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Fluorescence spectroscopy of synthetic melanin in solution
ARTICLE IN PRESS Journal of Luminescence 129 (2009) 44–49
Contents lists available at ScienceDirect
Journal of Luminescence journal homepage: www.elsevier.com/locate/jlumin
Fluorescence spectroscopy of synthetic melanin in solution G. Perna a, M.C. Frassanito a, G. Palazzo b, A. Gallone a, A. Mallardi c, P.F. Biagi d, V. Capozzi a, a
` di Foggia, Viale Pinto, 71100 Foggia, Italy Dipartimento di Scienze Biomediche, Universita ` di Bari, Via Orabona 4, 70126 Bari, Italy Dipartimento di Chimica, Universita c ICPS-CNR, Via Orabona 4, 70126 Bari, Italy d ` di Bari, Via Amendola 173, 70126 Bari, Italy Dipartimento Interateneo di Fisica, Universita b
a r t i c l e in f o
a b s t r a c t
Article history: Received 1 February 2008 Received in revised form 5 June 2008 Accepted 29 July 2008 Available online 6 August 2008
We report a detailed investigation of fluorescence properties of synthetic eumelanin pigment in solution. A complete set of fluorescence spectra in the near-UV and visible range is analysed. Excitation spectra at a few selected emission energies are also investigated. Our measurements support the hypothesis that fluorescence in eumelanin is related to chemically distinct oligomeric units that can be selectively excited. Fluorescence due to large oligomer systems is spectrally differentiated from that due to monomers and small oligomer systems. Fluorescence excitation measurements show the contribution of 5,6-dihydroxyndole-2-carboxylic acid and 5,6-dihydroxyndole monomers to the emission of small-size oligomers. & 2008 Elsevier B.V. All rights reserved.
Keywords: Eumelanin Fluorescence Physical properties Optical properties Biopolymers
1. Introduction The melanins are a class of biological pigments that provide coloration to animals and plants. In particular, eumelanin (a brown-black pigment formed from the oxidative polymerization of dihydroxyindolequinones) and pheomelanin (a brown-red pigment derived from cysteinyl-dopa) are the predominant forms of melanin in humans. Eumelanin is the more prevalent melanin pigment found in skin, hair and eyes, where it acts mainly as an excellent photoprotectant [1]. In addition, other roles have been proposed for eumelanin pigment: non-radiative converter of photoexcited electronic states [2], antioxidant and free radical scavenger [3]. However, melanin plays a dual role with respect to the sunlight’s UV radiation: on one hand, it is beneficial because it absorbs the UV radiation, thereby reducing the UV damage in cutaneous cells [4]; on the other hand, it is deleterious by acting as a photosensitizer that generates active oxygen species capable of causing DNA strand breaks, although such a role originates mostly in pheomelanin [4]. The balance of these two processes determines whether a beneficial action or a malignant transformation activated by the oxygen species occurs. The biological functions of the melanin pigment are strictly related to the physical properties. Although eumelanin has been widely investigated, some important questions about physical properties still remain open
Corresponding author. Tel.: +39 881 588053; fax: +39 881745887.
E-mail address: [email protected] (V. Capozzi). 0022-2313/$ - see front matter & 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jlumin.2008.07.014
[5]. In particular, an important open question is to better understand the structural organization of eumelanin: although it is accepted that eumelanin is an aggregate of indolic monomers, such as 5,6-dihydroxyindole (DHI), 5.6-dihydroxyndole-2-carboxylic acid (DHICA), 5,6-indolequinone (IQ) and semiquinone (SQ), it is still not well known how these monomer units are connected together. In higher organisms, natural eumelanin is formed in specialized organelles called melanosomes. The key enzyme of melanogenic pathway is the tyrosinase. This enzyme catalyzes the first rate-limiting steps of melanogenesis, the hydroxylation of L-tyrosine and the subsequent oxidation of the intermediate Ldopa to yield L-dopaquinone. Several other proteins residing in this specialized organelle, the melanosome, are involved in melanogenesis. Eumelanins are considered to be firmly bound to proteinaceous components, through covalent or ionic bonds [6]. It is important to remove the protein component in order to study the physical properties of the eumelanin biopolymer. A method to prepare eumelanin without protein is to synthesize it non-enzymatically, for example in a process starting from oxidation of tyrosine with hydrogen peroxide. The sample obtained in this way is called synthetic eumelanin. Fluorescence (FL) spectroscopy technique has been used over many years to investigate the physical properties of eumelanin [7,8]. In fact, eumelanin shows radiative emission if photoexcited by visible and UV radiation, although the radiative quantum yield is extremely low [2]. In addition, few differences exist between FL spectra of synthetic and natural eumelanin, apart from the presence of protein peaks in the latter [9]: so synthetic eumelanin
ARTICLE IN PRESS G. Perna et al. / Journal of Luminescence 129 (2009) 44–49
can be considered as a simple model to investigate the chemical and physical properties of eumelanin. However, the findings obtained for synthetic eumelanin cannot be directly generalized to the natural pigment, because of the presence of protein component in the latter. Instead, such findings can be used as starting points to investigate the physical properties (absorption, emission, etc.) of natural eumelanin and to study the role of proteins in the structural organization of the natural biopolymer. Recently, very accurate measurements of FL and fluorescence excitation (FLE) spectroscopy of synthetic melanin solutions have been performed by the group of Meredith and co-workers [2,10,11]. The FL spectra, consisting of a single broad band, were interpreted according to the ‘‘chemical disorder model’’ [12], by considering the eumelanin structure as composed of an ensemble of chemically distinct macromolecules (oligomers), each with a different highest occupied molecular orbital–lowest unoccupied molecular orbital (HOMO–LUMO) energy gap. The broad emission band was due to the convolution of many different narrower peaks, each one corresponding to the FL of a different macromolecule and, at the same time, the strong excitation wavelength dependence was explained by a selective excitation of differentsized oligomers [11]. However, the idea that eumelanin is a heterogeneous pigment containing many different oligomeric structures is present in scientific literature from several decades. In fact, a strong debate has been going on for many years on whether eumelanin is a highly cross-linked heteropolymer [13–16] or composed of oligomeric nanoaggregates [17–19]. In the last years, several theoretical [20,21] and experimental [22,23] studies have been published in support of the oligomeric structure. In Refs. [2,10,11] synthetic melanin was submitted to acid and basic treatments before spectroscopic measurements. In this work we report on the FL properties of chemically untreated commercial synthetic eumelanin in solution. The FL spectra present features related to large oligomer and monomers, and small oligomer systems. This proves the presence of strong chemical, size heterogeneity in the biopolymer, which are related to its specific photoprotective role.
