Physics and Chemistry of Melanins.

Physics and Chemistry of Melanins. 

The peculiar characteristics of the melanins are always the colour and their free radical nature (6). Scarse results have also been found with the most sophisticated apparatus (X-rays, MS, NMR, Laser) (Link 22).

There are three fundamental theories on the chromophores of melanins. The first is that which proposes that melanin is a mixture of chromophores which resonate in different parts of the spectrum. A mixture of red, blue, green etc. chromophores absorb the radiation of all the wave lengths of visible light and appear black. Another theory predicts that a highly conjugated system produces black, brown and red-brown depending to the gap value of the semiconductors. What is the extension of the conjugation that allows a body to appear black? Many natural substances possess extended systems of conjugated orbitals (for example the carotinoids) but none of these compounds appears black. The study of the electronic structure of an ordered and ideal polymer of 5,6-indolquinone which is a common component of the eumelanins (hair, eyes, skin, etc.), conducted using the theory of Hückel, predicts that the black colour appears in a polymeric unit with a number of monomers higher than 10 and up to a infinite polymer and that, besides, it is an amorphous semiconductor and a good conductor of electricity (3) . The theoretical model is not supported by few experimental data. The only experimental data which currently exist for polyarene and polyene structures are those relative to pyrrole- black and acetylene -black which, as has been said, are radical-polarone system. ( Link 9, 12, 21, 22 ) In the study of the melanins it has often been claimed that the different colours, yellow, orange, red, red-brown, black are due to the type and the size of the granules of the pigment and to the distribution of the granules in the tissue. A third theory proposes that the melanins are schemochromes, that is that the colour depends on particular particle structure. The melanins could be a sort of black body in which the light which penetrates is reflected and diffused until it is completely absorbed. The cause which produces the "black" must be studied using solid state theory and band model of semiconductors.

Melanin as stable free radical .

Despite the fact that every chemical and physical study of melanin must be interpreted with more care compared to what would usually be the case in the study of pure and crystaline substances, the black melanins (eumelanins), brown, red-brown, and yellow melanins (pheomelanin), the allomelanins and the humic acids present a characteristic EPR signal, sometimes with some hint of a hyperfine structure (Link 22). The origin of the paramagnetism is still a controversial problem. Several efforts, (nothing is ever clear and definitive in scientific research on the black substances), have been made to correlate the free radical nature of the melanin with certain biological functions, like the physiology of vision, photoprotection, threshold switching, the electret effect etc. Studies have been carried out on all the melanins in acqueous suspensions and almost always give an EPR signal at about 4-6 G. The spin concentration is around the value 4-10 x 1017 spin/g. In the "polymer" there would be one free radical every 200-1,000 "monomers". It would seem that there are two radicalic centres in the black products that originate from the o.phenols: one being essential (intrinsic), highly stable, generated in the course of melanogenesis and "trapped" in the product and the another being extrinsic, transient and reactive which can form in the melanin by the action of the different chemico-physical agents. Passing from black melanins to brown and red-brown products (pheomelanins) it is possible to observe radicals with better defined structures, at different pH, like those of semiquinonamine and semiquinone. EPR studies carried out on the hair and skin of several bovine races and on albinoes have mainly been used by geneticists and pathologists. Albino subjects, with the same phenotype character, have hair with differing electronic characteristics. In some albino subjects there is a weak EPR signal which is completely absent in others. There are, that is, true albinoes and false albinoes (6, l).

