6 eV the black cadmium 1,6 eV. Colour depends to the gap value.Inorganic pheomelanin and melanin.

Study concentrated more on the molecule  than on the particle leads us to the big scientific mess of the past century.  A long time is now required to get out from this difficult situation but sooner or later the truth will come to light.   Our ignorance of nanoscience was a negative element for our understanding.  Isolated attempts to introduce the concept of particle for the melanins were made in 1992 only.  (9).

Melanosome are acid-resistant hydrolysis (60) but easily degraded by physical ( LASER , MALDI, MALDI-TOF ) or chemical ( H2O2 ) methods.

Extraction of pigments is a difficult task. Many different methods are used (3), (4), (9), (16), (31), (67), (76), (77) but none can be recommended.

BCM is made up of small oligomeric units  (43-47), (11), (10),  and the structural morphology reflects the aggregation of  oligomeric molecules. 

Even though models of melanins as substances operating according to “solid state” mechanisms were proposed in the last decades of the last century, the biological implications of such ideas (9), (15-16) (20),  did not receive much attention because of the scarcity of reliable experimental confirmation and also because of the difficulty of accepting a somewhat unconventional point of view for the biochemical-physical sciences.  In more recent years, the study of the structure of the particles of melanin isolated from the cell or prepared synthetically (28), (29) has confirmed that both their photophysics and their photochemistry (17-20) need to be explained in terms of aggregated solids in which the geometrical and physical characteristics play an essential role in understanding the physiology of the pigment at a molecular level.

The results of x-ray diffraction studies (21-25) on granules of melanin are limited to confirming their amorphous structure in which there are ordered regions, and to the rather speculative existence of a sort of “fundamental unit” (26).  More recent studies of tunnel effect microscopy  have confirmed the previous results and introduced the concept of a heirarchical structure in the process of self-aggregation.  The electrical conductivity of a black substance may be notably increased by doping (on this see acetylene black and pyrrol black as examples) (28-30).  Melanins produced synthetically and isolated from biological systems act as amorphous semiconductors with threshold switching.  Switching is reversible at potential gradients two to three orders of magnitude lower than reported  for inorganic thin films  and comparable to gradients existing in some biological systems.   melanins give off a flash of light when they switch (electroluminiscence).  Of a number  of other biological materials  tested, only cytochrome C acted similarly, but at the high potential gradients reported for thin film  amorphous semiconductors.  (Science, 183, 853, (1974)  ;www.organisemicnductors.com

The values reported in literature  should eventually be revised with data obtained from working on integral BCM. 

This experimental situation inevitably brings one to consider the melanins as colloidal nanostructures (for the dimension of their granules) for which the relationship structure-function must be based on chemical processes in a heterogenous phase.  The effect of light radiation, for example on the processes of charge transfer, must be interpreted not at a molecular level, but rather as a “photocatalytic” action of the type studied in certain metallic oxides.  Briefly, as in the colloidal inorganic semiconductors, charges may be stored as band conduction states and as a consequence energy conversion must always involve multielectronic transfer processes.  The interesting thing is that the electronic structure of the melanins can give the possibility of such charge storage.

Often the study of melanins (BCM and BSM) is carried out on artefacts.  Artefacts of melanins, little known from a chemical point of view, form in the course of the synthesis or extraction from the biological sources, even in the blandest of conditions: processes of oxidation, decarborboxylation, condensation, or substitution of the natural cation may all take place during isolation

It should be remembered that all the ortho. diphenols may produce melanin, by an oxidative process, with a sufficient number of free positions (at least two) to make the Raper-Mason scheme difficult to interpret.  Black material (melanin) is formed by oxidation in atmospheric O₂ or by H₂O₂ especially at elevated pH.  The material always has one oxygen more than the number present in the precursor (catechol, 5,6-dioxyindoles, adrenaline,DOPA, dopammine, methyl-catecholes and  methyl-5,6-dihydroxyindoles etc. ).   (Fig. 5-8  )

Attempts of extraction and purification of melanins by preventative mixing of the various organelles of the melanocyte have been made (31).  The extraction of melanin from the various biological sources is, effectively, the extraction of granules and of active melanosomes and premelanosomes.  The melanin which is obtained by the various extractive processes used to date is a set of particles partly formed during the course of extraction.  The melanin which is obtained is, therefore, an artefact.  The subsequent purification process  produces:

a.  decarboxylation on heating

b.  oxidation of the benzenoid part (when the precursor is 5,6-dihydroxyindole, DHI) by theH2O2 present in atmospheric oxygen

c.  loss of the cation

d.    carbazole condensation processes with the use of hot HCl (formation of pyrroletetracarboxylic acid in the oxidative degradation). 

e.  conversion of the dopachromic unit of the”polymer” into a DHI and a DHICA structure.

