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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgement
  8. References

The key structural factors underlying the unique black chromophore of eumelanin biopolymers have so far defied elucidation. Capitalizing on the ability of 1% polyvinylalcohol (PVA) to prevent pigment precipitation during melanogenesis in vitro, we have investigated the visible chromophore properties of soluble eumelanin-like polymers produced by biomimetic oxidation of 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic (DHICA) in 1% PVA-containing buffer at pH 7. Upon dilution DHI-eumelanin solutions exhibited almost linear visible absorbance changes, whereas DHICA-eumelanin displayed a remarkable deviation from linearity in simple buffer, but not in PVA-containing buffer. It is suggested that in DHICA polymers, exhibiting repeated interruptions of interring conjugation due to lack of planar conformations, the black chromophore is not due to an overlap of static entities defined intrinsically by the conjugation length across the carbon frame, but results largely from aggregation-related intermolecular perturbations of the π-electron systems which are extrinsic in character.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgement
  8. References

Eumelanins, the black functional biopolymers of human skin and hair, have so far escaped detailed structural elucidation because of their marked insolubility and high molecular heterogeneity. [1] A most intriguing, still unsettled issue concerning eumelanins is why they are black. Early views [2, 3], envisaged the occurrence of low-lying conduction bands and/or charge transfer interactions resembling those in quinhydrones, but more recent theories were based on an amorphous semiconductor model.[4].

Direct investigations of the broad band absorption spectrum of eumelanins elucidated the relative contributions of absorption versus scattering effects, however, attempts at identifying the basic chromophoric units have so far met with failure. Besides the evident relevance to skin photoprotection, developing a picture of the nature and interaction mechanisms of the individual chromophores underlying eumelanin black color is of general relevance to natural multichromophoric light-harvesting systems and is extremely important to dissect crucial structure–property function relationships, to guide the rational design and application of eumelanin-based thin films and materials for optoelectronic devices.

Valuable insights into eumelanin chromophore have derived from investigation of the species produced by oxidative polymerization of 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA), the key monomer precursors of eumelanins,[5] and from some effective approaches to solubility issues. image

The first approach was based on the synthesis of a glycated 5,6-dihydroxyindole precursor leading on oxidation to a water-soluble dark polymer which exhibited a distinct and at 314 nm and a broad visible absorption resembling that of natural eumelanins.[6] Spectrophotometric investigations revealed the coexistence of catechol and o-quinone moieties as the key to visible light absorption and pointed to a sort of chromophoric dynamic disorder, based on effective π-electron delocalization within the diverse molecular constituents (intrinsic components) and intermolecular chromophore perturbations through aggregation effects (extrinsic contributions).

The second entry came from the discovery that as low as 1% polyvinylalcohol (PVA) can prevent precipitation of growing eumelanin polymers during melanogenesis in vitro, thus allowing investigation of the chromophoric phases accompanying oxidation of DHI without confounding scattering effects. [7] Additional insight into the relationships between oxidation level, π-electron delocalization and HOMO-LUMO gaps in eumelanins derived from integrated pulse radiolysis and computational investigations of the transient species generated by oxidation of DHI, DHICA and related oligomers;[8, 9] from studies of the chromophoric variations occurring on passing from DHI monomer to a tetramer;[10] from the characterization of organic-soluble pigments from DHICA benzyl and octyl esters, [11] as well as from a study of DHI oxidation using a Cu(II) mimic of tyrosinase at very low temperatures.[12].

Herein, we report a systematic spectrophotometric investigation into the oxidative polymerization of DHI and DHICA in PVA-containing buffer with a view to addressing key pending issues, including: (1) the analogies and differences in the absorption spectra in solution of the intermediate and final species produced by oxidation of DHI and DHICA, (2) the visible versus UV absorbance changes with dilution of the two solubilized polymers, (3) the role of PVA in the aggregation phenomena and in the prevention of precipitate formation.

Material and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgement
  8. References

Materials

5,6-Dihydroxyindole (DHI, 1) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA, 2) were synthesized as previously reported. [13] All solvents were reagent grade. All chemicals were purchased from Aldrich and Acros Chemical Co.

