J. Phys. Chem. 1995,99, 13028-13032
13028
Steady-State and Time-Resolved Study of the Proton-Transfer Fluorescence of 4-Hydroxy-5-azaphenanthrenein Model Solvents and in Complexes with Human Serum Albumin Alexander Sytnik* and Juan Carlos Del Valle? Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida 32306-3015 Received: April 13, 1995; In Final Form: June 12, 1999
The corrected fluorescence maxima of the proton-transfer (PT) tautomer of a novel protein-binding site probe, 4-hydroxy-5-azaphenanthrene(HAP),correlate with the static polarity of the environment. The FT fluorescence of HAP exhibits a maximum in water at 586 nm and at 623 nm in cyclohexane. This PT fluorescence is perturbed by either a strong base or acid, and the emission of the anion or cation shows up in the spectra, respectively. The fluorescence lifetime of the PT tautomer of HAP increases with solvent polarity, being 0.36 ns in cyclohexane and 0.50 ns in methanol. The steady-state and time-resolved data demonstrate the insensitivity of PT tautomer fluorescence of HAP to dipole-dipole relaxation of the environment and the high static polarity of the so-called hydrophobic binding site of human serum albumin (HSA). The overlap of the fluorescence spectra of HAP observed in water solution and in a complex with HSA suggests the similarity of the static polarities in these two environments.
Molecular spectroscopy has made great advances during the past two decades in the understanding and description of the excited-state intramolecular proton transfer (ESIPT).'-S This phenomenon has already found a practical application for a new type of dye molecular scintillators,8 and fluorescence probes for b i o m o l e ~ u l e s . ~The . ~ ~ ESIPT, which has a femtoseconds rise time,''.'* is followed by formation of the proton transfer (PT) tautomer in the excited state (S'I). In the ground state of this tautomer (S'O) the reverse PT occurs to the ground state of the normal tautomer (SO).If the molecule possessing an ESIPT is dissolved in sufficiently inert media (aprotic solvents, e.g., cyclohexane), which do not perturb the PT, the corresponding S'I S'O fluorescence dominates in the emission spectrum if nonradiative deactivation of the S'I proton transfer state does not interfere. In solvents which can interfere with PT,e.g., ethanol or dimethyl sulfoxide, several fluorescence bands may be observable: (a) the normal tautomer molecule fluorescence band (SI SO),whose onset overlaps with the onset of the normal molecule absorption; (b) the ground state anion or cation fluorescence (e.g., Sal Sa0) band that overlaps with the absorption of the anion or cation; and (c) the PT tautomer fluorescenceband (S'I SO), which may have a gross frequency shift, up to 13 000 cm-', from the normal molecule absorption. The molecule of interest here, 4-hydroxy-5-azaphenanthrene (HAP), exhibits an ESIPT which appears to be unaffected by hydrogen-bonding solvent interferences, and even in ethanol, with only PT tautomer f l u o r e s ~ e n c e observed ~.~~ as a unique emission. The present research is a continuation of an ESIPT exploration of HAP and the application of this probe for protein binding site study. The PT tautomer fluorescence of HAP appears in the orange-red spectral region and is clear of the UV excitation and natural emissions commonly observed in biomolecules. As shown elsewhere? by using the uncorrected steady-state fluorescence data for the detector sensitivity, the position of PT tautomer fluorescence for HAP correlates with the polarity of
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' On leave from Universidad Autdnoma de Madrid courtesy of a Fulbright scholarship. * To whom correspondence should be addressed. Abstract published in Advance ACS Absrracrs. August 1, 1995. @
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the environment, and it is free from the influence of solvent relaxation. Such a fluorescence probe, which is insensitive to the dielectric relaxation of the environment, could be very useful for the investigation of structural transitions 'in biopolymers, e.g., as a monitor of proteins folding and unfolding. Since the absorption spectrum of the normal tautomer of HAP (A,, 370 nm) overlaps with the fluorescence spectrum of tryptophan (Amax 350 nm), one can expect the Forster type intermolecular energy transfer from the intrinsic tryptophans of proteins to HAP. Such energy transfer will transform the UV excitation of protein tryptophans to the orange fluorescence of PT tautomer of HAP, which could be used for both distance and conformation transition determinations in proteins. In the present work we apply the corrected steady-state and the phase-modulation timeresolved fluorescence to further investigate the spectroscopic properties of HAP in different solvents and in complexes with human serum albumin (HSA). We are specially interested in unraveling the polar solvent relaxation independence of the PT tautomer fluorescence of HAP and in describing the static polarity properties of protein binding sites.