Wishing to summarise we may claim that to date nobody has managed to isolate a particle equal to that contained in a granule of melanocyte.  It follows that all the experimental data and chemo-physical parameters are uncertain and need to be revised. 

The application of physical methods, as for the heterogenous materials used, has not given good results.  Data from NMR and x-ray, for electrical and acoustic conducibility,  photochemistry and photobiology, are all not very reliable.  The interpretation of data obtained by MALDI and MALDI-TOF mass spectrometry is grotesque, leading to the definition of the melanins as a mixture of oligomers with low molecular weights.  Effectively the black particles built with oligomers of non-high molecular weight (11) explode (they break like a vase dropped from a height) under LASER action.  The explosion is similar to that (frantumation) of the graphite, which as is known, brought about the discovery of the fullerene C60.  It is possible that a similar reaction takes place because of the effect of an undocumented radiation or the collision of black interstellar particles. (Link 7.  Speculation on the chemistry of interstellar black matter.  or Link 8.  The dark secret of life). 

Fig.  2.   The BSM of interstellar spaces (Bok globules) and the BCM of Dr A. Satti a sudanese chemist of Khartoum. 

Chemo-biological researchers have paid little attention to the electrical conducibility of melanin (BCM) and the Proctor-McGinness effect, (www. organicsemiconductors. com).  Chemists have not considered the  new approach for organic chemistry.  Biologists have shown little interest in the extraordinary capacity-function of the melanin in linking ions and organic molecules, of forming charge transfer complexes, of transporting and storing gas (20) (common in fullerene, graphite and helicoid systems), of assembling molecules, of forming electromagnetic fields, of giving rise to communications systems between tissue and tissue, and between tissue and the CNS, of the role played in interstellar space and in the pre-enzymatic era. It is strange that all this has not stimulated new research on the function of melanin (BCM) in nature.

Considering the biogenesis of the cellular pigment, it is believed that the melanins are polymers of DHI and/or DHICA (initially believed to be artefacts by Raper).  The reactions, which lead to 5,6-dioxyindole (DHI) and to 5,6-dioxyindole-2-carboxylic (DHICA), would thus be under the rigid control of several enzymes and other factors.  As a consequence there should be an enzymatic system which regulates the construction of the black particle.  It should be noted that among other things the sepia may also yield a melanin from a precursor other than the tyrosine, which certainly compounds. 

The enzyme seems to be present not only in the melanocytes of melanoma but also in the melanocyte of sepia ink (34).  A different enzyme which catalyses the conversion of the dopachrome into DHI seems to be present in insects (4).  Analytic studies which demonstrate that in dopa-melanin the DHICA unit represents only 10% while in melanoma-melanin (35), only 20% are in contrast.  (36).  An attempt to reconcile the action of enzymes with the action of metals  has  been presented (4).