2. Materials and methods 2.1. Sample preparation
45
between 4.960 and 2.296 eV. A band pass width of 10 nm and an integration time of 1 s were used. FLE measurements were performed between 5 eV and the energies of the emission at 2.850, 2.638, 2.431 and 2.296 eV. The band pass width and integration time values were the same as for FL measurements. Background scans were performed under identical instrumental conditions using HPLC-grade water. The FL spectra have been also normalised to account for excitation beam penetration depth and emission reabsorption, according to the procedure described in Refs. [10,11].
2.3. Absorption spectroscopy Absorption spectra were measured by means of a JASCO V-530 double beam spectrophotometer in the 240–1100 nm spectral range, at a scan rate of 400 nm/min and with a slit band width of 2 nm.
3. Results and discussion 3.1. Absorption and fluorescence spectra The absorption spectrum at room temperature of synthetic eumelanin solution is shown in Fig. 1. Such spectrum is characterized by a strong absorption in the UV and visible spectral range, with a nearly featureless absorption lineshape and absorbance values monotonically increasing from near-IR to UV spectral region. A weak feature at about 4.6 eV is visible in the absorption spectrum: it could be due to absorption by residual tyrosine in the synthetic sample. FL spectra of synthetic eumelanin measured at different excitation energies are shown in Fig. 2. The features of particular interest are the following:
at lower (visible) excitation energies, a single broad band
characterizes the FL spectra, whereas a few bands are present in the spectra at higher (near-UV) excitation energies; the spectral position of the FL bands redshifts as the excitation energy decreases; the intensity and full-width at half-maximum (FWHM) of the FL emission decrease with excitation energy.
Commercial synthetic eumelanin powder produced by oxidation of tyrosine with hydrogen peroxide (Sigma-Aldrich) was used to prepare eumelanin samples. Briefly, an excess of eumelanin powder was dispersed in HPLC-grade water filtered through 0.2 mm pore size nylon membrane (Whatman) at the ratio of 1 mg of eumelanin to 1 mL of water. Since melanins are almost insoluble in most common solvents, such mixture was firstly sonicated for 30 min and then centrifuged (Heraeus sepatech ‘‘Biofuge 13’’) at 13,000 rpm for 30 min to remove large insoluble aggregates. The decanted eumelanin dispersion resulting from centrifugation was a homogeneous solution; such sample was used for FL measurements. The concentration value of the eumelanin was estimated, by means of the Lambert–Beer law, to be about 105 M. 2.2. Fluorescence spectroscopy FL and excitation spectra of eumelanin samples were measured by means of a Cary Eclipse FL spectrophotometer. All spectra were collected using a 1 cm 0.5 cm rectangular quartz cuvette. FL measurements were performed using several excitation energies
Fig. 1. Absorbance spectrum of the synthetic eumelanin solution at room temperature.
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G. Perna et al. / Journal of Luminescence 129 (2009) 44–49
Fig. 2. Fluorescence spectra at room temperature of a synthetic melanin solution at different excitation energies. The excitation energy value is reported on the right hand side of each spectrum and the sensitivity factor is reported on the left hand side of each spectrum.
linked to form an oligomer, delocalization of HOMO and LUMO wavefunctions occurs if the oligomer presents a planar structure. In this case, a redshift of the HOMO–LUMO gap of the oligomer species with respect to that of the monomer units results. In addition, larger the number of coplanar monomers forming the oligomer, lower the energy of the corresponding radiative emission. The authors of Ref. [12] introduced the name ‘‘chemical disorder model’’ to explain the nature of the broad band characterizing their FL spectra of melanin solution; such a broad band is due to the contribution of many different narrower peaks, each one corresponding to the HOMO–LUMO radiative recombination of slightly different chemical species, as monomer units and oligomer groups. We agree with such chemical disorder scheme to explain our FL spectra. In addition, the contribution of radiative emission due to monomeric units and small oligomer groups can be discriminated from the contribution of polymeric groups in the spectra shown in Fig. 2. In particular, the FL band observed at about 2.85 eV at higher excitation energies is due to FL from monomers and small oligomer groups. In fact, many monomers (DHI, IQ, DHICA), with different stable tautomeric forms, can coexist in melanin aqueous solution [25]. In contrast, the FL band at about 2.45 eV observed at higher excitation energies, which shifts to about 2.10 eV at lower ones, arises from radiative recombination of polymeric groups, i.e. entities made of a higher number of coplanar indolic units. In fact, redshift of the HOMO–LUMO gap occurs on oligomerization and stacking of oligomeric sheets [1]. The low-energy limit of melanin FL at about 1.8 eV is related to the emission from species having larger size planar polymeric groups (smaller HOMO–LUMO gap) and is capable of radiative emission. In addition, some monomeric and oligomeric indolic species can contribute to such low-energy FL, because some monomers and small oligomers have HOMO–LUMO gap in this spectral region [20,25,26].
