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J. Phys. Chem. B 2001, 105, 2864-2866
Ultrafast Nonradiative Relaxation Dynamics of Eumelanin J. Brian Nofsinger,† 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: October 31, 2000; In Final Form: January 9, 2001
Degenerate pump-probe femtosecond spectroscopy is used to examine the primary photodynamics of eumelanin from Sepia officinalis following UV-A excitation. Exciting and probing at 320, 350, and 380 nm produces a transient absorption that rises within the instrument response and decays with a lifetime less than approximately 50 fs. Following the rapid decay of this transient signal, the recovery dynamics associated with the bleaching of the ground-state absorption are observed. The transient pump-probe data are compared to reported photoacoustic and time-resolved emission data. This comparison shows the dominant nonradiative process in eumelanin is repopulation of the ground electronic state and that the time constants revealed by the nonexponential decays of the emission and absorption data are associated with chemically distinct species.
Melanins are a class of biological pigments found in hair, skin, eyes, brain, and the inner ear. Although melanin is one of the most ubiquitous natural pigments, its chemical structure is unknown and its biological role(s) are subject to debate.1 Discussions of melanins generally focus on two general pigment classes: eumelanin and pheomelanin. These are differentiated by the molecular building blocks of dihydroxyindoles and benzothiazines, respectively.2,3 It is important to realize that the term “melanin” is a generic name that imparts no specific structural and/or chemical information, nor does the use imply that the materials are the same in different types of tissue. In fact, a recent study by Prota and co-workers demonstates structural differences of mammalian eumelanins, which may result from the diversity of biosynthetic pathways and functions for this class of pigments.4 To date, little information exists concerning the time scales for the photophysical and photochemical processes that occur following excitation of eumelanin. Eumelanin is a weak emitter, and so radiative processes play a minor role in the photodynamics of the pigment. Time-resolved emission studies reveal nonexponential behavior. Four exponential time constants are required to describe the decay.5 These emission data do not provide information as to whether the competitive nonradiative pathway is ground-state repopulation, formation of excited electronic states (e.g., triplet states), or generation of nonemissive intermediates (e.g., semiquinones). In an effort to characterize the dynamics of the nonradiative decay in eumelanin, we carried out a series of photoacoustic experiments. From the photoacoustic waveforms, it is possible in principle to determine the percentage of absorbed light that is dissipated as heat and the rate constants for this dissipation as long as the dynamics occur within the response time of the experiment (10-100 ns). The photoacoustic signals following 350 nm excitation from Sepia officinalis eumelanin reveal that approximately 90% of the absorbed energy is released as heat, and that this relaxation occurs on a time scale faster than the instrument response.6 In this paper, we report the first femtosecond pump-probe absorption study of eumelanin. The data provide information † ‡
Duke University. Duke University Medical Center.
on the timescales on which eumelanin dissipates energy upon UV-A excitation. The transient pump-probe data are compared to reported photoacoustic and time-resolved emission data. Comparison of these data to the emission dynamics clearly shows that repopulation of the ground electronic state is the dominant nonradiative pathway following excitation. In fact, approximately 90% of the relaxation occurs in less than 20 ps. The preparation of the eumalenin from the ink sacs of S. officinalis for time-resolved optical measurements was similar to that reported elsewhere.6 The solution studied, which passed through a YM10 filter (Millipore-Amicon), was concentrated to a final volume of 5-10 mL using a YM1 membrane. (It is difficult to quantify the size distribution of the eumelanin aggregates created by this procedure. Studies on structurally characterized biomolecules suggest that the species present in solution range in size from raffinose (∼500 amu) to cytochrome c (∼12400 amu). The optical densities of the solutions at excitation wavelengths were between 0.1500 ( 0.0005 and 0.1920 ( 0.0005 in a 1 mm quartz cuvette. All samples were deoxygenated and flowed through the sample cell at a rate of 2 mL/min. Tunable femtosecond laser pulses were generated from a commercial 1 kHz repetition rate, regeneratively amplified, titanium-sapphire laser system (Spectra Physics). The output pulses of the regenerative amplifier were generally 120 fs (fwhm) and 0.8 mJ/pulse at 800 nm. These pulses were used to pump an OPA (Spectra Physics). The pulses from the OPA were 220 fs (fwhm) and approximately 3 µJ/pulse. The OPA pulse was split into pump and probe pulses for perfoming degenerate transient absorption measurements. The pump and probe traveled different paths and were focused and recombined at the sample. A stepper-motor-driven translation stage enabled the time of arrival of the pump and probe pulses at the sample to be varied; the minimum step size corresponded to a delay time of 17 fs. The probe pulses were split into two so that intensity could be monitored before and after passing through the sample, and subsequently the time-dependent transient absorption signal could be calculated. The intensity was measured using a photodiode (UDT UDT-020UV) interfaced to a computercontrolled lock-in amplifier (Stanford Research Systems SR850).