 To explain the chemical data, in contrast with the Raper-Mason  scheme         (www.tightrope.it/nicolaus/index.htm ) (Link 20)   , we suggested that the eumelanins originate from ortho. diphenol cyclodopa, that the fundamental unit is a hydrated quinonic unit  polymers having one oxygen more than the precursor  (Fig.  7), that the unit which constitutes the black particle may be described as a radical-polaronic system well represented by acetylene black (Fig.  6) and having a typical EPR signal.  This system explains the electrical conductivity, the colour of the amorphous semiconductor (gap 1,2-1,7 eV), and the easy breaking of the benzenoid rings (transformation into a substituted acetylene black).  The form of the black particle,( graphite sandwich, fullerene, and helical of oligomers),  rather than the molecular structure, explains properties like the affinity for ions, organic molecules and gases, the ability to reduce molecular oxygen, to superoxide ions and hydrogen peroxide.   Particle form may influence colour and conductivity. The concept of the particle changes the way of studying the melanins (BCM and BSM) and takes us instinctively to investigate the monomers and oligomers which contribute to the black particles.  The form and size of the particle may be a distinctive element of the property of different melanins. The era of nanochemistry, nanophysics and nanobiology has started.In conclusion compared to the past it seems that a new and more realistic picture of the black material BCM  and BSM may be presented. Oltre che per le sue proprietà assemblatrici tipiche delle BCM la forma idrata del indolchinone è atossica per la cellula e possiede molte delle proprietà di superfici del PLGA-collageno, le sue proprietà elettriche (effetto Proctor-McGinness) rappresentano un elemento di comunicazione fra tessuto e tessuto e fra tessuto e SNC.  Negli acidi umici la forma idrata reversibile può rappresentare una riserva di acqua da ridistribuire nel terreno.  In forma simile questa acqua solida può essere trasportata da meteoriti e comete.

                                                                                                                                         CONCLUSIONI

Con la formazione di un nero da ciclodopa diversi problemi chimici inerenti alla BCM vengono risolti o messi in condizione di essere risolti. Oligomeri radical polaronici a basso peso molecolare si assemblano per formare la particella. E' probabile che forma, grandezza, struttura possano influenzare la conducibilità elettrica e sonora, il trasporto di gas, ioni, molecole organiche sia della materia nera cellulare (BCM) che della materia nera sintetica (BSM).

Non è sicuro che la melanogenesi proceda attraverso fasi controllate dagli enzimi essendo più probabile l'azione esercitata da metalli quali Fe, Cu, Zn, Ni.   L'attività enzimatica è dosata sulla decolorazione, opinabile, della soluzione rossa.   Lo studio chimico è carente di analisi C, H, N, S, Cu, Fe, Zn, Ni, e degli spettri di massa.

 Per quanto riguarda la funzione della BCM (melanine),una questione assai dibattuta, non si è mai tenuto conto di quanto la chimica e la fisica venivano man mano  scoprendo.  La conducibilità elettrica e sonora, la proprietà, comune a tutta la materia nera, di legare ioni, molecole organiche,  gas, la proprietà di esplodere della melanina e del melanosoma  (59) per azione  del LASER, la proprietà di assemblaggio molecolare (principio della tensitegrità), la straordinaria  sensibilità alla acqua ossigenata.

Infine la conducibilità e la superconducibilita a vari valori del potenziale (effetto Proctor-Mc Ginness) www. organicsemiconductors. com.

Gli studi chimici sulle melanine patologiche sono scarsi.  Un vecchio lavoro fatto a Napoli (61) riferisce sulla estrazione da un melanoma di due melanine una nera brillante e l'altra matta, differenti per solubilità in alcali, che potrebbero essere una miscela di DHI-melanina e DHICA-melanina. E' possibile che DHI e DHICA siano coinvolti nella melanogenesi patologica e non in quella fisiologica? In altre parole la particella nera patologica (melanoma) è costruita in prevalenza con indoli  e quella fisiologica da dope e ciclodope come segnalato sperimentalmente dalle analisi centesimali ed  NMR.  

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39.  M. G. Peter, H. Foster, On the structure of eumelanins : identification of costitutional patterns by solid-state NMR spectroscopy, Angew.  Chem.  Int.  Ed.  English. 28,741-743, 1989.

40.  S.  Aime, M.  Fasano,  C. Croonbridge, '' Solid-state 13C NMR characterization of melanin free acids from biosynthetic and natural melanins. '' Gazz. Chim. Ital.  120, 663-664, 1990

41 Aime, S. Fasano, M. , Terreno, E. , Croonbridge, C. J. : NMR studies of melanins: Characterization of a solubile melanin free acid from Sepia ink.  Pigment Cell Res.  1991, 4, 216-221

42 Hervé, M. , Hirshinger, J. , Granger, P. ,Gilard, P. , Deflandre, A. , Goetz, N. : A 13C solid-state NMR study of the structure and auto-oxidation process of natural and synthetic melanin.  Biochim. Biophys.  Acta 1994 1204, 19-27.