3.2. Fluorescence excitation spectra The spectra at higher excitation energies are quite similar to those recently reported by Mosca et al. [24] for melanins from opioid peptides, whereas they are quite different from those reported by Meredith and Riesz [2] and Nighswander-Rempel et al. [10,11] for synthetic eumelanin. Indeed, the latter authors show FL spectra as mainly consisting of a single broad band. The spectral position of such band is blueshifted with respect to the low-energy band reported in Fig. 2. Further, this band redshifts with excitation energy, in agreement with our results. However, some differences exist between our preparation method of synthetic melanin samples and the method used by authors of Refs. [2,10,11]. In fact, our melanin samples were prepared without chemical treatments, whereas the melanin samples in Refs. [2,10,11] were submitted to chemical treatments. In particular, they first carried out acid precipitation, retained the pellet and discarded the surnatant (in order to remove monomers or small oligomers); the pellet was then fully dissolved in a basic NaOH solution (pH ¼ 10). Such a procedure results in a eumelanin solution containing essentially high-molecular-weight compounds. Instead, our melanin solution contains both oligomeric and polymeric macromolecules (with the exception of very large aggregates insoluble in neutral water). FL spectra of eumelanin are due to radiative transitions between energy levels of the molecular groups present in the biopolymer; hence, the energy of radiative emission depends on the HOMO–LUMO transition, which is also related to the specific molecular species and the degree of delocalization of HOMO and LUMO wavefunctions. For example, when monomeric units are
In order to gain further information about FL mechanism in synthetic eumelanin, FLE spectra have been obtained for emission at different energies; a few of these spectra are shown in Fig. 3. The excitation spectrum recorded at 2.850 eV is characterized by a well-defined peak at about 3.9 eV, which is superimposed to a lineshape similar to that of the absorption spectrum of eumelanin (monotonic increase with energy [20], as shown in Fig. 1, as expected because a rise of absorption coefficient causes an increase of emission intensity. The peak at 3.9 eV is also present in the other FLE spectra, but its intensity decreases by lowering the emission energy. This behaviour suggests that the emission at 2.850 eV, which corresponds to the energy of radiative band due to monomers and small oligomer units in Fig. 2, is mainly pumped by some specific fluorophores having molecular excited states at about 3.9 eV. This result is partly in disagreement with previously reported excitation spectra of synthetic melanin, which have revealed the presence of a spectral feature at about 3.4 eV, irrespective of the emission energy [10]. This feature has been associated with the presence of DHICA monomers, whose experimental HOMO–LUMO gap in solution is about 3.8 eV [12], linked at the edges of melanin oligomers. This monomer may also contribute to the peak at 3.9 eV in Fig. 3. In addition, several computational studies have found that the HOMO–LUMO gap of DHI monomer is approximately 4 eV [25,26]. Hence, DHI can also contribute to the peak at 3.9 eV in the FLE spectrum of Fig. 3. Since the emission energy at which the peak in FLE spectrum has the maximum intensity is at
ARTICLE IN PRESS G. Perna et al. / Journal of Luminescence 129 (2009) 44–49
47
Fig. 3. Fluorescence excitation spectra at room temperature of a synthetic melanin solution at different emission energies: (a) 2.850, (b) 2.638, (c) 2.431 and (d) 2.296 eV.
2.85 eV, it is reasonable that these two monomers, after having absorbed energy from the probe beam, are able to transfer it to other monomers and small oligomers, at which they are linked. Finally, these latter radiatively emit the received photon energy.
