SPECTROCHIMICA ACTA

PART A

ELSEVIER Spectrochimica Acta Part A 52 (1996) 1157 1162

Mini Review

Progress of infrared investigations of melanin structures

Barbara Bilifiska Department o/ Biophysical Chemistry, Silesian Medical Academy in Katowice, 41-200 Sosnowiec, Jagiellohska 4, Poland

Received 9 August 1995; revision accepted 12 February 1996

Abstract

The development over the past thirty years of IR spectroscopic investigations of natural and synthetic melanins is presented. Particular attention is paid to chemical modifications of melanins and interactions of melanins with metal ions and other chemicals in structural investigations. The role of IR spectral analysis during the different preparation processes of natural melanins is presented.

Keywords: Complexes; Interactions; IR spectroscopy; Melanins; Melanoproteins; Modifications; Structure

I. Introduction

Melanins are pigments widely distributed in the animal and plant world. They are irregularly built polymers, whose physical properties have not di- rectly determined their structures and all chemical properties.

According to Nicolaus [1] melanins, may be divided into three groups: eumelanins, phaeome- lanins and allomelanins. Eumelanins, which are black or brown pigments containing nitrogen are derived from tyrosine, dihydroxyphenylalanine (DOPA), dopamine and tyramine in the process of oxidative polymerization [2]. Phaeomelanins are pigments with lighter colours and are synthe- sized from tyrosine via the same pathway as eu- melanins, but in this process cysteine or glutathione is involved [2,3]. Allomelanins are present in the plant world. These pigments are formed during oxidative polymerization of pheno-

lic substrata in the presence of polyphenoloxidase [41.

Melanins are amorphous polymers which are insoluble in aqueous solutions and organic sol- vents and sometimes soluble in alkalis. Infrared spectroscopy is one spectroscopic method of structure investigation which has been applied to investigate the structures of natural and synthetic melanins. Unfortunately, the amorphous and widely varied melanin macromolecules are not interesting objects for classical spectroscopists, es- pecially in the IR spectral region.

2. IR spectroscopy of natural melanins

One can assume that the paper of Bonner and Duncan [5] gave the first information about the IR spectra of natural melanins isolated from Arenicola marine, Holothuria niger, ink sac of

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Sepia, beetle and bovine eye, the samples of which were prepared as films in CCI 4 suspensions. Inter- pretation was made on the basis of two bands at wavelength 3/~m and 6/~m which were attributed to the bonds of NH of OH groups and carbonyl groups, respectively. Ruban and Liakh [6] and Swan [2] did not find this spectroscopy technique suitable for determining melanin structure accord- ing to the assumption that the IR spectrum corre- sponds to the determined compound.

In the 1960s two papers which compared the properties of different melanins and melanin-like compounds appeared [7,8]. Although different physical methods were used for structural investi- gations, the interpretation of IR spectra was inad- equate. There is no relationship between the obtained spectra and the method of melanin isola- tion [7].

2.1. IR studies of fungal melanins and humic substances

Examples of characterization on the basis of IR spectra of melanin and melanin-like compounds isolated from fungi were presented by Chet et al. [9] and Malama et al. [10]. IR spectra presented in many papers have an informative character. In Ref. [10] we can analyse the spectra for the differ- ent samples of melanin pigments isolated from fungi. Malama et al. describe the spectra in detail and by comparison it is possible to interpret the bands Amide I, Amide II and Amide III. This indicates that some isolated pigments are melanoproteins. The bands near 1700 cm l are ascribed to the free carboxylic group COOH and the band at 1400 cm i to the ionized carboxylic group COO [10]. The bands in the spectral regions 1100-1050 cm -~ and 1240 1200 cm are ascribed to the hydroxylic, alcoholic and phe- nolic groups, respectively. The bands in the region 1600 cm ~ have been assumed to be due to the vibrations on the plane of the CH=CH bonds [10].

Malama et al. [10] show some similarities be- tween the melanins and the humic acids, which often both appear in the different systems, for example in soil. There are great similarities in the IR spectra of both groups of compounds. This method is able to indicate both the similarities

and differences of structures for these polymeric macromolecules [11-20].

2.2. Role of samples modification in the interpretation of IR spectra

The analysis of IR spectra connected with the interpretation of thermal properties of com- pounds, permits the detailed investigation of structural changes in melanin and humic com- pounds during heating [ 11,12,17,18,20,21 ].

