J. Phys. Chem. B 2001, 105, 11507-11512
11507
Primary Photophysical Properties of A2E in Solution Laura E. Lamb,† Tong Ye,† Nicole M. Haralampus-Grynaviski,† T. Richard Williams,† Anna Pawlak,‡ Tadeusz Sarna,*,‡ and John D. Simon*,†,§ Department of Chemistry, Duke UniVersity, Durham, North Carolina 27708, Department of Biophysics, Jan Zurzycki Institute of Molecular Biology, Jagiellonian UniVersity, Al. Mickiewicza 3, 31-120 Krakow, Poland, and Departments of Biochemistry and Ophthalmology, Duke UniVersity Medical Center, Durham, North Carolina 27710 ReceiVed: June 19, 2001; In Final Form: August 29, 2001
Time-resolved spectroscopic techniques are used to determine the primary photoprocesses of A2E in solution. Comparison of the absorption and excitation spectrum of A2E in methanol solution indicates excitation at 400 nm populates the S2 excited state. Transient absorption signals decaying with a time constant of 0.9 ps were observed probing around 800 nm. These signals are attributed to the S2fSn transition and reveal the S2fS1 relaxation occurs on the subpicosecond time scale. Transient absorption data probing at shorter wavelengths (480 and 550 nm) are attributed to the S1fSn absorption. These signals exhibit an exponential decay with a time constant of 11 and 13 ps, respectively. Time-resolved emission measurements of the corresponding S0rS1 decay reveal a nonexponential decay; however, >95% of the signal amplitude is described by an exponential decay with a time constant of 12.4 ps. Both time-resolved emission and absorption experiments therefore indicate repopulation of the ground electronic state occurs with a time constant of ∼12 ps. A weak transient absorption probing in the blue (400 nm) persists onto the nanosecond time scale and is attributed to the T1fTn absorption of A2E. Photoacoustic spectroscopy establishes the quantum yield for intersystem crossing of A2E in methanol solution is at most 0.03. The emission quantum yield of A2E in ethanol is determined to be 0.01, and so, nonradiative relaxation is the dominant primary event. The quantum yield for the generation of singlet molecular oxygen following 355 nm excitation of A2E in acetonitrile was determined to be 0.02, consistent with a low production of the excited triplet state. These results establish A2E is not an efficient photogenerator of reaction oxygen species in solution.
Introduction
SCHEME 1: Structures of A2E and iso-A2E
In 1988, Eldred and Katz examined chloroform:methanol extracts of human retinal pigment epithelium (RPE) cells and determined such cells contained a variety of fluorophores.1 When mass spectrometric techniques were used, one of the blueabsorbing and orange-red-emitting fluorophores was characterized and proposed to be N-retinylidene-N-retinylethanolamine.2 The molecular structure deduced from the mass spectral fragmentation patterns proved to be incorrect.3 In 1996, Sakai et al. established the emissive species was a bis-substituted pyridinium ring.4 In 1998, Parish and co-workers identified two isomers, the all-trans and 13-cis, which are now commonly referred to as A2E and iso-A2E, respectively.5 The structure of A2E and its iso-isomer are shown in Scheme 1. Both isomers are present in human RPE cells,5 and recent work shows both are contained in ocular lipofuscin,6,7 an age pigment, which accumulates within the lysosomal body of the RPE cell. While the exact biological mechanism for the formation of A2E and iso-A2E is not established, recent work by Mata and co-workers provides strong evidence that the first step is the Schiff base condensation of retinal with phosphatidylethanolamine in the rod outer segments.8 * To whom correspondence should be addressed. Work, (919)660-1506; fax, (919)660-1605;
[email protected]. † Duke University. ‡ Jagiellonian University. § Duke University Medical Center.