3.3. Deconvolution of fluorescence spectra A deconvolution procedure by using Gaussian functions has been performed to analyse the FL spectra better. A few of these deconvolutions are shown in Fig. 4. The fit parameters are the energy position of the band maximum, the maximum intensity and the FWHM of the fitting Gaussian band. The continuous lineshape is the sum of the Gaussian bands (dashed lines) and it is obtained by a least-squares fit procedure to the measured spectra (dots). It is clearly evident that in the spectra at higher excitation energies, three Gaussian bands (labelled as A–C in Fig. 4) are needed to fit the experimental data quite well, whereas the FL spectra at lower excitation energies are well fitted by using only the A Gaussian band. In agreement with the discussion above, the A band emission, persisting from higher to lower excitation energies, is due to radiative recombination from large oligomeric and polymeric species, whereas the C band is due to radiative recombination from monomers and small oligomer species. As for the B band at about 2.64 eV, which is present in the spectra at larger excitation energy, at present it is not possible to identify the related chromophores. Some chemical species having large quantum yields may be involved in such recombination band; in fact, it has been reported that melanin quantum yield presents a slight maximum at about 2.6 eV [2,10]. Since it has been calculated [25] that an IQ tautomer presents an absorption band at 2.64 eV, the B band at 2.64 eV may be due to such chemical
species, in agreement with the result of Nighswander-Rempel et al. [10]. The excitation energy dependence of peak energy, FWHM and intensity of each Gaussian band has been summarized in Fig. 5. The spectral position and the FWHM of the A band present a ‘‘plateau’’ at about 2.39 eV and 550 meV, respectively, for excitation energies in the UV region. On the contrary, the spectral position redshifts and the FWHM decrease on lowering the excitation energy in the visible region. This behaviour is in good agreement with the chemical disorder scheme, which involves the presence of a distribution of FL centres, due to radiative emission of planar polymeric chemical species having different sizes. When excitation in the UV region occurs, all the FL centres can absorb light, because the HOMO–LUMO gap energy of all polymeric species is lower than the energy of exciting light; consequently, all the FL centres are involved in the emission process and, therefore, the peak energy and FWHM remain almost constant, irrespective of the excitation energy. On the contrary, the excitation energies in the visible region are not sufficient to excite all the polymeric species and selective excitation of polymeric species with lower HOMO–LUMO gap occurs as the excitation energy decreases. The decrease of the number of species having larger HOMO–LUMO gap (i.e. smaller size) causes a redshift of the peak energy and a bleaching of the peak intensity. At the same time, by lowering the excitation energy, the number of species or radiative centres that can absorb the excitation light (i.e. the centres having lower HOMO–LUMO gaps) decreases and this effect causes a narrowing of the FL band. The same behaviour can be observed for the spectral position, FWHM and FL intensity of the C band. In this case, the distribution of FL centres, due to monomers and small oligomer groups, is centred at higher energy than that due to polymeric species, because of the reduced size. So the FL of this feature is possible
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G. Perna et al. / Journal of Luminescence 129 (2009) 44–49
Fig. 4. Deconvolution of a few fluorescence spectra (dots) of a synthetic melanin solution by means of Gaussian lineshapes (dashed lines).
only for excitation energy in the UV range. In fact, this emission is present only at the largest excitation energy. Also the B band at 2.64 eV can be observed only for the highest excitation energies. Its spectral position and FWHM remain almost constant, because of the reduced number of chemical species involved in such FL recombinations.
4. Conclusions In conclusion, we have presented an investigation of FL and FLE spectra of chemically untreated synthetic melanin solution. Comparison of our results with the published FL spectra shows some peculiar difference, due to chemical treatments performed on melanin solution before FL measurements. In particular, our measurements on untreated melanin indicate that the FL due to ensembles of large oligomer systems can be spectrally differentiated from that due to monomers and small oligomer systems when excitation with sufficiently high energy occurs. On the other
hand, FL spectra of other authors [2,10] are characterized by a single broad band, due to chemically distinct macromolecules (equivalently, monomers and oligomers having different sizes). FLE measurements reveal that the band at low energy is pumped by the disexcitation pathway of DHICA and DHI monomers linked to small-size oligomers. Deconvolution procedure of our FL spectra by means of Gaussian functions has revealed the presence of a further peak at 2.64 eV, whose nature is still unclear at present. The spectral position, FWHM and intensity of the two broad bands depend on the excitation energy, in agreement with the ‘‘chemical disorder model’’. This investigation can be used as a starting point to investigate the physical properties of eumelanin pigment by means of FL measurements. In fact, investigations about the natural eumelanin pigment are in progress to clarify how the FL properties of eumelanin change when protein content is considered. This is for answering a very important question: are the proteins a residual from the bio-synthesis process or are they fundamental for the biological functions?
ARTICLE IN PRESS G. Perna et al. / Journal of Luminescence 129 (2009) 44–49
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References
Fig. 5. Energy (a) and FWHM (b) of the three Gaussian FL bands, resulting from the deconvolution procedure (Fig. 4), as a function of the excitation energy. Error bars were calculated through the errors of the fit parameters provided by the fitting procedure.
Acknowledgements We are grateful to R. Cicero for stimulating discussion and to D. Fiorentino and D. Fiocco for technical cooperation.