43.  A. Pezzella, M. D'Ischia, A. Napolitano, A. Palumbo, G. Prota '' An integrated approach to the study of sepiomelanin:Evidence for a high proportion of degraded5,6-dihydroxyindole-2-carboxylic acid units in the pigment backbone ''   Tetrahedron, 53, 8281-8286,  (1997) ;   Rapid Communications in Mass Spectrometry, 11, 368-372, (1997).

44.  A. Napolitano,A. Pezzella, G. Prota, R. Seraglia, P. Traldi  '' Structural analysis  of synthetic melanins from 5,6-Dihydroxyindole by Matrix - assisted Laser desorption / ionization Mass Spectrometry '' Rapid Comm.  Mass Spectrom. , 10, 468-472 (1996).

45.  A. Pezzella, A. Napolitano, M. d'Ischia, G. Prota, R. Seraglia, P. Traldi '' Identification of partially degraded oligomers of 5,6-Dihydroxyindole-2-carboxylic acid in Sepia melanin by Matrix-assisted  LASER desorption/ionisation  Mass Spectrometry '' Rapid. Comm. Mass Spectrom.  4, 368-373, 1997

46.  A. Bertazzo, C. Costa, G. Allegri, R. Serraglia, P. Traldi '' Biosynthesis of melanin from dopamine.  An investigation of early oligomerization products ''  Rapid Comm.  Mass Spectrom.   9, 634-640      (1995).

47.  A. Bertazzo, C. Costa, G. Allegri, M. Schiavolin, D. Favretto, P. Traldi '' Enzymatic oligomerization of tyrosine  by tyrosinase and peroxidase studied by Matrix-assisted Laser Desorption/Ionization Mass Spectrometry ''  Rapid Comm.  Mass Spectrom, 13, 542-547,  (1999).

48.  A.  Korner, J. Pawelek, '' Mammalian tyrosinase catalyzes three reactions in the biosynthesis  of melanin '' Science, 217, 1163-1165,  (1982).

49.  A. Korner, J. Pawelek,  '' Dopachrome conversion  : a possible control point in melanin biosynthesis '', J. Invest. Dermatol. , 75, 192-195,  (1980).

J. Pawelek, A. Korner, A. Bergstrom, J. Bologna ''New Regulators of Melanin biosynthesis  and the autodestruction  of melanoma cell '', Nature, 286, 617-619, (1980).

49 bis.  P.  Aroca, F.  Solano, J. C.  Garcia-Borron, J. A.  Lozano "Specificity of dopachrome tautomerase and inhibition by carboxylate indoles.  Considerations on the enzyme active site" Biochem.  J.  277, 393, (1991).

50.  B.  Kagedal, P.  Konradsson, T.  Shibata, Y.  Mishima "High-performance liquid-chromatographic analysis of dopachrome and dihydroxyphenylalanine" Anal.  Biochem.  225, 264, (1995)

51.    P. Aroca, F. Solano, J. C Garcia-Borron, J. A. Lozano ''A new spectrophotometric assay for dopachrome  tautomerase ''J. Biol. Biophys. Methods, 21, 35-46  (1990)

52.  F. Solano, C. Jimenez-CervantesJ. H. Martinez-Liarte, J. C. Garcia-Borron, J. R. Jara, J. A. Lozano '' Molecular Mechanism for catalysis by a new zinc-enzyme,dopachrome tautomerase '' Biochem. J. , 313, 447-453,  (1996).

53.  J. L. Pennock, J. M. Behnke, Q. D. Bickle, E. Devaney, R. K. Grencis, R. E. Isaac, G. W. P. , Joshua, M. E. Selkirk, Y. Zhang, D. J. Meyer ''  Rapid purification and cheracterization of L-dopachrome-metyl ester tautomerase  (macrophage-migration-inhibitory factor) from Trichinella spiralis, Trichuris muris and Brugia pahangy ''  Biochem. J. , 335, 495-498, (1998).