Synthetic melanin preparation

Oxidation of the appropriate substrate (DHI, [14], DHICA, [15]; 12.5 mm) in 0.1 m phosphate buffer pH 7.0 was carried out with horseradish peroxidase (HRP, 25 U mL−1) and H2O2 (12.5 mm). After 20 min the solution was diluted 25- to 100-fold with 0.1 m phosphate buffer pH 7.0 for optical characterization.

PVA-added buffer was prepared by dissolving PVA in phosphate buffer in the appropriate weight ratio and warming to prevent cluster formation. PVA-containing buffers were thermostated at the desired temperature prior to use. When necessary, the mixture was treated with a solution of NaBH4 in methanol (20 mm).

Spectra were in a thermostated cell at 25°C with or without filtration through a membrane with 0.47 Millipores. In PVA-added medium the buffer used was prepared dissolving PVA in the appropriate weight ratio and warming to prevent cluster formation; prior starting oxidations, buffers were then thermostatted at the desired temperature.

Absorption spectra were taken on a spectrophotometer having the cell compartment thermostated at 25 ± 0.2°C.

Steady-state optical characterization

Fluorimetric measurements were performed with a fluorescence spectrophotometer Jasco FP 750. UV–Vis absorption spectra were recorded on a commercial UV-spectrophotometer Jasco V 650 vis dual-beam spectrophotometer.

Results and Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgement
  8. References

As reported previously[7], PVA is a most suitable additive for studies of chromophore development during eumelanin synthesis due to water solubility and good emulsifying and surfactant properties in neutral aqueous solution, allowing for efficient interactions with the growing synthetic melanin.

Based on the results reported in the preceding study, oxidation of DHI and DHICA was carried using peroxidase/H2O2 in 0.1 m phosphate buffer, pH 7, containing PVA (27000 Da) at 1 (w/w)% concentration. On visual inspection, in both cases the reaction led to the generation of dark brown species that persisted in solution over 24 h without apparent precipitation of solid material (as checked by filtration on a Millipore membrane). Kinetic analysis (not shown) plotting the rise in the visible absorbance (e.g. 530 nm) with time indicated that the maximum intensity was attained in the early hours followed by a plateau up to 24 h and more (as reported previously)[7, 16], during which time the overall absorption profile remained unchanged suggesting that melanization was complete. In control experiments carried out in the absence of PVA, the almost immediate precipitation of dark solid was observed with both indoles, with marked scattering effects partly covering chromophoric features (not shown).

Figure 1 shows the UV–Visible absorption spectra of the dark polymers produced by oxidation of DHI (Panel a) and DHICA (Panel b). These absorption profiles are not dissimilar from those of the insoluble DHI polymers and of intact natural eumelanin samples from Sepia [17] and hair, [18] in which, however, significant scattering contributions are present. Given the complete lack of scattering effects, the spectra reported herein can be taken to show the isolated visible light-absorbing oligomeric/polymeric chromophores contributing to eumelanin black appearance in the solid state.

image

Figure 1. UV–Visible absorption spectra taken at increasing dilutions of eumelanin-like polymers obtained by peroxidase/H2O2 oxidation of DHI (A) and DHICA (B) in phosphate buffer, pH 7.0 containing 1% PVA. Dilutions were obtained by adding fixed aliquots of the final oxidation mixture to phosphate buffer pH 7.0 containing 1% PVA w/w.

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Despite apparent similarities, e.g. in the UV band around 320 nm, the absorption profiles of the two polymers differed in the presence of a pronounced broad band around 500 nm in the DHI-derived eumelanin which was negligible in the case of the DHICA oxidation products. This difference, which accounts for the darker color of the DHI polymer, may denote either different intrinsic chromophores or the different participation of extrinsic contributions related to intermolecular (aggregation) interactions. To address this issue, the effects of dilution on the absorption properties of the soluble pigment were investigated and compared in the presence and in the absence of 1% PVA in the dilution medium (phosphate buffer at pH 7.0). The rationale of the experiments was that dilution in the absence of PVA would decrease both eumelanin and PVA concentrations without altering their relative ratio, thus allowing for intermolecular interactions and the possible rescuing of the PVA-segregated eumelanin chromophores. Conversely, dilution in PVA-containing medium decreases the eumelanin/PVA ratio, whereby eumelanin components a fortiori are caged into PVA and a fortiori cannot escape from the excess wrapping polymer. The above reasoning is founded on previous detailed investigations of solvent effects on the aggregation behavior of PVA, showing that water is a poor solvent for PVA molecules and that the polymer chains would be largely unperturbed coils, arranged midway between extended and collapsed conformations. They would therefore be capable of wrapping the growing indole chains thus affecting the oxidative polymerization process. Operation of effective hydrophobic interactions with growing eumelanin oligomers would shield these latter from both extensive water solvation and mutual interactions, permitting detection of stable eumelanin chromophores devoid of scattering effects.