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Materials and Methods Spectroscopic Measurements. The absorption spectra were recorded on a Shimadzu UV-2100 spectrophotometer. The fluorescence spectra and time-resolved phase-modulation experiments were performed with a Huorolog-2 spectrofluorometer (Spex Industries, Edison, NJ). Chemicals. All solvents were spectrograde quality and were checked for fluorescence impurities. HAP was a gift from Dr. Pi-Tai Chou (Department of Chemistry, National Chung Cheng University, Chia-Yi, Taiwan, R.O.C.). The commercial product (TCI America, Portland, OR) was twice recrystallized from methylcyclohexane and then vacuum-sublimed. HAP was dissolved in water, 1 x lod5M, using an ultrasonic vibrator Branson 2210 (Branson, Danbury, CT). Preparation of HAP-HSA Complexes. Essentially fatty acid-free HSA was a product from Sigma. HAP was dissolved in ethanol, and then 7 pM of the stock solution was added to the protein solution in 0.05 M Hepes buffer, pH 7.0 (2.2 mL). 0 1995 American Chemical Society
J. Phys. Chem., Vol. 99, No. 34, 1995 13029
Proton-Transfer Fluorescence of 4-Hydroxy-5-azaphenanthrene
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Proton-transfer tautomer Figure 1. Excited-state proton-transfer tautomerism in HAP.
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Figure 2. Absorption and fluorescence spectra of HAP in DMSO at 298 K: (1) normal tautomer absorption; (2) proton-transfer tautomer fluorescence, hex = 370 nm; (3) anion and proton-transfer tautomer fluorescence, hex = 400 nm.
After incubation and mixing for 3 h at room temperature, the solutions were centrifuged for 30 min at 23 OOOg.
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Figure 3. Absorption (left) and fluorescence spectra (right) of HAP in DMSO at 298 K: (upper) in the presense of 0.013 M NaOH; (lower) in the presence of 0.2 M HC1. hex = 370 nm (see text). 1 .o a, 0
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Results and Discussion Steady-State Fluorescence of PT Tautomer of HAP in Different Solvents. HAP has a strong six-membered ring intramolecular hydrogen bond between the 0 - H proton and the N atom (Figure 1). This hydrogen bond permits a highly efficient generation of the proton-transfer tautomer of HAP even in ethanol at both 298 or 77 K,9.'3eliminating the fluorescence of the normal tautomer of HAP. Even in dimethyl sulfoxide (DMSO), which is known as one of the most interfering solvents for intramolecular hydrogen bond formation,14 the absorption spectrum of HAP does not include the presence of the ground state anion band (Figure 2). Usually the absorption band of a corresponding anion is red shifted in comparison with the absorption band of the normal molecule. Excitation at the maximum of the first band of the absorption spectrum of HAP in DMSO is followed by the unique PT tautomer fluorescence with the A,, at 612 nm. Only the excitation at the red edge (400 nm) of the absorption spectrum of HAP in this solvent, where one can expect the contribution of the ground state anion, yields an additional peak at -500 nm in the fluorescence spectrum (Figure 2). By monitoring at Le,,, = 510 nm, the excitation spectrum of HAP in DMSO is shifted to the red by 50 nm compared to the spectrum monitored at Le, = 620 nm (not shown). This dependence of the excitation spectrum at the monitored wavelength proves the existence of new chemical species in the ground state. We attribute this species to the HAP anion. Figure 3 shows the absorption and fluorescence spectra of HAP in DMSO in the presence of the high concentrations of the base and acid. The base (0.013 M NaOH) causes the formation of the ground state anion which has the absorption maximum at 438 nm and the fluorescence maximum at 523 nm. The addition of a drop of HCl (concentrated) to the working solution restores the PT tautomer fluorescence, and some emission from the ground state cation (Amax = 500 nm) is seen. HAP has limited solubility in water at room temperature. However, an aqueous solution of HAP (1 x M) exhibited
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Figure 4. PT fluorescence of HAP in water (l), 1:l complex with HSA (2). and dioxane (3). de, = 370 nm, T = 298 K.
