J. Phys. Chem. B 2002, 106, 6133-6135
6133
Ultrafast Spectroscopic Study of Pheomelanin: Implications on the Mechanism of Superoxide Anion Formation Tong Ye† and John D. Simon*,†,‡ Department of Chemistry, Duke UniVersity, Durham, North Carolina 27708, and Department of Biochemistry, Duke UniVersity Medical Center, Durham, North Carolina 27710 ReceiVed: February 21, 2002; In Final Form: April 30, 2002
UV-A excitation of pheomelanin results in the activation of molecular oxygen through the production of the superoxide radical anion (O2•-). Formation of this species has been attributed to rapid reaction between O2 and solvated electrons produced by one-photon ionization of pheomelanin. Herein, ultrafast spectroscopy is used for the first time to directly examine the primary photodynamics of pheomelanin following UV-A excitation (320-400 nm). Immediately following photolysis (t ∼ 100 fs) a transient absorption centered at 720 nm is observed. This feature decays nonexponentially; the decay dynamics are described by a sum of three exponential lifetimes, 0.84, 6.3, and 120 ps. While this transient species has the same absorption maximum as the solvated electron, the absorption line shapes differ. The data establish O2•- production does not arise from oxygen scavenging of electrons produced by direct photoionization of pheomelanin. The transient species is assigned to the first excited singlet state of pheomelanin, and the nonexponentiality of the decay is attributed to different aggregated structures present in solution.
subsequent formation of the superoxide radical anion.6,7
Introduction Melanins are among the most widespread pigments in plants and animals. There are two major classes of natural melanins, the black-brown eumelanin and the yellow-red pheomelanin. Eumelanin and pheomelanin are differentiated by the molecular building blocks of dihydroxyindoles and benzothiazines, respectively.1,2 Epidemiological data reveal a higher rate of incidence of UV-induced skin damage among red-haired and fair-skinned individuals compared to those of darker skin type.3 This difference in the propensity for skin damage is hypothesized to result from, in part, increased photochemical activity of pheomelanin compared to eumelanin.4 However, for comparable skin and hair color, marked variations were observed in the minimum erythmal dose (the minimum amount of UVB exposure needed to observe burning of skin, MED) as well as in the hair melanin composition.5 In fact, no significant relationship between MED and pheomelanin, eumelanin, or total melanin (eumelanin plus pheomelanin) content was found. A relationship was found, however, between the eumelanin/ pheomelanin ratio and the MED values, suggesting UV sensitivity is associated with high pheomelanin and low eumelanin levels and that the eumelanin/pheomelanin ratio may be a chemical parameter for predicting individuals at high risk for skin cancer and melanoma. In contrast to eumelanin, pheomelanin undergoes rapid photodecomposition in the presence of oxygen upon exposure to UV light. Chedekel and co-workers proposed that the primary photochemical process in the degradation of pheomelanin by UV light involved the photoionization of electrons followed by * To whom correspondence should be addressed: Department of Chemistry, Duke University, Box 90346, Durham, NC 27708. Work: (919) 660-1506. Fax: (919) 660-1605. E-mail:
[email protected]. † Duke University. ‡ Duke University Medical Center.
pheomelanin + hν f e-aq + pheomelanin radical (1) e-aq + O2 f O2•-
(2)
The photoconsumption of oxygen by pheomelanin generates O2•-, and the quantum yield for this process is ∼7.1 × 10-4 for excitation at 360 nm.8 It is unlikely that the efficiency for scavenging of this species by molecular oxygen is unity and so that above quantum yield represents the minimum value for solvated electron production if the above mechanism is correct. While the quantum yield for the photodissociation of pheomelanin is not known, benzothiazole model compounds generate e-aq with an efficiency of ∼0.06, 2 orders of magnitude greater than the photoconsumption yields, supporting the possibility of the above mechanism.9 Herein the primary photoreactions of pheomelanin are examined using transient optical spectroscopy. An ultrafast transient species is observed on the picosecond time scale. While this transient species exhibits an absorption spectrum with a maximum intensity at 720 nm, the line shape differs significantly from that of solvated electrons. Analysis of the transient data clearly establishes O2•- production does not arise from oxygen scavenging of electrons produced by the photoionization pheomelanin. The transient species is attributed to the initially populated excited electronic state of pheomelanin. Experiments and Materials Experiments were conducted using a commercial regeneratively amplified, Ti:Sapphire laser system (Spitfire, Spectra Physics, 120 fs (fwhm), 0.9 mJ/pulse centered at 800 nm, 1 kHz repetition rate). Light pulses raging from 300 to 390 nm were generated using an OPA (Spectra Physics). Pulses of 400 nm were derived from second harmonic generation of amplified
10.1021/jp025672l CCC: $22.00 © 2002 American Chemical Society Published on Web 05/22/2002
6134 J. Phys. Chem. B, Vol. 106, No. 24, 2002
Figure 1. Transient absorption dynamics collected at 800 nm following photoexcitation of an aqueous solution of pheomelanin at 350 nm. The solid line is a fit of a multiexponential function to the dynamics; the time constants and amplitudes are as listed in the text. The inset shows the instantaneous rise followed by the fast decay of the transient absorption.