54.  

a.  J.  Pawelek " Is dopachrome tautomerase necessary to get DHICA from dopachrome? " Pigment.  Cell Res.  7, 126, (1994)

b.         F.  Solano, J. C.  Garcia-Borron, J. A.  Lozano "Is dopachrome tautomerase necessary to get DHICA from dopachrome? Pigment Cell Res.  7, 125, (1994)

c.         S.  Pavel "Dopachrome tautomerase is not essential for DHICA formation" Pigment Cell Res.  7, 123, (1994).

55.  H.  Sugimoto, M.  Taniguchi, A.  Nakagawa, I.  Tanaka, M.  Suzuki, J.  Nishihira "Crystallization and preliminary X-ray analysis of human D-dopachrome tautomerase" J.  Struct.  Biol.  120, 105, (1997).

56.  H.  Sugimoto, M.  Taniguchi, A.  Nakagawa, I.  Tanaka, M.  Suzuki, J.  Nishihira "Crystal Structure of human D-dopachrome tautomerase, a homologue of macrophage migration inhibitory factor, at 1,54 A resolution" Biochem.  38, 3268, (1999).

57.  H.  Yoshida, J.  Nishihira, M.  Suzuki, K.  Hikichi "NMR characterization of physicochemical properties of rat D-dopachrome tautomerase" Biochem.  Mol.  Biol.  Int.  42, 891, (1997).

58.  G.  Odh, A.  Hindemith, A. M.  Rosengren, E.  Rosengren, H.  Rorsman "Isolation of a new tautomerase monitored by the conversion of D-dopachrome to 5,6-dihydroxyindole" Biochem.  Biophys.  Res.  Commun.  197, 619, (1993).

59.  S. L. Jacques, D. J. McAuliffe '' The melanosome :Threshold temperature for explosive vaporization and internal absorption  coefficient during pulsed LASER irradiation ''   Photochem. Photobiol.  53, 769, (1991).

60.  J. Borovansky, P.  Hach, J. Duchon '' Melanosome  : an unusually resistant subcellular particle '' Cell Biology International Reports, Vol. 1, 549-554 (1977).

Melanosome are resistant to acid hydrolysis but they are easily degraded  by LASER or, as occur in vivo, by H2O2. 

61.  M. Bakunin, G. Dragotti '' Contributo alla conoscenza dei pigmenti melanici ''Rend.  Real.  Acc.  Sci.  Fis. Mat.  Vol.  X, 222, (1904)

62.  

a.  R. A. Nicolaus,M. Piattelli, G. Narni,, '' The structure of sepiomelanin'' Tetrahedron, Letters 21, 14-17, (1959). 

 b.  M. Piattelli,R. A. Nicolaus '' The structure of melanins and melanogenesis-I-The structure of melanin in Sepia '' Tetrahedron  15, 66-75, (1961).

c.  M. Piattelli, E. Fattorusso,S. Magno, R. A. Nicolaus '' The structure of melanins and melanogenesis-II-Sepiomelanin and synthetic pigments '' Tetrahedron 18, 941-949, (1962).

d.  M. Piattelli,E. Fattorusso, S. Magno,R. A. Nicolaus '' The structure of melanins and melanogenesis -III-The structure of sepiomelanin '' Tetrahedron, 19, 2061-2072 (1963)

e.  R. A. Nicolaus, M. Piattelli, E. Fattorusso ''The structure of melanins and melanogenesis -IV-On some natural melanins '' Tetrahedron, 20, 1163-1172 (1964).

63.  J. Medrano, D. Dudis ''Quasi-particles in polymeric conductors '' in Organic Superconductivity, pag. 275.  Ed.  V. Z. Kresin, W. A. Little, Plenum Press,New York,  (1990)

64.  R. Nicolaus '' Sugli acidi pirrolcarbonice. Nota I.  Acido 2,3,5-pirroltricarbonico '' Gazz. Chim. Ital.  83, 239-251, 1953

65.  R. Nicolaus,G. Oriente ''Sugli acidi pirrolcarbonici : acido 2,3,4,5 pirroltetracarbonico '' Gazz. Chim. Ital.  84, 230-241, 1954

66.  E. Fattorusso, L. Minale, S. De Stefano, G. Cimino, R. A. Nicolaus '' Struttura e biogenesi delle feomelanne. Nota V. -Sulla struttura della gallofeomelanina1 '' Gazz. Chim. Ital.  98, 1443-1463, 1968.