IR spectroscopic properties of melanins from soil fungi [17,18,20] which are important in struc- tural investigations of natural melanins, stimula- ted the research to some extent. The fungal mel- anin examinations of Filip et al. [17] were per- formed on the basis of IR spectral comparison of melanoproteins and different humic acids, and model systems synthesized from the supposed ini- tial compounds. Comparison of the IR spectra of isolated samples and their methylated, hydrolysed and heated forms has permitted the basic struc- tural similarities and differences of investigated macromolecules to be determined. Chemical mod- ifications of macromolecules were performed for the melanoprotein samples obtained during the isolation process, and hence difficulties of inter- pretation appeared in relation to samples of intact melanins. IR spectroscopy has been shown to be very useful for the examination of structural changes of melanin from fungi and humic acids during heating [11,12,18,20]. Destruction and de- cay processes observed during the heating of melanins and humic acids also permit to investi- gation of their structure and thermal stability. Aromatic systems of melanin subunits are the most stable structures. In similar studies, IR spec- tra of melanins isolated from natural sources (ba- nanas, human hair) and DOPA-melanin were compared with the analysis of thermal properties (DSC) [21].

Pierce and Rast [22] have shown that compari- son of IR spectra melanin from cultivated mush- room (Agaricus bisporus) and synthetic melanins obtained from presumed precursors in various environmental conditions permits the origin and structure of natural melanin to be determined. The interpretations of IR spectra of natural and

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synthetic melanins have been enriched by investi- gations of brown coal spectra [23].

2.3. IR spectra of melanins from eyes

A separate group of IR investigations is the analysis of IR spectra of melanins isolated from mammalian eyes and human hair, and chemically synthesized melanins. There are many sources of melanin in the human organism, but unfortu- nately not all are accessible to examination. A few papers have presented IR spectra of melanins isolated from cattle eyes [24-28] in which de- tailed interpretations have been presented. Analy- sis of IR for the melanins from cattle eyes and DOPA-melanin at different pH values permits establishment of the possibility of activation of functional groups of the macromolecule [26]. Among molecular groups which may interact with many metal ions and chemicals, the car- boxylic and hydroxyl phenolic groups are the most important. Knowledge of their reactions is very important for the analysis of interactions of melanins with different ligands, for example metal ions, drugs and dyes. IR spectroscopic investiga- tions of melanin isolated from human eyeballs are very difficult due to lack of access to the biological material. Menon et al. [29] in 1982 obtained IR spectra of melanins isolated from an iris, a choroid and a ciliary body of two human populations with blue and brown irises. They did not find any significant differences between these spectra, but differences between them and the spectrum of DOPA-melanin were demonstrated. This does not indicate that the melanins isolated from human eyes do not belong to the eumelanin group. This conclusion confirms the other investi- gations of Menon et al. [30], and analysis of IR spectra for the melanins isolated from human, swine and cattle eyes [27]. Examinations of redox properties and the association affinities of differ- ent compounds suggest there are structural differ- ences between them [29,30].

2.3.1. Problem of melanin hydration Investigations of melanin hydration by Bridelli

et al. [26] for melanin samples in KBr pellets do not seem to have given valid results. Interpreta-

tion of the presence of water in macromolecules such as melanin is very difficult because amor- phous compounds of this type adsorb water molecules on the surface and can locate them in the interlayer spaces, not on the basis of structural bonding; furthermore the dispersion phase is very hygroscopic. Such behaviour does not facilitate the drawing of suitable conclusions. Investigations of the thermal properties of natural and synthetic melanins have confirmed these facts [21,31]. The problem of the hydration of melanin is an inter- esting one which has been addressed before [2,32]; from complicated structural assemblies and mac- romolecular architecture one has to take into acc- ount the physicochemical properties of melanin molecules, which complicates a unified methodo- logical approach.

2.4. IR spectra of melanin from human hair: role of &olation method

The analysis of IR spectra of melanin isolated from human hair has been carried out by Bolt [33], Hackman and Goldberg [34], Menon et al. [35] and Bilifiska and co-workers [28,36]. Bolt [33] reported the IR spectrum of melanoprotein from women with black hair, whilst Bilifiska et al. [28] presented and analysed the spectra of melano- proteins which had been isolated according to the method of Bolt [33] from dark, red and fair human hair, and melanins obtained after hydroly- sis of their melanoproteins.

In this latter paper a detailed analysis of the spectra of melanoproteins and melanins was pre- sented and the results were compared with model systems of melanoproteins and melanins produced from the precursors DOPA, adrenochrome and, dopamine. The IR spectra of natural and model melanoproteins were similar and showed charac- teristic strong bands at about 1540, 1650 and 3300 cm-l. Generally, after hydrolysis of these melanoproteins the IR spectra of all samples demonstrated the shapes which had been observed for the melanins. Menon et al. [35] obtained IR spectra of melanins isolated from black and red human hair according to Bolt's method [33] with some modifications. The spectra showed differ- ences between natural melanins and DOPA-

1160 B. Bilihska / Spectrochimica Acta Part A 52 (1996) 1157 1162

melanin, and variations depending on hair colour [35]; however, their interpretation of these spectra seems improbable. Hackman and Goldberg [34] have shown IR spectra of melanins isolated from black human hair, banana skin and flies; their interpretation is close to that of Tollin and Steel- ink [8].

