The pigment of Sepia

Nota di Marco Olivieri e del socio Rodolfo A. Nicolaus

Key words: melanin, eumelanin, dopachrome, cuttlefish, melanosome.


Sepiomelanin is not the most elusive and enigmatic pigment found in Nature but just a particle waiting for a rational study.

Sepiamelanin Na+ form occurs in the ink sac of Sepia officinalis as Ca and Mg salt. The free acid is obtained by action of dilute HCl: the sepiomelanic acid. Sepiamelanin is a black insoluble, infusible, hygroscopic powder; in fresh preparations, on the contrary, loss of weight (loss of CO2? ) is observed. The pigment is a mixture of particles in varying stages of development as showed by ultracentrifugal density-gradient methods [1]. Working with fresh ink a post mortem melanogenesis in still active organelles occurs, resulting possibly in an artificial melanogenesis of dopachrome. Heating of sepiamelanin produce loss of CO2 and of H2O. Melanin is sensitive to oxygen, oxidants, pressure, light; pulsed laser irradiation produce an explosive fragmentation of melanosome similar to those what occurs in the skin [ 2 ] and to other black particles.

The pigment has the property to bind many organic products, drugs, ions, gas, recalling the beaviour of charcoal. Some properties like the EPR spectrum are in agreement with a radical-polarone polymer structure.

Sepiamelanin-film in doped or undoped form acts as an amorphous semiconductor. As a Little's structure it may conduct electricity without resistance like certain metals [3], [4], [5]. Chemical and physical studies show that sepiamelanin, in contrast with general view, is derived from 5,6-dihydroxyindole ( DHI ).

 

Polymerization through all free positions of DHI produces a polycondensed structure and further assembling of intermediates build up the particle [6]. The presence of polycondensed structure is in agreement with X-rays diffraction spectra and, chemically, with the isolation of pyrroletetracarboxylic acid among the oxidative degradation products of sepiamelanin.

More recent studies have shown that the ejected ink of Sepia officinalis contains an enzyme, which promotes the decarboxylative rearrangement of dopachrome to DHI [7], [8].

The study of sepiamelanin isolated and purified with incorrect methods produced results contrasting the assumption that the black particle is constructed with DHI units. For example, some results have shown that sepiomelanin is a copolymer of DHI and DHICA (5,6-dihydroxyindole-2-carboxylic acid ) [9].

More recently, it has been suggested that sepiamelanin is a mixture of oligomeric structures incorporating over 75% of DHICA-derived units and only 20% of DHI-derived units, occuring for the most part as pyrrole carboxylic acid degraded form [10]. It should be noted that such a structure cannot be black, would not show the typical EPR spectrum and would be easily soluble in dilute alkaline solutions.

These conflicting and surprising results are caused probably by the incorrect methods of isolation and purification adopted. In order to avoid these inconvenients we present a protocol for isolation and purification of sepiamelanin based chiefly on Chedekel method [11].

Considering that all sepiomelanin samples examined so far are artifacts, all literature data should be reexamined. Some papers, particularly, [11], [9], [12], [13], [10], [14], [15], [16], [17], [18], require a large critical discussion.

Sepiomelanin protocol of isolation and purification starting from fresh ink is given in the following table 1. Variable amounts of H2O2containing melanosomes are present in fresh ink. How and when to eliminate the H2O2 has not been considered so far.

Reagents are analytical grade. Distilled water was used. All operations are conducted at room temperature working in the dark. Solids are separated from liquids by centrifuging. Samples for analysis are dried on P2O5 in vacuum at room temperature.

Working at high centrifuge speed a large amount of immature melanosomes can be obtained. The mature/immature melanosome ratio was found to change in stored and fresh ejected ink. Centrifuge speed (2000 - 4000 rpm) is adjusted with the ink fluidity.

Melanosomal fractions of different maturation grade, shape and size may be obtained by following methods:

a) the ''sieve'' procedure of Chedekel [11]

b) the ''sugar'' procedure of Hearing [19]

c) the ''surfactants'' procedure of Ito-Prota [10].

MALDI (matrix assisted laser desorption ionization ) may be a good distinctive criterium between the different melanosomal fractions. In the MALDI spectrum of immature melanosome, precursors and oligomers are present, whereas they are absent in mature melanosome.

With some modification, the above mentioned procedure (table 1) could be applied to the study of others melanins.

 

Table 1

 

An ink sac was cut open, emptied and centrifuged: wash with H2O x 3

                                                                             ¯
                                                                           SM
                                                                             ¯

Wash HCl 1N x 3, keep 1 night HCl 6N, wash HCl 1N x 3, H2O x 3, acetone x 3, H2O x 3. Dry on P2O5 in vacuum.

                     
Treatment with H2O2 in Na2CO3 2N. Filter if necessary. Acidify with HCl 2N. Filter and centrifuge.

 

Legend for table 1

SM = Ca, Mg salt of SMA, homogeneous (size, density, mass spectrometry ) fraction of mature melanosomes (granules) from Sepia ink. Samples for analysis are dried at room temperature over P2O5. Analysis for C, H, N, Ca, Mg, Na, Fe, Cu, Zn.

Physical determination:


SMA = Sepiomelanic acid obtained from SM. Samples for analysis are dried at room temperature over P2O5. Analysis for C, H, N, Ca, Mg, Na, Fe, Cu, Zn.

Physical determination:


SMA ester = methyl ester of SMA obtained by esterification with CH3OH + HCl gaseous [9].

 

SMA ether = methyl ester (ether) of SMA obtained by CH2N2 treatment [9].


SMAI = Insoluble sepiomelanic acid obtained after oxidation with H2O2 in alkali of fine powdered SMA and acidification of alkaline solution [20]. Simple and complexe pyrrolepolycarboxylic acids may be present in this precipitate. SMAI was obtained in 70% yield w/w. This acid is indicated for chemical and physical studies thanks to its solubility in alkali.

 

SMAS = Sepiomelanic acid solution obtained from SMA after oxidation with H2O2 in Na2CO3 2N, acidification and separation from SMAI [20]. Simple acids, carbonilic compounds, simple and complexe pyrrolepolycarboxylic acids are present in this solution.


Mail and questions to:

 

R. Nicolaus
Rampe Brancaccio 9, I-80132 Napoli
rnicolaus@tightrope.it

M. Olivieri
olivieri@chemna.dichi.unina.it

 

Naples 28 June 1999


Bibliography


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