[1] P. Meredith, B.J. Powell, J. Riesz, S.P. Nighswander-Rempel, M.R. Pederson, E.G. Moore, Soft Matter 2 (2006) 37. [2] P. Meredith, J. Riesz, Photochem. Photobiol. 79 (2004) 211. [3] T. Sarna, B. Pilas, E.J. Land, T.G. Truscott, Biochim. Biophys. Acta 883 (1986) 162. [4] S. Takeuchi, W. Zhang, K. Wakamatsu, S. Ito, V.J. Hearing, K.H. Kraemer, D.E. Brash, Proc. Natl. Acad. Sci. 101 (2004) 15076. [5] P. Meredith, T. Sarna, Pigment Cell Res. 19 (2006) 572. [6] L. Zeise, Analytical methods for characterization and identification of eumelanins, in: L. Zeise, M.R. Chedekel, T.B. Fitzpatrick (Eds.), Melanin: Its Role in Human Photoprotection, Valdenmar Publishing, Overland Park, KS, 1995, pp. 65–79. [7] J.M. Gallas, M. Eisner, Photochem. Photobiol. 45 (1987) 595. [8] J.B. Nofsinger, J.D. Simon, Photochem. Photobiol. 74 (2001) 31. [9] G. Perna, M.C. Frassanito, G. Palazzo, A. Gallone, A. Mallardi, P.F. Biagi, V. Capozzi, Unpublished results. [10] S.P. Nighswander-Rempel, J. Riesz, J. Gilmore, J.P. Bothma, P. Meredith, J. Phys. Chem. B 109 (2005) 20692. [11] S.P. Nighswander-Rempel, J. Riesz, J. Gilmore, P. Meredith, J. Chem. Phys. 123 (2005) 194901. [12] M. Linh Tran, B.J. Powell, P. Meredith, Biophys. J. 90 (2006) 743. [13] H.C. Longuet-Higgins, Arch. Biochim. Biophys. Acta 186 (1960) 231. [14] A. Pullman, B. Pullman, Biochim. Biophys. Acta 54 (1961) 384. [15] D.S. Galvao, M.J. Caldas, J. Chem. Phys. 88 (1988) 4088. [16] D.S. Galvao, M.J. Caldas, J. Chem. Phys. 92 (1990) 2630. [17] J. Cheng, S.C. Moss, M. Eisner, P. Zcshack, Pigment Cell Res. 7 (1994) 255. [18] J. Cheng, S.C. Moss, M. Eisner, Pigment Cell Res. 7 (1994) 263. [19] G.W. Zajac, J.M. Gallas, J. Cheng, M. Eisner, S.C. Moss, A.E. Alvarado-Swaisgood, Biochim. Biophys. Acta 1199 (1994) 271. [20] K.B. Stark, J.M. Gallas, G.W. Zajac, M. Eisner, J.T. Golab, J. Phys. Chem. B 107 (2003) 11558. [21] K. Bochenek, E. Gudowska-Nowak, Chem. Phys. Lett. 373 (2003) 532. [22] J.M. Gallas, K.C. Littrel, S. Seifert, G.W. Zajac, P. Thiyagarajan, Biophys. J. 77 (1999) 1135–1142. [23] C.M.R. Clancy, J.D. Simon, Biochemistry 40 (2001) 13353. [24] L. Mosca, C. De Marco, M. Fontana, A.M. Rosei, Arch. Biochem. Biophys. 371 (1999) 63. [25] Y.V. Il’ichev, J.D. Simon, J. Phys. Chem. B 107 (2003) 7162. [26] B.J. Powell, T. Baruah, N. Bernstein, K. Brake, R.H. McKenzie, P. Meredith, M.R. Pederson, J. Chem. Phys 120 (2004) 8608.
Contents lists available at ScienceDirect
Journal of Luminescence journal homepage: www.elsevier.com/locate/jlumin
Fluorescence spectroscopy of synthetic melanin in solution G. Perna a, M.C. Frassanito a, G. Palazzo b, A. Gallone a, A. Mallardi c, P.F. Biagi d, V. Capozzi a, a
` di Foggia, Viale Pinto, 71100 Foggia, Italy Dipartimento di Scienze Biomediche, Universita ` di Bari, Via Orabona 4, 70126 Bari, Italy Dipartimento di Chimica, Universita c ICPS-CNR, Via Orabona 4, 70126 Bari, Italy d ` di Bari, Via Amendola 173, 70126 Bari, Italy Dipartimento Interateneo di Fisica, Universita b
a r t i c l e in f o
a b s t r a c t
Article history: Received 1 February 2008 Received in revised form 5 June 2008 Accepted 29 July 2008 Available online 6 August 2008
We report a detailed investigation of fluorescence properties of synthetic eumelanin pigment in solution. A complete set of fluorescence spectra in the near-UV and visible range is analysed. Excitation spectra at a few selected emission energies are also investigated. Our measurements support the hypothesis that fluorescence in eumelanin is related to chemically distinct oligomeric units that can be selectively excited. Fluorescence due to large oligomer systems is spectrally differentiated from that due to monomers and small oligomer systems. Fluorescence excitation measurements show the contribution of 5,6-dihydroxyndole-2-carboxylic acid and 5,6-dihydroxyndole monomers to the emission of small-size oligomers. & 2008 Elsevier B.V. All rights reserved.
Keywords: Eumelanin Fluorescence Physical properties Optical properties Biopolymers
1. Introduction The melanins are a class of biological pigments that provide coloration to animals and plants. In particular, eumelanin (a brown-black pigment formed from the oxidative polymerization of dihydroxyindolequinones) and pheomelanin (a brown-red pigment derived from cysteinyl-dopa) are the predominant forms of melanin in humans. Eumelanin is the more prevalent melanin pigment found in skin, hair and eyes, where it acts mainly as an excellent photoprotectant [1]. In addition, other roles have been proposed for eumelanin pigment: non-radiative converter of photoexcited electronic states [2], antioxidant and free radical scavenger [3]. However, melanin plays a dual role with respect to the sunlight’s UV radiation: on one hand, it is beneficial because it absorbs the UV radiation, thereby reducing the UV damage in cutaneous cells [4]; on the other hand, it is deleterious by acting as a photosensitizer that generates active oxygen species capable of causing DNA strand breaks, although such a role originates mostly in pheomelanin [4]. The balance of these two processes determines whether a beneficial action or a malignant transformation activated by the oxygen species occurs. The biological functions of the melanin pigment are strictly related to the physical properties. Although eumelanin has been widely investigated, some important questions about physical properties still remain open
Corresponding author. Tel.: +39 881 588053; fax: +39 881745887.