67.  S. Ito, K. Fujita '' Microanalysis of eumelanin and pheomelanin in hair and melanomas by chemical dgradation and liquid chromatography '' Analytical Biochemistry  144, 527-536, 1985

68.  P. Manini, M. D'Ischia, M. Milosa, G. Prota '' Acid-promoted competing pathways in the oxidative polymerization of 5,6-Dihydroxyindoles and related compounds : straightforward cyclotrimerization routes to diindolocarbazole  derivatives '' J. Org. Chem, 63, 7002-7008, 1998.

 69) 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 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. 

70.  M. Seiji, T. B. Fitzpatrick, R. T. Simpson, M. S. C. Birbeck  ''Chemical composition and terminology of specialized organelles (melanosomes and melanin granules) in mammalian melanocytes '' Nature 197, 1082-1084, 1963.

71.  M. Seiji, K. Shimao, M. S. C. Birbeck, T. B. Fitzpatrick  ''Subcellular localization of melanin biosynthesis '' in The Pigment Cell : Molecular, Bioogical and Chemical Aspects, Ed.  P. Riley, J. C. Fortner, New York Academy of Sciences, 100, 497, 1963.

72.  A. Pezzella, A. Napolitano, M. D'Ischia, G. Prota,R. Seraglia, P. Traldi '' Identification partially degraded oligomers of 5,6-dihydroxyindole-2-carboxylic acid in Sepia melanin by matrix-assisted LASER desorption/ionization mass spectrometry '' Rapid Comm. Mass Spectr.  11, 368-372, 1997.

73.  H. Wyler, J. Chiovini '' Die Synthese von Cyclodopa (leucodopachrome) '' Helv. Chim. Acta    51, 1476-1482, 1968.

74. A. Aroca, F. Solano, C. Solinas, J. C. Garcia-Borron, J. A. Lozano, Eur. J. Biochem.  208, 155-  163, 1992

75.  H. Wyler, '' The dopachrome-dihydroxyindole '' in   Melanogenesis. Its chemistry as a therapeutic strategy in melanoma.  Manchester Meeting   1991

76. M.Benathan et al., Yale J.Biol.Med. 53, 389,1980 cited by (4)

77. Y.Liu et al., Pigment Cell Research 16, 355-365, 2003.

78. F. E. Barr, Med Hypothese 11, 1-139, 1983.

79. E.J.Land et al., Pigment Cell Research 16, 397-406, 2003 .

The particle

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

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

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

Papers (1-72)with comment and figures useful for research  :

 (To improve quality of research any reference should be followed by comment and critique)

Index

( number = reference )

Allomelanin  43

Albino, melanoma  5

Biology  17,18, 31, 37, 39, 50, 51,52, 57, 62, 67

Binding  7

Cosmo  63, 64,

Conductivity  9, 20, 22, 25, 26, 27, 48, 49, 54, 59, 66

Eye  6

Evolution  8

Humic acids 73

LASER   23, 24, 58

MALDI    74

Neuromelanin  1,10, 11,12,13,

Nanoscience   18, 30, 42, 44,45, 46, 53, 55, 56, 60, 61

Physics   28,65

Structure    33, 34, 35, 36, 38, 40, 41, 47, 61, 68, 69, 70, 71

Skin      2,15,16

Sepiomelanin   3,4,32

###################################################################

Melanins are classified into two main groups :

BCM = Black Cell Matter ( animals,plants, fungi )

BSM = Black synthetic Matter ( the most famous precursors are DHI, DHICA, Pyrrole,Acetylene, )

BCM are divided into three groups  :

Eumelanins   ( precursors until now found DOPA, Cyclodopa, DHI,  DHICA )

Pheomelanins ( precursors until now found the aminoacid cysdopa, with Dibenzothiazine and Dibenzothiazinonie chromophores )

Allomelanins ( precursors until now found catechol, 1,8-dihydroxynaphtalene

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To be read before :

!. R.A.Nicolaus ‘’ Melanins ‘’  pag 1-310, Hermann, Paris, 1968

2. R.A.Nicolaus  ‘’ Melanins ‘’ in Methodicum Chimicum Vol.11, part 3, pag 190-199, Ed.F.Korte, AP, 1978

3. G.Prota ‘’  Melanins and melanogenesis ‘’  pag 1-290, AP, San Diego 1992

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

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

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

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

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

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

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

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

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

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

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

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

 
8) Melanins as a matrix for chemical evolution .