10.1021/jp004045y CCC: $20.00 © 2001 American Chemical Society Published on Web 03/13/2001
Relaxation Dynamics of Eumelanin
Figure 1. Absorption spectrum of an aqueous solution of eumelanin from S. officinalis.
Figure 2. Degenerate 350 nm pump-probe transient absorption data for aqueous eumelanin from S. officinalis (solid line). The dashed line is the calculated curve using data from time-resolved emission measurements; see the text for details.
All experiments were performed with the relative polarization of the pump and probe beam being set at the magic angle. Data were recorded using time increments of 850, 50, and 17 fs. Traces reported herein represent a combination of time scales using all three step sizes. The emission quantum yield of deoxygenated eumelanin following 350 nm excitation was measured using a Fluorolog-3 spectrometer (Spex, ISA). The spectrum was corrected for PMT quantum efficiency and grating reflectivity effects using a correction file supplied by Spex, ISA. Quinine sulfate dihydrate (Fluka) was used as a standard (Φ ) 0.577 for excitation at 350 nm).7 Previously reported photoacoustic data reveal rapid nonradiative relaxation following UV-A excitation. Approximately 90% of the absorbed energy is released as heat on a subnanosecond time scale.6 Figure 1 shows the absorption spectrum of an aqueous solution of Sepia eumelanin. Figure 2 shows degenerate pump-probe data collected at 350 nm. Following excitation, a rapid ground-state bleach is observed. Taking into account the incomplete bleach recovery observed in the transient absorption experiements, it follows that the dominant reaction pathway revealed by the photoacoustic data is nonradiative relaxation to the ground electronic state. This is supported by the low emission quantum yield of 0.003 for these samples. Only a small fraction of the initially excited molecules emit. Therefore, the emission dynamics reflect competition between radiative and nonradiative relaxation to the ground state. Structural properties of eumelanin must govern the mechanisms by which photoexcited decay to the ground state occurs.
J. Phys. Chem. B, Vol. 105, No. 14, 2001 2865 Unfortunately, little is actually known about the molecular and electronic structure of eumelanin. The bleach recovery shown in Figure 2 is nonexponential. Like these transient absorption data, time-resolved emission studies of eumelanin reveal nonexponential decays. Four time constants are needed to describe the emission data. In the case of S. officinalis eumelanin, we previously reported that the time constants (amplitudes) for the decay of emission at 520 nm following 335 nm excitation are 58 ps (0.54), 0.51 ns (0.22), 2.97 ns (0.16), and 7.0 ns (0.08).5 Consider a model in which the time constants revealed in the emission data reflect a discrete distribution of noninteracting molecular species present in solution. There is substantial experimental evidence that supports the conclusions that natural eumelanins are formed from the aggregation of small oligomeric units derived from the oxidative polymerization of dihyroxyindole (DHI) and dihydroxyindolecarboxylic acid (DHICA).8-10 On the basis of microscopy and induced aggregation studies, it is reasonable to associate the noninteracting molecular species with different aggregation states of the constituent oligomers.11-15 Within such a model, the dynamics of ground-state repopulation can be predicted using the emission quantum yield and parameters derived from a fit to the emission dynamics. Specifically, the amplitudes derived from the emission data correspond to relative absorption cross sections of the different aggregation states. Therefore, the relative amplitudes are the same in the emission and bleach recovery signal observed in the transient absorption data. If we assume a constant radiative quantum yield (φr) of 0.003, the nonradiative time constants are derived in a straightforward manner from the emission time constants using the following equation:
τnr ) [1/(φrτr) - 1/τr]-1
(1)
where τnr is the nonradiative lifetime that is in direct competition with the radiative lifetime, τr. This results in four nonradiative lifetimes: 166 fs, 1.5 ps, 6.2 ps, and 19.8 ps. Using the same relative amplitudes from the emission data, a calculated pumpprobe trace convoluted with a 280 fs Gaussian reproduces the experimental data as illustrated in Figure 2. The multistate model proposed in this paper does not take into account ground-state repopulation lifetimes longer than 19.8 ps. However, the bleach recovery in Figure 2 does not fully recover to the initial absorption within 100 ps. Furthermore, the transient signal is constant for delay times longer than 50 ps. This suggests that there are competing relaxation pathways beyond the initial excited state for each or a subset of the aggregation states. This is supported by other time-resolved experiments that have measured longer lifetime decay mechanisms. For example, time-resolved electron spin resonance dynamics for the photoinduced decay of radicals from eumelanins on millisecond and longer time scales have been measured. Therefore, there are other photochemical processes that ultimately lead to free radical production that have not been revealed by time-resolved optical techniques used to date. In addition to the bleach recovery dynamics revealed in Figure 2, a transient absorption signal is present at early probe delay times. Figure 3 shows data collected with 320 and 350 nm excitation and probe. This feature is also observed at 380 nm but is not observed in a degenerate 400 nm experiment. If the magnitude of the bleach is normalized for the two data sets shown in Figure 3, then the amplitude of the transient absorption component increases with decreasing excitation wavelength. There are two reasonable origins of this signal: electronic dephasing and population relaxation. Wavelength-dependent
2866 J. Phys. Chem. B, Vol. 105, No. 14, 2001
Nofsinger et al. There are two important conclusions that can be drawn from this work. First, comparison of the pump-probe data to photoacoustic data clearly shows that the dominant nonradiative process in eumelanin is repopulation of the ground electronic state. Second, the ability to predict the absorption data from the emission data has the implication that the time constants revealed by the nonexponential decays of the emission and absorption data are associated with chemically distinct species. Acknowledgment. This work is supported by a grant from the National Institute of General Medical Sciences.