800 nm pulses with a 0.5 mm BBO crystal. Probe pulses in the visible were generated using interference filters to select the desired wavelength from a white light continuum, which was produced by focusing 800 nm pulses on a 2 mm sapphire plate. The pump-probe transient absorption measurement setup was described previously in detail.10 The beam diameters of both the pump and probe at the region of spatial overlap within the sample were determined by enabling quantitative analyses on observed signal intensities. Pheomelanin was synthesized according to published procedures,11 dissolved in sodium phosphate buffer (pH of 7.2). The optical densities of the samples at excitation wavelengths were about 1.0 o.d. in a 1 mm quartz cuvette. All transient optical experiments were performed using the magic angle between the polarization of the pump and probe pulses in a 1 mm path length flowing cuvette. During photolysis, a peristaltic pump circulated the solution. Results and Discussion The sample of synthetic pheomelanin was excited at 350 nm and probed at a series of wavelengths from 490 to 800 nm. Figure 1 shows the transient dynamics observed by probing at 800 nm following 350-nm excitation. An instantaneous rise of transient absorption is observed within the instrument time response. This transient then decays nonexponentially; a sum of three exponentials with time constants (amplitudes) of 0.84 ps (61%), 6.3 ps (27%), and 120 ps (12%) is needed to describe the experimental data. Several other wavelengths from 300 to 400 nm were used to excite the sample. For this entire range of excitation wavelengths, the transient dynamics observed at 800 nm were identical. The initial intensity of the transient absorption was examined as a function of excitation pulse energy. As shown in Figure 2, a linear relationship is observed, supporting the conclusion the intermediate is formed by a one-photon process. As mentioned in the Introduction, UV excitation of pheomelanin results in the production of superoxide anions, hydrogen peroxide, and hydroxyl radicals.8,12,13 Previous literature reported that photolysis pheomelanin with both UV and visible light leads to the production of solvated electron, which in aerated solutions can give rise to superoxide radicals.14 Although formation of solvated electrons in aqueous solution is extremely fast, the geminate recombination and scavenging of oxygen are slower kinetic events.15 Given the quantum efficiency for oxygen photoconsumption by pheomelanin, ∼7.1 × 10-4 for excitation at 360 nm, we should be able to observe the signal from solvated electrons, if this intermediate is involved in the mechanism of superoxide production. In principle, the observed transient signal
Letters
Figure 2. Transient absorption dynamics at 800 nm following photoexcitation of an aqueous solution of pheomelanin at 400 nm are recorded at various powers of excitation. The dynamics are invariant with pulse energy. The inset shows the maximum signal (b) as a function of the excitation pulse energy (Ep). A linear relationship is observed between these variables, establishing that the formation of the transient species is a one-photon process.
Figure 3. Transient spectra following photoexcitation of an aqueous solution of pheomelanin at 350 nm plotted for delay times of 400 fs (b), 1 ps (9), 10 ps (O), and 100 ps (0). The solid lines are spline interpolations of data points.
in Figure 1 is consistent with this mechanism. If this transient were assigned to the solvated electron, then the decay dynamics would reflect recombination processes with pheomelanin (geminate and perhaps reactive with other moieties in the pheomelanin structure). Oxygen scavenging will not occur on the picosecond time scale and so the yield of solvated electrons available for scavenging is manifested by the incomplete signal recovery observed at long delay times. This offset would then reflect the concentration of electrons that escape into the surrounding solvent and are then available for reaction with oxygen. The oscillator strength of the solvated electron is well characterized (800 ) 15 000 M-1 cm-1).16 Given the excitation energy, the concentration of solvated electron generated can be obtained by the transient data, from which a quantum yield can be determined. For the experimental conditions used, the initial signal intensity (t ∼ 200 fs) would correspond to a quantum yield of 9.1 × 10-3. As shown in Figure 1, the signal decays rapidly and if the transient signal reflects the solvated electrons, the dynamics indicate that less than 5% of those initially formed persist onto the nanosecond time scale and would then be available for scavenging by oxygen. This gives a maximum quantum yield of 4.5 × 10-4 for superoxide formation, which is close to that determined by photoconsumption measurements (7.1 × 10-4). With multiple probe wavelengths, the transient absorption spectrum following excitation at 350 nm was constructed. Figure 3 shows the transient absorption spectra at several delay times after excitation. The spectral feature exhibits an absorption maximum at ∼720 nm, similar to that of the solvated electron in water. The dynamics at various probe wavelengths are
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J. Phys. Chem. B, Vol. 106, No. 24, 2002 6135 the nonexponential dynamics is attributed to relaxation by the same chromophore in different environments (e.g., aggregation state). We are in the process of determining the action spectrum for formation of this transient for different mass fractions of pheomelanin as well as carrying out more extensive transient absorption studies in an effort to quantify the complete photophysical events that ultimately lead to the activation of oxygen by this pigment. Conclusions
Figure 4. Transient spectra following photoexcitation of an aqueous solution of pheomelanin at 350 nm for a delay time of 100 ps (0) normalized and compared to the absorption spectrum15 of solvated electrons in water. While both spectra have essentially the same wavelength of maximum absorbance, the line shapes differ significantly.