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

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

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

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

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

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

 E.Hirsch et al., 334, 345, (1988).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

22) All black materials are electroactive .

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

23) Explosive vaporization during pulsed laser irradiation of melanosome.

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

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

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

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

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

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

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

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

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

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

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

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

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

www.tightrope.it/nicolaus/index.htm

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

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

Apparently  sepiomelanin is similar to DHI- melanin.

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

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

The radical polarone system of melanins..Structure proposed for melanin oligomers taking in account the electric semiconductor property.The acethylene-black system is shown.

(Link 5,6,9,12,19,21,22) 

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

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

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

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

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

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

 www.tightrope.it/nicolaus/index.htm

35) Cyclodopa precusor of eumelanin (BCM).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

42) Melanin particle ( L.Zeise, B.L.Murr, M.R.Chedekek, Pigment Cell Research, 5, 132-142, 1992 ) is formed by mixture of oligomers species with molecular weight in the range of 500-1500  Da

A.Napolitano et al.,  Rapid Comm. Mass Spectrom., 10,204-208, 1996 ; 10,468-472, 1996 ; A.Pezzella et al. Tetrahedron, 53, 8281-8286, 1997. 

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

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

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

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

Some peculiar properties known until now  are:

1. Sound and electrical conductivity    ( BCM and BSM )
2. Storage of gas, water,  liquids ( Earth and Interstellar spaces ).

     c.   Binding of ions and organic molecules

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

e. All chemical and physical data may change with form and size of the particle, preparation and purification methods,  binding effect, doping.

45 ) Melanin standard method : particle description.

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

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

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

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

48    ) Electrical conductivity of DOPA-melanin.

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

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

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

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

49)  Semiconductor properties of natural melanins.

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

50 ) Melanogenesis in the ink gland of Sepia.

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

51)…the color loci of mice……

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

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

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

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

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

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

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

 BW=  band width  of valence band.

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

IP = ionization potential . Low value indicated easy oxidable

  Melanins are considered material with a little gap.

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

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

Time-dependent current-vs-voltage curves show that the time to traverse the negative - differential-resistance (NDR) segment is much slower than would be expected  from electronic switching mechanism.Double-pulse measurements add to the evidence that thermal effects dominate electronic effects in melanin.A pseudomemory was found in melanin  ( C.H.Culp et al. J.Appl.Phys. 46,3658-3660 ,1975 )

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

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

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

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

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

MALDI spectra show complex pyrrole acids of obscure origin.

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

60 )  Chemical and physical data may change with form and size of the particle, methods of synthesis,   purification and doping.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

71 )  It was found that about 96% of the CO₂ evolved  in the reaction DOPA-----Melanin  arise from the breakdown of the benzene nucleous.

G.A.Swan et al., J.Chem.Soc.381-384, 1954.

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

72 ) Composition of melanin samples depends on  centrifuge speed

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

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

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

 .

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

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

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

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

The conclusions about the structure of BCM ( eumelanin ) in 1992.

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

76 ) Pheomelanin and Pheochrome ( other names trichosiderin,trichochrome ) derive from the new aminoacid  5-S-cysteinil-dopa  ( Cysdopa ) .Chromophores are dibenzothiazine and dibenzo thiazinone.The structure of the pigment of red haior was established

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

Link 14 of www.tightrope.it/nicolaus/indexc.htm

bruno.nicolaus@virgilio.it   

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rnicolaus@tightrope.it 

www.tightrope.it/nicolaus/index.htm 

crippa@fis.unipr.it 

perchardj@hotmail.com

bologne@unina.it

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Ringraziamo il personale della Biblioteca della Stazione Zoologica di Napoli per la assistenza data.

Warning :  Various teaching tools  ( school,  journals, books, encyclopedias, Internet, etc. )  still quote  descriptions  of  black matter ( BCM and BSM ) and melanogenesisi  which are not up to date and do not comply with those reported by here..

Naples May 2002

Naples April 2004