Figure 3. Degenerate 320 nm (solid line) and 350 nm (dashed line) pump-probe transient absorption data for aqueous eumelanin from S. officinalis. If these two data sets are normalized at the bleach maximum, the relative amplitude of the early absorption component increases with decreasing laser wavelength.
data offer insight into this issue. For example, the effect of wavelength detuning on the amplitude of the electronic dephasing signal was extensively investigated in a study of the dye molecule HITCI in ethylene glycol. With increasing detuning from the absorption maximum, the relative amplitude of the transient signal to the net bleach signal increases.17 This behavior is also consistent with theoretical calculations based on the formalism developed by Mukamel and co-workers for modeling the molecular response of dissolved molecules when probed in nonlinear laser experiments.18 Using the shape of the eumelanin absorption spectrum shown in Figure 1, detuning from the absorption maximum increases as the excitation wavelength increases. Therefore, if the origin of the transient absorption signal is electronic dephasing, the amplitude of the signal should increase with increasing wavelength, opposite that exhibited by the data. Furthermore, this transient absorption feature vanishes when the wavelength is changed from 380 to 400 nm, which represents an increase in detuning. We conclude that this dynamic feature is not a result of coherent interactions, but is a reflection of population dynamics following UV-A excitation. Therefore, temporal studies carried out with a delay increment of 17 fs reveal a transient absorption component that decays with a time constant less than approximately 50 fs, the smallest lifetime our system can resolve. This absorption signature is possibly due to excitation during vibrational cooling of an excited state.
References and Notes (1) Sealy, R. C.; Felix, C. C.; Hyde, J. S.; Swartz, H. M. In Free Radicals in Biology; Pryor, W. A., Ed.; Academic Press: New York, 1980; Vol. IV, pp 209-259. (2) Ito, S. Biochim. Biophys. Acta 1986, 883, 155-161. (3) Ozeki, H.; Ito, S.; Wakamatsu K.; Ishiguro, I. Biochim. Biophys. Acta 1997, 1336, 539-548. (4) Novellino, L.; Napolitano, A.; Prota, G. Biochim. Biophys. Acta 2000, 1475, 295-306. (5) Forest, S. E.; Lam, W. C.; Millar, D. P.; Nofsinger, J. B.; Simon, J. D. J. Phys. Chem. 2000, 104, 811-814. (6) Nofsinger, J. B.; Forest, S. E.; Simon, J. D. J. Phys. Chem. 1999, 103, 11428-11432. (7) Eastman, J. W. Photochem. Photobiol. 1967, 6, 55-72. (8) Chio, S. X-ray diffraction and ESR studies on amorphous melanin. Ph.D. Dissertation, University of Houston, Houston, TX, 1977. (9) Napolitano, A.; Pezzella, A.; Prota, G.; Seraglia, R.; Traldi, P. Rapid Commun. Mass Spectrom. 1996, 10, 468-472. (10) Pezzella, A.; Napolitano, A.; d’Ischia, M.; Prota, G.; Seraglia, R.; Traldi, P. Rapid Commun. Mass Spectrom. 1997, 11, 368-372. (11) Zeise, L.; Murr. B. L.; Chedekel, M. R. Pigm. Cell Res. 1992, 5, 132-142. (12) Vitkin, I. A.; Woolsey, J.; Wilson, B. C.; Anderson, R. R. Photochem. Photobiol. 1994, 59, 455-462. (13) Gallas, J. M.; Littrell, K. C.; Seifert, S.; Zajac, G. W.; Thiyagarajan, P. Biophys. J. 1999, 1135-1142. (14) Nofsinger, J. B.; Forest, S. E.; Eibest, L. M.; Gold, K. A.; Simon, J. D. Pigm. Cell Res. 2000, 13, 179-184. (15) Clancy, C. M. R.; Nofsinger, J. B.; Hanks, R. K.; Simon, J. D. J. Phys. Chem. 2000, 104, 7871-7873. (16) Felix, C. C.; Hyde, J. S.; Sealy, R. C. Biochem. Biophys. Res. Commun. 1979, 88, 456-461. (17) Cong, P. J.; Yan, Y. J.; Deuel, H. P.; Simon, J. D. J. Chem. Phys. 1994, 100, 7855-7866. (18) Mukamel, S. Principles of Nonlinear Optical Spectroscopy; Oxford University Press: New York, 1995.