For the sake of convenience, polymer concentration was referred to as the starting monomer concentration under conditions of complete oxidation. Moreover, the dilution effect was investigated under normalized conditions against a reference wavelength in the UV region, supposedly less affected by intermolecular interactions. Figure 2 shows plots of absorbance intensity ratios Ax/A307 at variable visible wavelengths for DHI-eumelanin as a function of dilution.

image

Figure 2. Normalized plots of the absorbance ratios Ax/A307 of DHI eumelanin at selected wavelengths (■ 384 nm, 420 nm, + 550 nm, * 690 nm) as a function of dilution in the presence (A) and in the absence (B) of PVA in the dilution buffer. For all wavelengths normalization was obtained by dividing all absorbance ratios by the relevant value at 425 μm concentration.

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Inspection of the curves in Fig. 2 revealed variable wavelength-dependent changes in the PVA-containing diluting medium with no well-defined trend. However, a slight but statistically significant increase in the Ax/A307 ratio was observed at the lowest concentrations (highest dilutions) in the absence of additional PVA (Panel B). The maximum effect was a 15% enhancement at 690 nm under the maximum dilution conditions examined which indicated a significant increase in the visible absorption versus the reference UV wavelength with dilution. In a control check it was consistently found that the absorbance at 307 nm (A307) decreased linearly with dilution, whereas visible absorbance values (Ax) deviated significantly from linearity.

Quite surprisingly, a remarkably greater enhancement in the visible absorbance, up to 80% increase in the A700/A307 ratio at the highest dilution tested, was observed in the case of DHICA eumelanin (Fig. 3).

image

Figure 3. Normalized plots of the absorbance ratios Ax/A307 of DHICA eumelanin at selected wavelengths (■ 384 nm, ▲ 420 nm, + 550 nm, * 690nm, - 750 nm) as a function of dilution in the presence (A) and in the absence (B) of PVA in the dilution buffer. For all wavelengths normalization was obtained by dividing all absorbance ratios by the relevant value at 300–400 μm concentration.

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Two most important observations regarding the chromophoric features of the soluble eumelanin polymers stem from the above results. The first point is that a variable yet well detectable increase in the visible absorption with dilution versus the reference UV maximum (307 nm) occurs in the absence of added PVA. This effect could be ascribed to intermolecular interactions between eumelanin chromophores that ensue when the high dilution conditions weaken the antiaggregating effects of PVA. By contrast, dilution in PVA-containing buffer does not results in significant enhancements in visible absorption, indicating that PVA is still effective in preventing intermolecular interactions between eumelanin chromophores. The finding that the absorbance increase affects selectively the visible region is consistent with aggregation or stacking effects involving quinonoid moieties, as the latter are apparently the main determinants of visible absorption.[7] Second, and most significantly, the enhancement of visible absorbance relative to reference UV maximum with dilution is much more pronounced in the case of DHICA-eumelanin than the DHI polymer. The latter exhibits a stronger visible band around 500 nm. It is suggested that DHI-eumelanin chromophore is due mainly to intrinsic effects relating to efficient π-electron delocalization within the largely planar oligomeric scaffolds, where the HOMO and LUMO can extend over significant portions of the molecular frame. On the contrary, DHICA-eumelanin visible chromophore would be largely extrinsic in character, reflecting mainly chromophoric perturbations by intermolecular aggregation-dependent effects which have been possible to demarcate and rescue under conditions of high PVA dilution.