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Figure 5. Dependence of the PT tautomer fluorescence maximum of HAP on the dielectric constant of solvent at 298 K for aprotic (filled circles) and protic (hollow circles) solvents.
the pure PT tautomer fluorescence with A,, =586 nm on excitation at A,, = 370-410 nm (Figure 4). The position of HAP fluorescence Amax in water has the shortest wavelength in comparison with the other solvents studied. Figures 5 and 6 show the dependence of PT fluorescence Qmax of HAP on the dielectric constant and the empirical solvent polarity parameter EN^ of R e i ~ h a r d t . 'Both ~ figures clearly show the influence of polarity on the position of the HAP fluorescence maximum. The minor deviations from the linearity, which are observed for the Reichardt scale, may be caused by the fact that this scale includes the mutual dependence of fluorescence maximum on both nonspecific (polarity and polarizability) and specific solvent
Sytnik and Del Valle
13030 J. Phys. Chem., Vol. 99, No. 34, 1995
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Figure 6. Dependence of the PT tautomer fluorescence maximum of HAP on the empirical solvent polarity parameter EN^ of ReichardP at 298 K.
effects (e.g., acidity and basicity).I6 In our previous work? we observed the linear dependence of the A,, position of the PT tautomer fluorescence of HAP on the EN^ parameter of Reichardt. The linearity was the result of plotting Amax in nanometers as well as the use of uncorrected data for the detector sensitivity. Catalan and collaborators have proposed a scale which reflects only the dipolarity and polarizability of the solvent.I6 In the present work we do not discuss the relation among polarity, basicity and acidity of the environment. HAP as a fluorescence probe could be sensitive to the effects of basicity and acidity of solvent, but at any rate these two effects are minor in comparison with the influence of static polarity. In this paper we are concerned with unraveling the dependence of PT fluorescence of HAP on solvent relaxation. Comparison of the Environment Relaxation Sensitivity of Fluorescence Probes (HAP with Dansyl) Bound with the Protein. As shown in Figure 4, the fluorescence spectrum of HAP forming the 1:l complex with HSA (A,, = 588 nm) almost overlaps the fluorescence spectrum of HAP in water. This suggests that the HAP environment in the protein binding site has a polarity comparable with that of water. This conclusion is drawn from the PT tautomer fluorescence of HAP insensitivity to the solvent rela~ation.~ Dansyl, another fluorescence probe, is sensitive to both polarity and relaxation of the surrounding molecules. If the relaxation time of the environment is longer than the fluorescence lifetime of this probe, its emission occurs before the solvent relaxation, and the emission maximum shifts to a shorter wavelength in comparison with the fluorescence occurring after solvent relaxation. For dansyl one can predict the blue shift of fluorescence with the decrease of temperature and polarity of the environment. From previous studies it is known that high affinity binding sites of HSA are rigid on the nanosecond time scale.”.’* Because of this rigidity, the fluorescence of probes sensitive to the environment relaxation, and possessing a fluorescence lifetime shorter than the relaxation of surrounding groups in protein matrix, does not represent the actual polarity of the environment. We chose dansylphenylalanine (DP) as a dansyl derivative probe for HSA, because it binds with albumin much more strongly than dan~y1amide.I~Figure 7 shows the fluorescence spectra of DP in water, dioxane, and 1: 1 complex with HSA. It is worthy of mention that the PT fluorescence of DP-HSA is closer to the DP fluorescence in dioxane. In the case of HAP, the HAP-HSA fluorescence maximum is shifted by 33 nm from the fluorescence of HAP in dioxane (Figure 4). The relaxation dynamics of water molecules, -50% of which occurs in less than 50 fs for one relaxation component and somewhere between 100 and 1000 fs for the remaining relaxation components,20 has a great contribution to the shift of dansyl fluorescence in water compared with dioxane. HAP
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Wavelength [ n m ] Figure 7. Fluorescence of dansylphenylalanine in water (I), 1: 1 complex with HSA (2), and dioxane (3). Le, = 330 nm, T = 298 K.