E-mail address: [email protected] (V. Capozzi). 0022-2313/$ - see front matter & 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jlumin.2008.07.014
[5]. In particular, an important open question is to better understand the structural organization of eumelanin: although it is accepted that eumelanin is an aggregate of indolic monomers, such as 5,6-dihydroxyindole (DHI), 5.6-dihydroxyndole-2-carboxylic acid (DHICA), 5,6-indolequinone (IQ) and semiquinone (SQ), it is still not well known how these monomer units are connected together. In higher organisms, natural eumelanin is formed in specialized organelles called melanosomes. The key enzyme of melanogenic pathway is the tyrosinase. This enzyme catalyzes the first rate-limiting steps of melanogenesis, the hydroxylation of L-tyrosine and the subsequent oxidation of the intermediate Ldopa to yield L-dopaquinone. Several other proteins residing in this specialized organelle, the melanosome, are involved in melanogenesis. Eumelanins are considered to be firmly bound to proteinaceous components, through covalent or ionic bonds [6]. It is important to remove the protein component in order to study the physical properties of the eumelanin biopolymer. A method to prepare eumelanin without protein is to synthesize it non-enzymatically, for example in a process starting from oxidation of tyrosine with hydrogen peroxide. The sample obtained in this way is called synthetic eumelanin. Fluorescence (FL) spectroscopy technique has been used over many years to investigate the physical properties of eumelanin [7,8]. In fact, eumelanin shows radiative emission if photoexcited by visible and UV radiation, although the radiative quantum yield is extremely low [2]. In addition, few differences exist between FL spectra of synthetic and natural eumelanin, apart from the presence of protein peaks in the latter [9]: so synthetic eumelanin
ARTICLE IN PRESS G. Perna et al. / Journal of Luminescence 129 (2009) 44–49
can be considered as a simple model to investigate the chemical and physical properties of eumelanin. However, the findings obtained for synthetic eumelanin cannot be directly generalized to the natural pigment, because of the presence of protein component in the latter. Instead, such findings can be used as starting points to investigate the physical properties (absorption, emission, etc.) of natural eumelanin and to study the role of proteins in the structural organization of the natural biopolymer. Recently, very accurate measurements of FL and fluorescence excitation (FLE) spectroscopy of synthetic melanin solutions have been performed by the group of Meredith and co-workers [2,10,11]. The FL spectra, consisting of a single broad band, were interpreted according to the ‘‘chemical disorder model’’ [12], by considering the eumelanin structure as composed of an ensemble of chemically distinct macromolecules (oligomers), each with a different highest occupied molecular orbital–lowest unoccupied molecular orbital (HOMO–LUMO) energy gap. The broad emission band was due to the convolution of many different narrower peaks, each one corresponding to the FL of a different macromolecule and, at the same time, the strong excitation wavelength dependence was explained by a selective excitation of differentsized oligomers [11]. However, the idea that eumelanin is a heterogeneous pigment containing many different oligomeric structures is present in scientific literature from several decades. In fact, a strong debate has been going on for many years on whether eumelanin is a highly cross-linked heteropolymer [13–16] or composed of oligomeric nanoaggregates [17–19]. In the last years, several theoretical [20,21] and experimental [22,23] studies have been published in support of the oligomeric structure. In Refs. [2,10,11] synthetic melanin was submitted to acid and basic treatments before spectroscopic measurements. In this work we report on the FL properties of chemically untreated commercial synthetic eumelanin in solution. The FL spectra present features related to large oligomer and monomers, and small oligomer systems. This proves the presence of strong chemical, size heterogeneity in the biopolymer, which are related to its specific photoprotective role.
2. Materials and methods 2.1. Sample preparation
45
between 4.960 and 2.296 eV. A band pass width of 10 nm and an integration time of 1 s were used. FLE measurements were performed between 5 eV and the energies of the emission at 2.850, 2.638, 2.431 and 2.296 eV. The band pass width and integration time values were the same as for FL measurements. Background scans were performed under identical instrumental conditions using HPLC-grade water. The FL spectra have been also normalised to account for excitation beam penetration depth and emission reabsorption, according to the procedure described in Refs. [10,11].
2.3. Absorption spectroscopy Absorption spectra were measured by means of a JASCO V-530 double beam spectrophotometer in the 240–1100 nm spectral range, at a scan rate of 400 nm/min and with a slit band width of 2 nm.
3. Results and discussion 3.1. Absorption and fluorescence spectra The absorption spectrum at room temperature of synthetic eumelanin solution is shown in Fig. 1. Such spectrum is characterized by a strong absorption in the UV and visible spectral range, with a nearly featureless absorption lineshape and absorbance values monotonically increasing from near-IR to UV spectral region. A weak feature at about 4.6 eV is visible in the absorption spectrum: it could be due to absorption by residual tyrosine in the synthetic sample. FL spectra of synthetic eumelanin measured at different excitation energies are shown in Fig. 2. The features of particular interest are the following:
at lower (visible) excitation energies, a single broad band
characterizes the FL spectra, whereas a few bands are present in the spectra at higher (near-UV) excitation energies; the spectral position of the FL bands redshifts as the excitation energy decreases; the intensity and full-width at half-maximum (FWHM) of the FL emission decrease with excitation energy.
Commercial synthetic eumelanin powder produced by oxidation of tyrosine with hydrogen peroxide (Sigma-Aldrich) was used to prepare eumelanin samples. Briefly, an excess of eumelanin powder was dispersed in HPLC-grade water filtered through 0.2 mm pore size nylon membrane (Whatman) at the ratio of 1 mg of eumelanin to 1 mL of water. Since melanins are almost insoluble in most common solvents, such mixture was firstly sonicated for 30 min and then centrifuged (Heraeus sepatech ‘‘Biofuge 13’’) at 13,000 rpm for 30 min to remove large insoluble aggregates. The decanted eumelanin dispersion resulting from centrifugation was a homogeneous solution; such sample was used for FL measurements. The concentration value of the eumelanin was estimated, by means of the Lambert–Beer law, to be about 105 M. 2.2. Fluorescence spectroscopy FL and excitation spectra of eumelanin samples were measured by means of a Cary Eclipse FL spectrophotometer. All spectra were collected using a 1 cm 0.5 cm rectangular quartz cuvette. FL measurements were performed using several excitation energies
Fig. 1. Absorbance spectrum of the synthetic eumelanin solution at room temperature.