It was recently shown that visible chromophores can hardly be generated from reduced (catecholic) indolic oligomers however extended the molecular size.[19] The reason lies in the peculiar mode of coupling of the indole units which is often incompatible with linear or rod-shaped oligomeric structures allowing fully extended π-electron delocalization. Thus, significantly red-shifted HOMO-LUMO gaps and visible light absorption can be achieved only via oxidized quinonoid moieties. The marked deviation from linearity of the visible absorbance decrease in DHICA eumelanin with dilution would provide now evidence that reversible aggregation phenomena inducing charge transfer or redox processes crucially affect absorption features in solution. Although it is difficult at present to predict the exact dynamics by which oligomeric DHICA-derived species approach each other, and the exact mechanism of external chromophore perturbation, it is worth noting that known oligomer structures from DHICA feature interring bondings that would hinder coplanar conformations and extended interring conjugation over a high number of units. Ensuing breaks of electronic communication between monomer units would account for a predictably poor visible extinction coefficients of individual chromophores and relatively less significant intrinsic contributions. Based on the new data, which confirm previous observations [6] by a different approach, it is now clear that eumelanin black chromophore and basic structure–property correlationship can be best explained by an improved paradigm which integrates the chemical disorder model [20] with dynamic contributions of interchromophoric interactions.[6] The basic underpinning of the proposed dynamic disorder model is that in solution eumelanin components can be thought of as chains of extended π-electron systems interrupted at irregular intervals, depending on monomer composition, oligomer structure and coupling patterns. Within each oligomer/polymer component the chromophoric entities may be dynamically equilibrated, e.g. by intramolecular tautomerism and by intermolecular perturbations, thus giving rise to chromophore coupling and mixing. Within this variable and dynamic scenario, control of aggregation by PVA or other additives may offer valuable and unexplored options to tailor eumelanin properties to well-defined functionality and application targets. As shown in previous chemical studies, PVA affects only to a limited extent the course of 5,6-dihydroxyindole polymerization in aqueous phosphate buffer, leading to minor qualitative alterations in the oligomer composition of the resulting eumelanin-like material relative to the same species produced in the absence of PVA, but causing no detectable effect on key structural units and mode of growth [7]. Dynamic light scattering (DLS) and small angle neutron scattering (SANS) analysis also demonstrated that PVA does not induce significant modifications in the mode of growth and shape of the aggregates, with the sole exception of size control [16]. Thus, although further work is required to better clarify this issue, available evidence would allow to conclude that PVA does not affect substantially the basic physicochemical properties of the eumelanin-type polymers produced by oxidative polymerization of 5,6-dihydroxyindoles. Aside from minor effects on medium polarity, the main role of PVA would be simply to create a net-like system thwarting DHI oligomer chain lengthening by confining and hindering the growth process within restricted regions of the medium.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgement
  8. References

Eumelanin black color appears to be intimately associated with the multichromophoric heterogeneous mixtures of different species that are produced by oxidation of DHI and DHICA.[2, 5, 15, 21] Herein, we report new data supporting the view that eumelanin chromophores in solution cannot be defined exclusively on an intrinsic basis by the conjugation length of the isolated absorbing species, but also by the external perturbations of conjugated chromophoric units caused by interactions between the oxidized and reduced polymer chain domains. This would entail a dynamic composition of intrinsically and extrinsically defined light-absorbing species rather than a mixture of static entities. Prevalence of DHI units would favor intrinsically absorbing components with extended intramolecular conjugation, whereas large proportions of DHICA units may enhance aggregation-dependent visible chromophore development.

Overall, these results provide novel breakthroughs into eumelanin chromophore under scattering-free conditions and offer a valuable methodological approach to differentiate directly between static and dynamic disorder in complex black chromophores. Whereas static disorder directly reflects molecular properties of individual components, the chromophoric dynamic disorder model introduces new options to tailor properties via fine modulation of aggregation processes, providing the basis for future applications of eumelanin-based optoelectronic devices.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgement
  8. References

This work was carried out within the aims of the EuMelaNet special interest group and was supported in part by grants from Italian Ministry of University (MIUR), PRIN 2008 projects.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgement
  8. References