TABLE 1: HAP Proton-Transfer Tautomer Fluorescence Mean Lifetimes at 298 K sample A,,, nm rem. ns le,, nm X’ cyclohexane 623 370 0.365 0.428 0.421 0.491 612 370 acetonitrile 0.449 0.734 glycerol triacetate 390 61 1 612 0.502 1.188 370 dimethyl sulfoxide 0.497 0.495 600 methanol 370 HSA-HAP complex (1 :1) 0.760 0.579 637 370 HSA-HAP complex ( 1 5 ) 0.643 0.432 637 370 is insensitive to the solvent relaxation effect and the fluorescence spectrum of the HAP-HSA complex (Figure 4) reflects the polarity of the PT probe environment in the protein binding site. One can suggest that HAP and DP bind with HSA in different sites, and therefore they represent polarities of different environments. Our experiments with the 5:l HAP-HSA complex demonstrate the high polarity of the HAP environment (PT fluorescence A,,, is situated at 590 nm). Fluorescence Decay Time Studies of HAP. The main idea for the study of time resolved spectroscopy of HAP was to probe the solvent relaxation insensitivity of PT tautomer fluorescence and to determine the time window for structural study of proteins given by the HAP emission. Table 1 presents the fluorescence decay mean lifetimes of H A P in the various solvents, all observed decays being monoexponential, since the fitting of experimental data in the double-exponential model was worse than for a single exponent. For instance, the modeling of fluorescence decay times of H A P presented in Table 1 by two components provides the following parameters for acetonitrile: a1 = 0.970, rem.l= 0.421 ns; a2 = 0.03, rem,2= 0.418 ns; x2 = 0.549. For glycerol triacetate, al= 0.977,rem.l= 0.449 ns; a2 = 0.023, ~ e m . 2= 0.441 ns; x2 = 0.804. The PT tautomer fluorescence lifetime increases with the polarity of the environment. For example, in cyclohexane re, is 0.365 ns and is 0.497 ns in methanol. For the 1:1 HAP-HSA complex the decay of PT tautomer fluorescence is also monoexponential, with re, = 0.760 ns. This fact can suggest that all molecules of HAP are bound in the same affinity binding site with the 1:1 stoichiometry. One could propose that HAP could be bound to different sites in HSA, which possess very similar environmental properties, but that does not seem to be the case. The modeling of fluorescence decay for the HAP-HSA 5:l complex gave the best fit for the single exponent with the re, = 0.643 ns. A trial modeling was carried out using three fluorescence decay constants and led to no improvement over single decay constant fit to the rate law. It is well known that, for chromophores undergoing strong interaction with solvent dipoles in the SI state during the fluorescence lifetime, one could observe a strong dependence of rem on the &,,,.*I If the medium is nonpolar, solute-solvent
Proton-Transfer Fluorescence of 4-Hydroxy-5-azaphenanthrene
J. Phys. Chem., Vol. 99, No. 34, 1995 13031 1.0
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Figure 8. PT fluorescence (right scale) and remvs I , , dependence for HAP (left scale) in cyclohexane. Lex= 370 nm, T = 298 K.
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Figure 10. FT fluorescence (right scale) and remvs I,, dependence for HAP (left scale) in glycerol triacetate. I,, = 390 nm, T = 298 K.
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interaction is very weak, and r e m does not depend on Aem. Figure 8 shows the fluorescence spectrum and the rem vs Aem dependence for HAP in cyclohexane. In a solvent with a low polarity like cyclohexane, remfor the PT fluorescence of HAP does not change when A,, is increased, as expected. In contrast, in acetonitrile the PT fluorescence of HAP at 298 K exhibits an increment of remof 24% with increasing A,, from 567 to 671 nm (Figure 9). Such a behavior was not expected, because the relaxation time of acetonitrile is within the range 0.4-0.9 x and 3 orders of magnitude faster than re, of HAP at room temperature. Since the resolution of our phase-modulation instrument is limited to 20 x ns, which is 20-50 times longer than the relaxation of acetonitrile, we assume that the difference in the remat the short and long Aem range can be caused by the static distribution of solute-solvent interaction configurations in the S’, state. In addition, a rem vs Aem dependence for PT tautomer of HAP in DMSO was observed at 298 K. In this case, r e m increases from 0.474 ns at Aem 568 nm to 0.577 ns at &m 690 nm. Glycerol triacetate is an aprotic polar solvent with a relaxation time of 0.820 ns at 284 K,**which is long enough to be resolved by the Fluorolog-2 spectroflourometer. Figure 10 shows the PT fluorescence spectrum of HAP and the r e m vs A,, dependence in glycerol triacetate at 298 K. The re, of HAP (-0.470 ns) in glycerol triacetate is comparable with the solvent relaxation time; however, a change in the remvs A,,,,dependence is not markedly evident. The value of the PT fluorescence lifetime of HAP by monitoring at 581 nm is 0.453 ns, while by monitoring at 672 nm the rembecomes 0.492 ns. The sensitivity of our phase-modulation instrument is in the range of 20 ps, and the estimated error is 2%, so the data presented in the Figure 10 are examined as a small dependence of rem on Aem in glycerol triacetate at room temperature. These results support our prior conclusion on the independence of PT tautomer fluorescence of HAP of solvent relaxation.