Following the availability of synthetic methodologies for preparing A2E,5 researchers began to study the effects of this fluorophore on cellular function. The current literature on A2E emphasizes two types of possible toxic action of A2E in cells. First, A2E may play an adverse role in the cell because of its ability to act as an amphilphilic detergent and thereby disrupt membrane integrity.2,9,10 In a recent study by Holz and coworkers, A2E is found to inhibit the function of the ATP-driven proton pump located within the membrane of lysosomes.11 Second, A2E could be phototoxic.12 Sparrow, Nakanishi, and Parish demonstrated blue-light photoexcitation of A2E-enriched human RPE cells can lead to apoptosis.13 The degree of cellular damage depends on A2E concentration and is only observed if
10.1021/jp0123177 CCC: $20.00 © 2001 American Chemical Society Published on Web 10/27/2001
11508 J. Phys. Chem. B, Vol. 105, No. 46, 2001 A2E is present above a critical concentration. In a recent study by Davies et al., the concentration of A2E in lipofuscin-fed cells was found to be at least 100-fold less than that needed to cause phototoxicity.6 These cells were still damaged upon exposure to blue light, and so, those workers concluded there must be other blue-absorbing compounds, in addition to A2E, responsible for oxygen activation and the phototoxicity of lipofuscin. While the mechanism of cellular damage remains unresolved, there is convincing evidence that photoexcitation of lipofuscin results in the generation reactive oxygen species. Experiments by Rozanowska et al. on solvent extracts of lipofuscin establish that photoexcitation of the extracted molecules can generate singlet oxygen.14,15 It has been proposed that photoproduction of this reactive oxygen species contributes to the phototoxicity of lipofuscin.12,14-21 However, the ability of A2E to photogenerate this species has not been addressed. Surprisingly, there are only a few studies on the photophysics and photoreactivity of A2E.6,13,22-25 Sparrow and co-workers published steady state spectroscopic properties of A2E (absorption, emission, and excitation spectra).10 Cubeddu and coworkers examined the emission dynamics of A2E in solution and in RPE cells using time-correlated single photon counting.22 They reported nonexponential decays for the emission of A2E in both cases. The emission of A2E in solution and A2E in loaded RPE cells exhibited exponential components with time constants of 6.6 ns, 1.9 ns, 330 ps, and 5.8 ns, 1.7 ns, 400 ps, respectively. The time constants and relative amplitudes of these components differed for the solution and cellular environment. In addition to these time constants, a component of 120 ps, which comprised about one-third of the amplitude of the emission signal, was reported for A2E in loaded RPE cells. Corresponding previous studies on lipofuscin reveal complicated nonexponential decays; for granules extracted from eyes greater than 50 years of age, the four exponential components reported are 6.6 ns, 2.0 ns, 730 ps, and 210 ps, similar to those reported for A2E.26 Because of the unresolved status of the potential phototoxicity of A2E and the lack of explanation for the previously reported complicated emission dynamics, we have carried out a systematic examination of the primary photochemical processes using steady state and time-resolved spectroscopic techniques. In this paper, the lifetime of the excited singlet state of A2E is shown to be significantly shorter than previously reported. Furthermore, in disagreement with previous reports, we find the emission dynamics of A2E in solution is nearly single exponential. Here, we also report the quantum yield for singlet oxygen generation following blue-light excitation. The combined data show A2E relaxes back to the ground state on the picosecond time scale in solution and A2E is inefficient at activating oxygen because of its low yield of intersystem crossing under these conditions.