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Fig. 2. Fluorescence spectra at room temperature of a synthetic melanin solution at different excitation energies. The excitation energy value is reported on the right hand side of each spectrum and the sensitivity factor is reported on the left hand side of each spectrum.
linked to form an oligomer, delocalization of HOMO and LUMO wavefunctions occurs if the oligomer presents a planar structure. In this case, a redshift of the HOMO–LUMO gap of the oligomer species with respect to that of the monomer units results. In addition, larger the number of coplanar monomers forming the oligomer, lower the energy of the corresponding radiative emission. The authors of Ref. [12] introduced the name ‘‘chemical disorder model’’ to explain the nature of the broad band characterizing their FL spectra of melanin solution; such a broad band is due to the contribution of many different narrower peaks, each one corresponding to the HOMO–LUMO radiative recombination of slightly different chemical species, as monomer units and oligomer groups. We agree with such chemical disorder scheme to explain our FL spectra. In addition, the contribution of radiative emission due to monomeric units and small oligomer groups can be discriminated from the contribution of polymeric groups in the spectra shown in Fig. 2. In particular, the FL band observed at about 2.85 eV at higher excitation energies is due to FL from monomers and small oligomer groups. In fact, many monomers (DHI, IQ, DHICA), with different stable tautomeric forms, can coexist in melanin aqueous solution [25]. In contrast, the FL band at about 2.45 eV observed at higher excitation energies, which shifts to about 2.10 eV at lower ones, arises from radiative recombination of polymeric groups, i.e. entities made of a higher number of coplanar indolic units. In fact, redshift of the HOMO–LUMO gap occurs on oligomerization and stacking of oligomeric sheets [1]. The low-energy limit of melanin FL at about 1.8 eV is related to the emission from species having larger size planar polymeric groups (smaller HOMO–LUMO gap) and is capable of radiative emission. In addition, some monomeric and oligomeric indolic species can contribute to such low-energy FL, because some monomers and small oligomers have HOMO–LUMO gap in this spectral region [20,25,26].
3.2. Fluorescence excitation spectra The spectra at higher excitation energies are quite similar to those recently reported by Mosca et al. [24] for melanins from opioid peptides, whereas they are quite different from those reported by Meredith and Riesz [2] and Nighswander-Rempel et al. [10,11] for synthetic eumelanin. Indeed, the latter authors show FL spectra as mainly consisting of a single broad band. The spectral position of such band is blueshifted with respect to the low-energy band reported in Fig. 2. Further, this band redshifts with excitation energy, in agreement with our results. However, some differences exist between our preparation method of synthetic melanin samples and the method used by authors of Refs. [2,10,11]. In fact, our melanin samples were prepared without chemical treatments, whereas the melanin samples in Refs. [2,10,11] were submitted to chemical treatments. In particular, they first carried out acid precipitation, retained the pellet and discarded the surnatant (in order to remove monomers or small oligomers); the pellet was then fully dissolved in a basic NaOH solution (pH ¼ 10). Such a procedure results in a eumelanin solution containing essentially high-molecular-weight compounds. Instead, our melanin solution contains both oligomeric and polymeric macromolecules (with the exception of very large aggregates insoluble in neutral water). FL spectra of eumelanin are due to radiative transitions between energy levels of the molecular groups present in the biopolymer; hence, the energy of radiative emission depends on the HOMO–LUMO transition, which is also related to the specific molecular species and the degree of delocalization of HOMO and LUMO wavefunctions. For example, when monomeric units are
In order to gain further information about FL mechanism in synthetic eumelanin, FLE spectra have been obtained for emission at different energies; a few of these spectra are shown in Fig. 3. The excitation spectrum recorded at 2.850 eV is characterized by a well-defined peak at about 3.9 eV, which is superimposed to a lineshape similar to that of the absorption spectrum of eumelanin (monotonic increase with energy [20], as shown in Fig. 1, as expected because a rise of absorption coefficient causes an increase of emission intensity. The peak at 3.9 eV is also present in the other FLE spectra, but its intensity decreases by lowering the emission energy. This behaviour suggests that the emission at 2.850 eV, which corresponds to the energy of radiative band due to monomers and small oligomer units in Fig. 2, is mainly pumped by some specific fluorophores having molecular excited states at about 3.9 eV. This result is partly in disagreement with previously reported excitation spectra of synthetic melanin, which have revealed the presence of a spectral feature at about 3.4 eV, irrespective of the emission energy [10]. This feature has been associated with the presence of DHICA monomers, whose experimental HOMO–LUMO gap in solution is about 3.8 eV [12], linked at the edges of melanin oligomers. This monomer may also contribute to the peak at 3.9 eV in Fig. 3. In addition, several computational studies have found that the HOMO–LUMO gap of DHI monomer is approximately 4 eV [25,26]. Hence, DHI can also contribute to the peak at 3.9 eV in the FLE spectrum of Fig. 3. Since the emission energy at which the peak in FLE spectrum has the maximum intensity is at
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Fig. 3. Fluorescence excitation spectra at room temperature of a synthetic melanin solution at different emission energies: (a) 2.850, (b) 2.638, (c) 2.431 and (d) 2.296 eV.