The chemistry of free radicals, which has almost always been associated to the processes of polymerisation and oxidation, has developed in isolation from the context of organic chemistry. The chemistry and biochemistry of natural substances have always considered the free radicals with diffidence because of their reactivity which makes them difficult to control. It is probable that different radicalic reactions intervene in melanogenesis. Melanogenesis has never been considered as an essentiallly radicalic process. The EPR signal, present in all the melanins, has been attributed to a system like the cyaninic colourants (merocyanines) which give, as is known, EPR signals similar to those of the melanins even though they do not have unpaired electrons. This explanation of the meaning of the EPR signal by the merocyanines seems strange? The hypothesis that the signal comes from an inert radical of the copolymerisation material of the melanin cannot be accepted. A somewhat surprising observation is that the radical is little present in the melaninin granule and therefore of little significance , while it is highly probable that the important properties of the melanin (solubility, colour, reactivity, conductivity) are linked to this electron which lives alone in a gap of the granule. When the melanin lightens from black it becomes brown, red-brown, yellow, that is one passes to pheomelaninic granules (hair, fur, feathers, eyes), not only is the EPR signal always present but it appears in a more and more structured form, to the point that it is possible to distinguish the black melanin from the red-brown (pheomelanin). The EPR signal is present both in melanins prepared in the laboratory under most varied conditions and in pheomelanins of very varied origins. For the humic acids of the hydrosphere and of the lithosphere, so important for life, one has a very similar general picture even if the heterogeneousness of the material makes the granules of the humic acid much more difficult to study. The results obtained with IR, NMR, X-rays and MS cannot be believed significant. The various spectra which often present absorption characteristics, may not be due to the humic acid but to incorporated substances (which can be used for the qualitative and quantitative analysis of terrains and of waters). The EPR signal is pure, clear and very similar to that of the melanins. In general one obtains the EPR signal of two types of stable radical which must be attributed either to the pyrocatechine-resorcine type or the quinhydrone type. In general one has signals of various intensity according to the origin of the humic acid (humic acids from micro-organisms, soluble humic acids, black carbon humic acids, grey humic acids, brown humic acids, marshland, manure, peat). Notable paramagnetism is present in the fungal humic acid (Cephalosporium gordoni) which is also darker: the blacker the composite the more intense the EPR signal. It is calculated that for a concentration of 1017 spin/g in a "composite" of molecular weight 10,000 there is 1 radical for every 600 monomers. It is plausible that, from a structuralis chemical point of view, the part of the "macromolecule", perhaps the chromatic part (acetylene-black), responsible for the paramagnetic phenomenon is assimilable to that of the more typical melanins present in the animals or produced by micro-organisms. Therefore the history of the EPR signal is identical for all the melanins regardless of their origins. As often happens in research the failure of an objective (identification, for example, of the structure responsible for paramagnetism in the melanin) produces new and useful knowledge. The discovery that the humic acids of various origins and natures are free radicals has given useful information on all the existing relationships between the EPR signal and the properties of these substances. In the soil there are all the conditions for the genesis of free radicals: water, light, heat, organic and inorganic catalysts (Fenton reactive types). The soil is all a melting pot of radicals; in the soil the primordeal matrix can operate with ease .

The hypothetical radical melanogenes of the soil can form through the action of oxygen and light on products deriving from micro-organisms, from the oxidative demolition of the lignin, and from other biological polymers. In the physiology of plants humic acids act both as growth factors and as activators of cellular respiration. The semiquinonic radicals of the humic acids influence germination and, linking to atmospheric oxygen, transport active oxygen in the soil. The humic acids as polycations fix and transfer precious counteranions to the life of the soil and the waters.They can be good conductors when opportunely prepared and doped. In conclusion, polymers like melanin from DOPA, melanin from sepia, humic acids, melanin from phenols, like pyrocatechine and pyrrole blacks, etc. , apparently differing among themselves, exhibit an identical signal at EPR which can be reinforced up to even 100 times if the measurement is made on sodium salts. The melanins of the soil (humic acids) form by a fluctuating and non-reproducible mechanism where time must be considered as an intrinsic parameter of the dynamic of the natural process and where the activation of water is a fundamental parameter (also cellular liquid). The concept of time is a problem of fundamental importance for the biologist of melanin, in that in its genesis this is not free from environmental and cosmic forces and influences or from the activation of the water under the action of electromagnetic fields or from solar phenomena in general. To show that some reactions cannot be located in the current logic of science, Piccardi stressed that accepting time as a coordinate negates the fundamental dogma according to which only reproducible experiments are valid. In the case of melanin, in fact, it is not possible to control the conditions in which an experiment is conducted without taking into account the time coordinate, because during its course the conditions in which an experiment, a chemical or biochemical reaction, is conducted change. In experiments on melanin the time may not be considered an isotrope in every direction in space, nor homogeneous for every successive instant. Melanin cannot be represented by a formula, with a complete melanogenetic scheme in that it is not representable in terms of linear equations because it is not possible to hypothesise a correspondence between cause and effect in the temporal succession of the relationships. A negligible reaction in a fixed instant can become a determining reaction in a succeeding instant.

In melanogenesis many classical concepts, definitions, chemical dogmas are obsolete, they are lost in the past. If the experimentor looks at the scientific explanation of the natural processes and is not content with the measurements of the immediately measurable quantities then he understands that he is working in an imperfect way. The analysis (combustion) of the melanins coming from the sack of the sepia are non-reproducible even with the same method of preparation of samples coming from the same source. To the chemist the synthesis of black content in the sack of the sepia must seem a non-reproducible process and is perhaps fluctuating because of the interference of magnetic activity and radiation of the sun on the sea, of the atmospheric electrical potential, of the variations in the Earth’s magnetic field.

The spirit of research in the field of the melanins must be renewed, accepting the hypotheses works some researchers, sometimes students, most of the time young emarginated researchers, would like to introduce into the world of research.