Lamb et al. Absorption spectra were recorded on a diode array spectrophotometer (Hewlett-Packard 8452A). Emission and excitation spectra were recorded using a fluorimeter (Spex Fluorolog-3). All spectra were corrected to account for the wavelengthdependent response of various components of the instrument. Fluorescence quantum yields were determined relative to Rhodamine 590 (φF ) 0.94 in ethanol solution at room temperature).27 Femtosecond absorption data were collected using equipment previously described.28,29 Emission lifetimes were determined using time-correlated single photon counting. The excitation beam at 400 nm was generated by frequency doubling of the 800 nm output of a regeneratively amplified femtosecond titanium-sapphire laser system (Spectra Physics Millenia/ Tsunami amplified by a Coherent REGA 9000 pumped by a Coherent 16W Sabre Ar-Ion laser). The excitation pulses were ∼200 fs (full width at half-maximum) in duration, 0.4 µJ in energy, and the repetition rate was 250 kHz. Count rates were kept below 10% of the excitation repetition rate with a variable neutral density filter to ensure temporal distortions did not occur. The emission was detected at magic angle with respect to the polarization of the excitation source. Fluorescence from this excitation was passed into a double subtractive monochromator (MK Photonics, Inc. DK242) and detected by a microchannel plane (Hamamatsu R3809U-50). The voltage signal generated by the fluorescence was amplified (EG&G Ortec model 9306) and passed through a constant fraction discriminator (EG&G Ortec model 9307) and into a picosecond time analyzer (EG&G model 9308) for a start pulse. A fraction of the 800 nm light from the regeneratively amplified cavity was sent to an inverted photodiode (Electrooptics Technology ET 2010) for a stop pulse. This signal was passed through a discriminator and two delays (EG&G Ortec model 425A) and into the time analyzer. The experiment is controlled by a Pentium III computer system (Dell). The instrument response function had a typical width of 35 ps. Fluorescence decays were fit using the program Ffit. This program deconvolutes the instrument response function from experimental decays. Pulsed-laser photoacoustic measurements were collected as previously described except that the excitation pulse was produced by an OPA (Continuum Panther) pumped by a nanosecond Nd:YAG laser (Continuum Surlite 3).30 The photogeneration of singlet oxygen was determined by monitoring the 3Σr1∆ emission of O2 using an apparatus similar to that previously described.15 The quantum efficiency for the production of O2(1∆) by energy transfer, φΕΤ, was determined by comparing the signal amplitudes of the solution of A2E and retinal (φΕΤ ) 0.4)31 with matched optical density at the excitation wavelength of 355 nm. Results
Experimental Section A2E was synthesized and purified using published procedures.5 Specifically, a 2:1:1 mixture of retinal (Sigma) and ethanolamine (Sigma) and acetic acid (VWR) in ethanol was stirred at room temperature in the dark for 2 days. The mixture was run on a silica gel column. The crude A2E isolated from the column was purified by reverse phase high-performance liquid chromatography (HPLC) using a C-18 column (Vydac 201TP5415). Previously reported purification methods were adapted for our specific instrument and column.5,8 The A2E and iso-A2E peaks were completely resolved, and the absences of other impurities were confirmed using fast atom bombardment mass spectroscopy (JEOL XS102A).
Figure 1 shows the absorption, emission, and excitation spectra of A2E in methanol solution at room temperature. These data are similar to those reported by Sparrow and co-workers.10 Note the excitation and absorption spectra differ; specifically, the excitation spectrum is red-shifted by ∼40 nm. These data indicate the emissive state of A2E is not the same as that initially excited upon absorption. Figure 2 shows the laser-induced photoacoustic signal following the 430 nm excitation of a nitrogen-saturated methanol solution of A2E. The signal for a matched optical density solution of bromocresol purple (BCP) is shown as a standard. The data for BCP corresponds to the complete conversion of the absorbed energy into heat (278 kJ mol-1). The fact the
Primary Photophysical Properties of A2E in Solution
J. Phys. Chem. B, Vol. 105, No. 46, 2001 11509
Figure 1. The absorption (s), emission excited at 434 nm (s), and excitation spectra monitored at 650 nm (‚‚‚) of A2E in room temperature methanol solution. The intensity shown is as a function of energy.
Figure 2. The laser-induced photoacoustic signal following the 430nm excitation of a nitrogen-saturated methanol solution of A2E at room temperature (‚‚‚). The signal for a matched optical density solution of BCP (s) is shown as a standard and corresponds to the complete conversion of the absorbed energy into heat (278 kJ mol-1).
Figure 4. Pump-probe time-resolved absorption following the excitation of A2E in methanol at 400 nm (s). The probe wavelengths are 480 (A), 550 (B), and 800 nm (C). All three probe wavelengths reveal a transient absorption that appears immediately upon photolysis and then decays to zero on the picosecond time scale. Table 1 presents the time constants and amplitudes obtained from exponential fits (‚‚‚) to the absorption data.