Summing up the new data on the steady-state and decay time fluorescence of HAP, we interpret these to indicate a sensitivity of the PT tautomer fluorescence of HAP on solvent static polarity and the insensitivity of this fluorescence on the solvent rela~ation.~ The comparison of PT tautomer fluorescence of the HAP-HSA complex with the HAP proton transfer fluorescence in different organic solvents and water suggests that the probe binding site of HSA has a static polarity comparable with the polarity of water. In our previous research the fluorescence maximum of the PT tautomer of HAP-apomyoglobin (apo-Mb) complex was blue-shifted by 5 nm in comparison with the fluorescence maximum of HAP bound with HSAS9This fact can suggest that probe binding site in apo-Mb has a polarity higher than the static polarity of HSA binding site and even that of water. The observation of very high polarity of probe binding sites in HSA and apo-Mb shows that the questions of hydrophobicity and hydrophobic interactions in proteins remain to be resolved.
Acknowledgment. We are pleased to acknowledge Prof. Michael Kasha and Prof. Javier Catalan for valuable discussions and comments on this work. Prof. Pi-Tai Chou is also acknowledged for the gift of a sample of HAP. This research was supported under Contract DE-FG05-87ER60517 between the Office of Health and Environmental Research, U.S. Department of Energy, and Florida State University. J.C.V. thanks the Fulbright Commission and Ministerio de Educacion y Ciencia de Espaiia for a Fulbright fellowship. References and Notes (1) Klopffer, W. Intramolecular proton transfer in electronically excited molecules. In Advances in Photochemistry; Pitts, Jr., J. N., Hammond, G. S., Gollnick, G., Eds.; John Wiley and Sons: New York, 1977; Vol. 10, pp 311-358. (2) Kasha, M. J. Chem. Soc., Faraday Trans. 2 1986, 82, 2379. (3) Barbara, P. F., Trommsdorff, H. P., Eds. Spectroscopy and dynamics of elementary proton transfer in polyatomic systems, Chem. Phys. 1989, 136, 153-360 (special issue). (4) Barbara, P. F., Nicol, M., El-Sayed, M. A., Eds. Photoinduced proton transfer in chemistry, biology and physics. J. Phys. Chem. 1991, 95, 10215- 10524 (special issue). (5) Formosinho, S. J.; Amaut, L. G.J. Phorochem. Photobiol. A: Chem. 1993, 75, 2 1. (6) (a) Khan, A. U., Kasha, M. Proc. Natl. Acad. Sci. U.S.A. 1983.80, 1767. (b) Chou, P. T.; McMonow, D.; Aartsma, T. J.; Kasha, M. J. Phys. Chem. 1984, 88, 4596. (7) Acuiia, A. U.; Amat-Guem, F.; Catalan, J.; Costela, A,; Figuera, J. M.; Muiioz, J. M. Chem. Phys. Lett 1986, 132, 567. (8) Sytnik, A,; Kasha, M. Radiat. Phys. Chem. 1993, 41, 331. (9) Sytnik, A.; Kasha, M. Proc. Natl. Acad. Sci. U.S.A. 1994.91. 8627. (10) Sytnik, A,; Gormin, D.; Kasha, M. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 11968. (11) Schwartz, B. J.; Peteanu, L. A.: Harris, C. B. J. Phys. Chem. 1992, 96, 3591. (12) Laermer, F.; Elsaesser, T.; Kaiser, W. Chem. Phys. Lett. 1988, 148, 119.
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(14) (a) Catalan, J.; Gomez, J.; Couto, A.; Laynez, J. J. Am. Chem. SOC. 1990, 112, 1678. (b) Catalan. J.; Perez, P.; Fabero, F.; Wilshire, J. F. K.; Claramunt, R. M.; Elguero, J. J. Am. Chem. Soc. 1992, 114, 964. (15) Reichardt, C. Solvents and Solvent Eflects in Organic Chemistry, VCH: Weinheim, Germany, 1988; pp 365-371. (16) Catalan, J.; Lopez, V.; Perez, P.; Martin Villamil, R.; Rodriguez, J. G. Liebigs Ann. 1995, 241. (17) Demchenko, A. P.; Sytnik, A. I. Proc. S f I E 1992, 1640, 191. (18) Demchenko, A. P. Biophys. Chem. 1982, 15, 101.
(19) Sytnik, A.; Kasha, M. Test of excitonic coupling of protein tryptophans as photon-antenna to tryptophan-dansyl as an energy-trap pair in binding site studies of serum albumins. Manuscript in preparation. (20) Jimenez, R.; Fleming, G. R.; Kumar, P. V.; Maroncelli, M. Nature (London) 1994, 369, 47 1. (2 1) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Plenum Press: New York, 1983; pp 217-253. (22) Kang, T. J.; Kahlow, M. A.; Giser, D.; Swallen, S.;Nagarajan, V.; Jarzeba, W.; Barbara, P. F. J. fhys. Chem. 1988, 92, 6800. Jp95 10709