The IR spectra of melanins isolated from hair and other natural sources discussed above with their interpretations, suggest that the isolation method may influence the structure of the melanin samples obtained. This problem was addressed for melanins isolated from human hair by different methods [36]. Infrared spectra were presented for the melanin isolated from dark, red and fair hair according to Bolt's method [33], and for melanin isolated from fair hair using the concentrated HC1 treatment. All methods used to isolate melanin from natural sources involve the danger of induc- ing structural changes in these very complicated macromolecules.

The early IR spectral analysis of Suzuki [7] showed that melanin structure modification re- sulted from the applied isolation method. The analysis of phaeomelanin structure using samples isolated from red hair (and feathers) was very difficult on the basis of IR studies [37]. Phaeome- lanin macromolecules have more complicated structures than eumelanins because of their differ- ent melanogenesis process.

Recently there has been increased interest con- cerning lipofuscins and their complexes with melanin. IR spectra of melanins isolated from the lipofuscins of human plasma have been reported by Hegedus et al. [38].

the investigation of the structure of synthetic melanins which uses IR spectral analysis applied to the natural melanins before and after chemical modification.

IR spectra of melanins synthesized from do- pamine and serotonin, and those isolated from the central nervous system have been presented by Ule et al. [39], whilst spectra of melanin obtained from Substantia nigra were reported by Mikulski [25] and Zecca et al. [40].

An interpretation of the DOPA-melanin spec- trum can be found in the paper of Von H6fs et al. [41]. Spectra of DOPA-melanin, melanin from dopamine, adrenochrome and model systems with protein were given in our paper [28]. An investiga- tion of the physico-chemical properties of natural and synthetic melanins and synthetic copolymers using IR analysis was performed by Miyake et al. [42]. IR spectroscopy was used to examine the macromolecular structure of natural and synthetic melanins after chemical modifications such as oxi- dation, reduction, methylation [43,44] and after treatment with sodium hydroxide, hydrochloric acid and heat [45].

IR spectroscopy has been applied to study the synthetic model melanins by Stepiefi and co-work- ers [46,47]. The IR spectra presented in these papers enabled us to estimate the possibility of copper ion binding to the macromolecular groups of the melanins created from catecholamines in the presence of metal. Metal ions interaction with natural and synthetic melanins has been investi- gated.

4. IR studies of melanin-metal ions complexes

3. IR studies of synthetic melanins

Melanins synthesized from their precursors are model systems for natural melanins. Sometimes, protein and metal ions are present in these sys- tems. Generally, there are two types of investiga- tion:study of the structure of oxidation products of the precursor with or without protein, metal ions or other substances, and study of complexes or systems consisting of synthetic melanin and added components. There is a third approach to

Complexation of Cu 2 + ions to a melanin has been shown by Khalifa [48] using IR spec- troscopy. The dependence on pH value of metal ion interactions with DOPA-melanin has been reported by Korytowski [43] together with the possibility of interaction of carboxylic and semi- quinone groups. Iron ions are very interesting, although they involve the limits of the methodol- ogy. Investigations of interactions of DOPA- melanin, melanin from dopamine and Sepia melanin with Fe 3 + ions depending on metal con-

B. Bilihska / Spectrochimica Acta Part A 52 (1996) 1157 1162 1161

tent using IR and M6ssbauer spectroscopy have been presented by Bardani et al. [49]. The possi- bilities of interactions between iron and car- boxylic, phenolic hydroxylic, amine groups and indolic systems have been realized and the pres- ence of paracrystalline subunits in melanin struc- tures was indicated [49].

The interaction between natural melanins iso- lated from human hair and cattle eyes and ferric ions has been investigated by our group [50,51]. It was shown that at low pH there is a significant possibility of interaction of metal with carboxylic groups [50,51], but phenolic hydroxylic groups may also participate [46]. Probably, the character of interactions depends not only on ions concen- tration and pH value, but also on the neighbour- ing groups of the ion.

5. Conclusion

IR spectroscopic analysis is a very satisfactory method by which to investigate isolation proce- dure for natural melanins and the oxidation reac- tions which lead to production of the melanin sample from its precursor. As yet, we do not have a proper analytical method, which is better than IR spectroscopy for the study of melanin molecu- lar groups and bonds. It is possible to resolve difficulties resulting from the molecular hetero- geneity of melanin granules during analysis of melanin IR spectra after chemical or thermal modification of the melanin samples. This ap- proach is very helpful for the investigations of different interactions of melanins with metal ions, drugs and also other chemicals.

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