Figure 3. Degenerate pump-probe time-resolved absorption following the excitation of A2E in methanol at 400 nm (s). The data can be fit to a kinetic process with a time constant (see text for details).
magnitude of the A2E signal is the same as that for BCP indicates the heat release by A2E is nearly quantitative. The relative signals are not affected by temperature and the presence of oxygen. Figure 3 shows degenerate pump-probe time-resolved absorption data collected at 400 nm. Figure 4 shows additional transient absorption data using an excitation wavelength of 400 nm and probe wavelengths of 480 (A), 550 (B), and 800 nm (C). Examination of the data in Figure 3 shows following excitation, a bleach is observed at 400 nm. With increasing delay time, the signal recovers to show a transient absorption at this wavelength, which persists onto the hundreds of picosecond time
scale. The data in Figure 4 reveal transient absorptions that appear concomitant with photolysis and then decay completely on the picosecond time scale. Table 1 presents the time constant(s) and amplitude(s) obtained from fits to the absorption data. Time-correlated single photon counting data for the emission from A2E at 650 nm following excitation at 400 nm are shown in Figure 5 and described by a multiexponential function. The time constants and amplitudes obtained from exponential fits to these data are also presented in Table 1. The emission quantum yield for A2E in ethanol was determined to be 0.01. Comparison of the 3Σr1∆ emission intensities following 355 nm excitation of optically matched samples of retinal and A2E reveals a quantum efficiency φΕΤ ) 0.02 for the production of O2(1∆) by A2E. Discussion Before the spectroscopy and photoreactivity of A2E are discussed, it is important to recall the molecule is present as
11510 J. Phys. Chem. B, Vol. 105, No. 46, 2001 TABLE 1: Time Constants and Amplitudes Obtained from Fits to Transient Absorption, Stimulated Emission, and Time-Correlated Single Photon Counting Emission Dynamics Transient Absorption λprobe (nm)
τ (ps)a
amplitude
400 480 550
9.8 13 11 3.8 0.9 13
0.46 0.54 -0.01
800
Time-Resolved Emission τ (ps)
amplitude
12.4 ( 2.1 90 ( 14 700 ( 150
0.954 0.043 0.001
a The time constants derived from the analysis of the transient absorption data are estimated to have an error of (15%.
Figure 5. Time-resolved emission decay of A2E at 650 nm in methanol with excitation at 400 nm (s). The instrument response (‚‚‚) was produced by the scatter of 400 nm light. The best fit (shown in gray) was generated by convolution of the instrument and a multiexponential function. The resulting time constants and amplitudes are given in Table 1.
two different isomers in solution, A2E and iso-A2E. These two species interconvert photochemically, and the photostationary state is given by an A2E to iso-A2E ratio of 4:1.5 To date, the photoisomerization rates and quantum yields have not been determined for either isomer. In addition, the extinction coefficient is only known for A2E and is 31 000 mol-1 dm2 at 430 nm.5 The data presented here were recorded on samples, which contained A2E and iso-A2E at photostationary state. Both isomers are present in ocular lipofuscin samples,6,7 and so, it is relevant to study the naturally occurring mixtures. First, we address the steady state spectroscopic properties of A2E. A2E is a conjugated polyene, which as a class of molecules is known to have closely spaced excited states.32-35 For many polyenes, the absorption band corresponds to the S0fS2 transition.36-38 Examination of the data in Figure 1 suggests this is also the case for A2E. The excitation spectrum is shifted ∼40 nm to lower energy from the absorption spectrum, indicating that the S1 state lies at least ∼1650 cm-1 below S2. Examination of the structures in Scheme 1 shows that 11 is the maximum number of conjugated double bonds. Andersson and Gillbro examined the photophysical properties of long chain conjugated carotenoids and showed the absorption energy (E) for the S0fS2 transition could be well-parametrized by an empirical function E/cm-1 ) 10 980 + (1.11 × 105N-1) - (9.60 × 104N-2), where N is the number of conjugated double bonds.39 While the molecular structures of conjugated carotenoids are