2.85 eV, it is reasonable that these two monomers, after having absorbed energy from the probe beam, are able to transfer it to other monomers and small oligomers, at which they are linked. Finally, these latter radiatively emit the received photon energy.
3.3. Deconvolution of fluorescence spectra A deconvolution procedure by using Gaussian functions has been performed to analyse the FL spectra better. A few of these deconvolutions are shown in Fig. 4. The fit parameters are the energy position of the band maximum, the maximum intensity and the FWHM of the fitting Gaussian band. The continuous lineshape is the sum of the Gaussian bands (dashed lines) and it is obtained by a least-squares fit procedure to the measured spectra (dots). It is clearly evident that in the spectra at higher excitation energies, three Gaussian bands (labelled as A–C in Fig. 4) are needed to fit the experimental data quite well, whereas the FL spectra at lower excitation energies are well fitted by using only the A Gaussian band. In agreement with the discussion above, the A band emission, persisting from higher to lower excitation energies, is due to radiative recombination from large oligomeric and polymeric species, whereas the C band is due to radiative recombination from monomers and small oligomer species. As for the B band at about 2.64 eV, which is present in the spectra at larger excitation energy, at present it is not possible to identify the related chromophores. Some chemical species having large quantum yields may be involved in such recombination band; in fact, it has been reported that melanin quantum yield presents a slight maximum at about 2.6 eV [2,10]. Since it has been calculated [25] that an IQ tautomer presents an absorption band at 2.64 eV, the B band at 2.64 eV may be due to such chemical
species, in agreement with the result of Nighswander-Rempel et al. [10]. The excitation energy dependence of peak energy, FWHM and intensity of each Gaussian band has been summarized in Fig. 5. The spectral position and the FWHM of the A band present a ‘‘plateau’’ at about 2.39 eV and 550 meV, respectively, for excitation energies in the UV region. On the contrary, the spectral position redshifts and the FWHM decrease on lowering the excitation energy in the visible region. This behaviour is in good agreement with the chemical disorder scheme, which involves the presence of a distribution of FL centres, due to radiative emission of planar polymeric chemical species having different sizes. When excitation in the UV region occurs, all the FL centres can absorb light, because the HOMO–LUMO gap energy of all polymeric species is lower than the energy of exciting light; consequently, all the FL centres are involved in the emission process and, therefore, the peak energy and FWHM remain almost constant, irrespective of the excitation energy. On the contrary, the excitation energies in the visible region are not sufficient to excite all the polymeric species and selective excitation of polymeric species with lower HOMO–LUMO gap occurs as the excitation energy decreases. The decrease of the number of species having larger HOMO–LUMO gap (i.e. smaller size) causes a redshift of the peak energy and a bleaching of the peak intensity. At the same time, by lowering the excitation energy, the number of species or radiative centres that can absorb the excitation light (i.e. the centres having lower HOMO–LUMO gaps) decreases and this effect causes a narrowing of the FL band. The same behaviour can be observed for the spectral position, FWHM and FL intensity of the C band. In this case, the distribution of FL centres, due to monomers and small oligomer groups, is centred at higher energy than that due to polymeric species, because of the reduced size. So the FL of this feature is possible
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Fig. 4. Deconvolution of a few fluorescence spectra (dots) of a synthetic melanin solution by means of Gaussian lineshapes (dashed lines).
only for excitation energy in the UV range. In fact, this emission is present only at the largest excitation energy. Also the B band at 2.64 eV can be observed only for the highest excitation energies. Its spectral position and FWHM remain almost constant, because of the reduced number of chemical species involved in such FL recombinations.
4. Conclusions In conclusion, we have presented an investigation of FL and FLE spectra of chemically untreated synthetic melanin solution. Comparison of our results with the published FL spectra shows some peculiar difference, due to chemical treatments performed on melanin solution before FL measurements. In particular, our measurements on untreated melanin indicate that the FL due to ensembles of large oligomer systems can be spectrally differentiated from that due to monomers and small oligomer systems when excitation with sufficiently high energy occurs. On the other
hand, FL spectra of other authors [2,10] are characterized by a single broad band, due to chemically distinct macromolecules (equivalently, monomers and oligomers having different sizes). FLE measurements reveal that the band at low energy is pumped by the disexcitation pathway of DHICA and DHI monomers linked to small-size oligomers. Deconvolution procedure of our FL spectra by means of Gaussian functions has revealed the presence of a further peak at 2.64 eV, whose nature is still unclear at present. The spectral position, FWHM and intensity of the two broad bands depend on the excitation energy, in agreement with the ‘‘chemical disorder model’’. This investigation can be used as a starting point to investigate the physical properties of eumelanin pigment by means of FL measurements. In fact, investigations about the natural eumelanin pigment are in progress to clarify how the FL properties of eumelanin change when protein content is considered. This is for answering a very important question: are the proteins a residual from the bio-synthesis process or are they fundamental for the biological functions?
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References
Fig. 5. Energy (a) and FWHM (b) of the three Gaussian FL bands, resulting from the deconvolution procedure (Fig. 4), as a function of the excitation energy. Error bars were calculated through the errors of the fit parameters provided by the fitting procedure.
Acknowledgements We are grateful to R. Cicero for stimulating discussion and to D. Fiorentino and D. Fiocco for technical cooperation.
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