essentially identical. Figure 4 compares the transient spectrum obtained at 100 ps following photolysis and that of the solvated electron. These data clearly show that the transient observed following UV-A excitation of pheomelanin differs from that of the solvated electron. The spectrum shows essentially no broadening within the first 130 ps (Figure 3), and this demonstrates that no solvated electron is detected as this species decays. Taking the sensitivity of the experimental apparatus into account, if formed, the quantum yield for solvated electron production by this melanin is less than 2 × 10-5, which is a factor of ∼30 lower than the yield for O2•- production. We conclude that solvated electron generation by photoionization following the UV-A excitation of pheomelanin is at most negligible, and therefore the formation of superoxide must occur by a different mechanism. Lack of information on the molecular structure of pheomelanin makes it hard to assign the observed ultrafast species. Ambient and nitrogen-purged samples show the same dynamics so decay of the primary species does not involve reaction with oxygen. At this point it is reasonable to assign this feature to the excited singlet state of pheomelanin. Recent chemical analyses of pheomelanin have identified specific molecules present in the pigment17 and we are actively examining whether such molecules exhibit transient photophysical properties as those reported herein. Finally, we note that while there is no change in shape of the observed transient for time delays up to 130 ps, the decay of this species is nonexponential. Recently we reported degenerate pump-probe absorption experiments on eumelanin that revealed the nonradiative ground-state repopulation of the pigment.10 We observed nonexponential ground state repopulation and attributed this feature to the presence of a distribution of aggregation states in solution, concluding that different aggregation states relaxed with different time constants. Preliminary AFM and optical spectroscopy studies of mass-selected fractions of pheomelanin solutions also show this pigment to be an aggregated structure. At this point
Ultrafast absorption spectroscopy of pheomelanin reveals a transient species (λmax ∼ 720 nm) following UV-A excitation. The line shape of this transient differs from that of solvated electrons and is assigned to the excited-state absorption of the first excited singlet state of pheomelanin. Relaxation from this state is nonexponential and is attributed to the presence of different size molecular aggregates in solution. The collective data establish that the primary process following UV-A photoexcitation of pheomelanin is not photoionization, and so such a mechanism cannot be responsible for the activation of oxygen (photoformation of superoxide radical anion) by pheomelanin. Acknowledgment. This work is supported by a grant from the National Institute of General Medical Sciences and Unilever Research US. References and Notes (1) (1)Ito, S. Biochim. Biophys. Acta 1986, 883, 155. (2) Ozeki, H.; Ito, S.; Wakamatsu, K.; Ishiguro, I. Biochim. Biophys. Acta 1997, 1336, 539. (3) Melanin: its role in human photoprotection; Zeise, L., Chedekel, M. R., Fitzpatrick, T. P., Eds.; Valdenmar Publishing Co.: Overland Park, KS, 1995. (4) Wenczl, E.; Van Der Schans, G. P.; Roza, L.; Kolb, R. M.; Timmerman, A. J.; Smit, N. P. M.; Pavel, S.; Schothorst, A. A. J. InVest. Dermatol. 1998, 111, 678. (5) Vincensi, M. R.; d’Ischia, M.; Napolitano, A.; Procaccini, E. M.; Riccio, G.; Monfrecola, G.; Santoianni, P.; Prota, G. Melanoma Res. 1998, 8, 53. (6) Chedekel, M. R.; Smith, S. K.; Post, P. W.; Pokora, A.; Vessell, D. L. Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 5395. (7) Chedekel, M. R.; Post, P. W.; Deibel, R. M.; Kalus, M. Photochem. Photobiol. 1977, 26, 651. (8) Sarna, T.; Menon, I. A.; Sealy, R. C. Photochem. Photobiol. 1984, 39, 805. (9) Lambert, C.; Sinclair, R. S.; Truscott, T. G.; Land, E. J.; Chedekel, M. R.; Liu, C. T. Photochem. Photobiol. 1984, 39, 5. (10) Nofsinger, J. B.; Ye, T.; Simon, J. D. J. Phys. Chem. B 2001, 105, 2864. (11) Ito, S. Pigm. Cell Res. 1989, 2, 53. (12) Sarna, T.; Menon, I. A.; Sealy, R. C. Photochem. Photobiol. 1985, 42, 529. (13) Krol, E. S.; Liebler, D. C. Chem. Res. Toxicol. 1998, 11, 1434. (14) Chedekel, M. R.; Land, E. J.; Sinclair, R. S.; Tait, D.; Truscott, T. G. J. Am. Chem. Soc. 1980, 102, 6587. (15) Peon, J.; Hess, G. C.; Pecourt, J.-M. L.; Yuzawa, T.; Kohler, B. J. Phys. Chem. A 1999, 103, 2460. (16) Hart, E. J.; Anbar, M. The Hydrated Electron; John Wiley & Sons: 1970. (17) Napolitano, A.; Di Donato, P.; Prota, G. J. Org. Chem. 2001, 66, 6958.