Lamb et al. different than A2E (e.g., the lack of a pyridinium ring near the middle of the chain), a conjugation length of n ) 11 is predicted to have a 0-0 excitation energy of S0fS2 transition of ∼493 nm. This is in excellent agreement with the A2E spectrum. Relaxation from S1 can, in principle, occur by emission, isomerization, internal conversion, and intersystem crossing. At room temperature, the emission quantum yield of A2E in ethanol is found to be 0.01. Thus, the dominant relaxation process(es) from S1 must be nonradiative. While the photoisomerization yield remains to be determined, insight into the relative contribution of intersystem crossing can be ascertained through photoacoustic measurements on photostationary mixtures of A2E and iso-A2E. Figure 2 shows matched solutions of BCP (standard) and A2E produce indistinguishable photoacoustic signals. The data for BCP corresponds to the complete conversion of the absorbed energy into heat (278 kJ mol-1) and so the data for A2E indicates the heat release is nearly quantitative. While the photoacoustic waveforms appear to be identical, the precision of these measurements is ∼2% and so it is possible that ∼6 kJ mol-1 is stored by A2E upon excitation. While the triplet state energy of A2E is not known, the triplet state energies of retinoids are on the order of 150 kJ mol-1.40 Assuming this value for the triplet state energy of A2E results in a storage of 6 kJ mol-1, which would correspond to an intersystem crossing quantum yield of φISC ) 0.03. The intersystem crossing yields for alltrans-retinal and N-all-trans-retinylidene-n-butylamine are 0.4 and 0.008, respectively.31 A2E contains a Schiff base linkage, and it is reasonable to expect that its intersystem crossing yield will be comparable to that of N-all-trans-retinylidene-n-butylamine. The lack of a phase shift in the waveform indicates nonradiative relaxation occurs on the nanosecond or faster time scale.41 It is important to point out net volume changes contribute to the photoacoustic signals. The relative contribution of volume changes and nonradiative energy release to the photoacoustic signal depend on the temperature of the sample.41 The signal for A2E is invariant from room temperature to ∼4 °C. Thus, net volume changes are negligible. There is clearly a volume change associated with the photoisomerization of A2E to isoA2E, and one might be misled to interpret that this result means the photoisomerization efficiency is low. The samples under study are already at photostationary state, and so, excitation does not result in any change in the A2E:iso-A2E ratio; so, no net volume change occurs. The relative ground state energies of A2E and iso-A2E are not known, and only by using a photostationary mixture in these experiments can the energy difference between the ground states of the two isomers be ignored in the analysis. The photoacoustic data enable the definitive conclusion that the dominant process is nonradiative relaxation back to the ground state on a fast time scale. To address the mechanism and dynamics of the nonradiative relaxation processes of A2E, both femtosecond transient absorption and time-correlated single photon counting experiments were performed. Figure 3 shows pump-probe time-resolved absorption data collected using an excitation wavelength of 400 nm. Examination of the data in Figure 3A shows that following excitation an initial bleach is observed at this wavelength. With increasing delay time, the signal recovers to show a transient absorption, which persists onto at least the hundreds of picosecond time scale. In modeling these data, the transient species, which give rise to the long time absorption is assumed to be generated from the S1 state in competition with nonradiative decay to the S0 ground state. The kinetics of such a model
Primary Photophysical Properties of A2E in Solution would be described by an expression of the form A(1 - e(-t/τ)) + B where the constants A and B are functions of the extinction coefficients of the ground state and transient species and the quantum efficiency of each process. The data are well-fit by this expression and reveal a time constant of 9.8 ( 1.5 ps (Table 1). The data in Figure 4 show transient absorption signals are observed probing to the red of the excitation wavelength. Transient absorptions are observed at all wavelengths examined, including those that overlap with the emission spectrum. When observed at 480 and 550 nm, the absorption signals decay to zero. The data shown are averages of multiple scans. While the signal-to-noise implies a small error, examination of individual data sets shows the accuracy of the measurement decreases as the signal decreases. For example, in the 480 nm data, averaging the individual scans reveals such a decrease. The range of signals for a particular value of the transient absorption was 0.1 ( 0.001, 0.02 ( 0.0025, 0.01 ( 0.005, and 0.005 ( 0.01. The figures present single exponential fits (dotted lines) to the data. The time constants obtained are 13 and 11 ps for the decays at 480 and 550 nm, respectively. While a better visual fit to the 480 nm data is obtained using a sum of exponentials, such fits are not statistically more significant than the fit obtained using a single exponent. The observed dynamics at 480 and 550 nm are essentially the same as the repopulation of the bleach observed at 400 nm, and so, we assign this transient to the excited singlet state of A2E. The transient data at 800 nm, however, are nonexponential and are described by a sum of three exponentials with time constants 0.9, 3.8, and 13 ps. The 0.9 and 3.8 ps time constants reflect the decay of transient species, and the 13 ps constant is associated with stimulated emission. The corresponding amplitudes are given in Table 1, and the fit is shown as a dotted line. Stimulated emission is not observed at either 480 or 550 nm. The initial signal at all wavelengths probed, including 800 nm, is dominated by the absorbance of the transient species. At 480 and 550 nm, however, the transient absorption decays with the same time constant as the stimulated emission masking its presence. This conclusion is further supported by the emission dynamics. Time-correlated single photon counting data for the emission from A2E at 650 nm following 400 nm excitation are described by a multiexponential function; however, the decay is dominated by a 12.4 ( 2.1 ps component that accounts for >95% of the amplitude (Figure 5). This time constant is significantly faster than that reported in previous studies22 and may have been missed because of the limited time resolution of those studies. It is important to stress that this time constant is significantly faster than that observed previously and that the observed decay is nearly single exponential, in contrast to previously reported studies on the emission of A2E. If we compare the emission decays collected at different purification steps in the synthesis of A2E, we find that the relative proportion of the ∼12 ps component, and therefore the applicability of a single exponential function to describe the decay dynamics, increases with purification. Mass spectral analysis shows that the purification steps remove various contaminants; however, even after extensive HPLC separation, the sample still contains trace amounts of contaminants. It is likely that the ∼4% (τ ) 90 ps) and ∼0.1% (τ ) 700 ps) components of the emission decay arise from the inability to remove trace impurities. The combined transient absorption and emission data (both stimulated and spontaneous) establish the lifetime of the S1 excited state of A2E is ∼12 ps. The transient absorption data
J. Phys. Chem. B, Vol. 105, No. 46, 2001 11511 reveal decreasing time constants with longer wavelengths. Specifically, the decay times observed at 480, 550, and 800 nm are 13, 11, and 3.8 ps (the longer component of the absorption decay observed), respectively. We ascribe this trend to vibrational relaxation within the excited S1 state. Similar results have been observed in both small polyenes and large conjugated carotenoids.35 In analogy to these related studies, the short time component of 0.9 ps revealed in the transient data for A2E at 800 nm is attributed to the S1rS2 relaxation. The emission decay occurs with a time constant that is over an order of magnitude slower than the S1rS2 relaxation process and therefore establishes that emission originates from the S1 state. We now turn to discuss the transient absorption observed probing at 400 nm for delay times greater than 30 ps. While the absorption spectrum for the excited triplet state of A2E has not been reported, the T1fTn absorption spectra are known for several related retinoid molecules peak in the vicinity of 400480 nm and these spectra have extinction coefficients on the order of 50 000-80 000 mol-1 dm2.40,42 The transient signal observed for pump-probe measurement to a maximum delay time of 150 ps shows no decrease in signal amplitude, supporting the conclusion the lifetime for the triplet state is at least on the nanosecond time scale. It is interesting to take the amplitude of the transient absorption on the picosecond time scale and the maximum quantum yield for intersystem crossing derived from the photoacoustic data to estimate the extinction coefficient of the T1fTn absorption at 400 nm. The extinction coefficient of the ground state absorption of A2E at 400 nm is 22 820 mol-1 dm2.5 If we take ΦISC ) 0.03, then the relative magnitudes of the initial bleach and transient absorption signal (>50 ps) give a value of ∼20 000 mol-1 dm2 for T at 400 nm. This is well within the range of observed values for the extinction coefficient of triplet spectra for retinoid molecules.42 A low quantum yield for triplet generation is confirmed by measurements of the quantum yield for formation of singlet oxygen (1O2) following photolysis of A2E at 355 nm. The efficiency for generation of 1O2 was determined by monitoring the emission of 3Σgr1∆g emission of 1O2 at 7880 cm-1. The emission intensity from matched optical density acetonitrile solutions of A2E and retinal were compared. The quantum yield for formation of 1O2 by photoexcited retinal in acetonitrile is 0.4.31 From the comparison of the signal amplitudes, the corresponding quantum yield for A2E was found to be 0.02. This quantum yield is consistent with the range for the quantum yield for formation of the triplet state of A2E as deduced from photoacoustic measurements. Triplet state lifetime for retinoids is fairly constant; that for all-trans-retinal, all-trans-retinol, and N-all-trans-retinylidene-n-butylamine is 11,31 17,43 and 17 µs,31 respectively. Assuming the lifetime of the triplet state of A2E is also on this time scale, then we would expect nearly quantitative quenching by O2, and in that case, the quantum yield for singlet oxygen formation is a measure of the intersystem crossing yield. Conclusions Excitation of A2E into its first absorption band results in the population of the S2 excited state. Following excitation, S1rS2 relaxation occurs within 0.9 ps. The lifetime of the S1 state is ∼12 ps, as reflected by the dynamics of both the S1fSn absorption and the S0rS1 emission dynamics. Evidence of vibrational relaxation within S1 is reflected by the time constants observed at the different probe wavelengths examined in the femtosecond absorption experiments. In contrast to previous
11512 J. Phys. Chem. B, Vol. 105, No. 46, 2001 reports, but in accord with the femtosecond absorption data, the emission decay is nearly exponential and decays with a ∼12 ps time constant. The combined photoacoustic, transient absorption, and oxygen sensitization data indicate the quantum yield for triplet formation upon blue-light excitation of A2E in solution is ∼0.02. This establishes A2E is essentially unreactive in solution and suggests A2E may not be a major phototoxic component of ocular lipofuscin. Acknowledgment. This work is supported by Duke University (J.D.S.), the State Committee for Scientific Research (T.S.), and the Wellcome Trust (T.S.). We thank Professor Michael C. Fitzgerald for access to the HPLC used to purify A2E. We thank Dr. N. Tkachenko for providing the Ffit program. References and Notes (1) Eldred, G. E.; Katz, M. L. Exp. Eye Res. 1988, 47, 71-86. (2) Eldred, G. E.; Lasky, M. R. Nature 1993, 361, 724-726. (3) Eldred, G. E. Nature 1993, 364, 396. (4) Sakai, N.; Decatur, J.; Nakanishi, K. J. Am. Chem. Soc. 1996, 118, 1559-1560. (5) Parish, C. A.; Hashimoto, M.; Nakanishi, K.; Dillon, J.; Sparrow, J. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 14609-14613. (6) Davies, S.; Elliott, M. H.; Floor, E.; Truscott, T. G.; Zareba, M.; Sarna, T.; Shamsi, F. A.; Boulton, M. Free Radical Biol. Med. 2001. (7) Sarna, T.; Burke, J. M.; Clancy, C. M. R.; Lamb, L. E.; Pawlak, A.; Rozanowska, M.; Simon, J. D.; Zareba, M. InVest. Ophthalmol. Visual Sci. 2001, 42, S764. (8) Mata, N. L.; Weng, J.; Travis, G. H. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 7154-7159. (9) Eldred, G. E. Gerontology (Basel) 1995, 41, 15-26. (10) Sparrow, J. R.; Parish, C. A.; Hasimoto, M.; Nakanishi, K. InVest. Ophthalmol. Visual Sci. 1999, 40, 2988-2995. (11) Holz, F. G.; Bergmann, M.; Schuett, F.; Kopitz, J. InVest. Ophthalmol. Visual Sci. 2001, 42, S943. (12) Beatty, S.; Koh, H. H.; Henson, D.; Boulton, M. SurV. Ophthalmol. 2000, 45, 115-134. (13) Sparrow, J. R.; Nakanishi, K.; Parish, C. A. InVest. Ophthalmol. Visual Sci. 2000, 41, 1981-1989. (14) Rozanowska, M.; Jarvis-Evans, J.; Korytowski, W.; Boulton, M. E. J. Biol. Chem. 1995, 270, 18825-18830. (15) Rozanowska, M.; Wessels, J.; Boulton, M.; Burke, J. M.; Rodgers, M. A. J.; Truscott, T. G.; Sarna, T. Free Radical Biol. Med. 1998, 24, 11071112. (16) Boulton, M.; Jarvisevans, J.; Rozanowska, M.; Korytowski, W.; Burke, J. M.; Sarna, T. Vision Res. 1995, 35, S157.
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