Chemical and Biological Caging Effects on the Relaxation of a Proton

Castilla La Mancha, AVenida Carlos III, S.N., Campus Tecnológico de Toledo, 45071 Toledo, Spain. ReceiVed April 22, 2008. ReVised Manuscript ReceiVed...
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Langmuir 2008, 24, 10352-10357

Chemical and Biological Caging Effects on the Relaxation of a Proton-Transfer Dye Juan Angel Organero, Cristina Martin, Boiko Cohen, and Abderrazzak Douhal* Departamento de Quı´mica Fı´sica, Seccio´n de Quı´micas, Facultad del Medio Ambiente, UniVersidad de Castilla La Mancha, AVenida Carlos III, S.N., Campus Tecnolo´gico de Toledo, 45071 Toledo, Spain ReceiVed April 22, 2008. ReVised Manuscript ReceiVed July 4, 2008 We report studies of the interaction between a proton-transfer dye (1′-hydroxy,2′-acetonaphthone, HAN), with the human serum albumin (HSA) protein and a β-cyclodextrin derivative (DM-β-CD) in neutral water solutions. We used steady-state and picosecond time-resolved emission spectroscopy to follow the structural changes of HAN due to the hydrophobicity and confinement effect of these nanocavities. Upon encapsulation, the fluorescence intensity of the 1:1 inclusion complex in both cavities increases, and the emission lifetimes become longer. For the DM-β-CD complexes, we obtained 430 and 920 ps, whereas for the HSA complexes we obtained 630 ps and 2 ns. Picosecond anisotropy measurements show strong confinement due to protein docking. The rotational time for the CD complex is 660 ps, whereas for the protein comples we find 6 ns. The process of energy transfer from the excited triptophan 214 (Trp214) of HSA to the trapped HAN occurs with high efficiency (71%), and the calculated distance between both chromophores is 17 Å. We believe that the results are important for a better understanding of the processes occurring in inclusion complexes such as those in nanopharmacodynamics.

1. Introduction Studying the effects of confinement on the behavior of molecules provides valuable information on the nature of the nanoenvironment and the dynamical properties of the nanosystems.1-4 Among these systems are the inclusion complexes of dyes and drugs encapsulated by cyclodextrins (CDs) and proteins, with special interest being given to the human serum albumin (HSA) complexes.5-8 For CDs, the change in size of its nanocavity offers a nanochemical tool for studying the size-dependent photophysics of the confined dye in their hydrophobic interiors.1,9-12 Furthermore, the understanding of molecular recognition in protein-ligand complexes at the atomic level needs to elucidate the role of the molecular interactions (hydrophobic, electrostatic, van der Waals, and hydrogen bonding) that govern the mechanism of the complexation process.13-17 HSA protein is the most abundant one in the circulatory system. Its principal function is * Corresponding author. E-mail: [email protected]. (1) Douhal, A. Chem. ReV. 2004, 104, 1955–1976. (2) Kiba, T.; Kasajima, T.; Nishimura, Y.; Sato, S.-i. ChemPhysChem 2008, 9, 241–244. (3) Ghosh, S.; Mandal, U.; Adhikari, A.; Dey, S.; Bhattacharyya, K. Int. ReV. Phys. Chem. 2007, 26, 421–448. (4) Mallick, A.; Purkayastha, P.; Chattopadhyay, N. J. Photochem. Photobiol., C 2007, 8, 109–127. (5) El-Kemary, M.; Gil, M.; Douhal, A. J. Med. Chem. 2007, 50, 2896–2902. (6) Banerjee, D.; Pal, S. K. Chem. Phys. Lett. 2008, 451, 237–242. (7) Zhong, D.; Douhal, A.; Zewail, A. H. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 14056–14061. (8) Douhal, A.; Sanz, M.; Tormo, L. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 18807–18812. (9) Monti, S.; Koehler, G.; Grabner, G. J. Phys. Chem. 1993, 97, 13011– 13016. (10) Pastor, I.; Di Marino, A.; Mendicuti, F. J. Phys. Chem. B 2002, 106, 1995–2003. (11) Douhal, A. Acc. Chem. Res. 2004, 37, 349–355. (12) Harada, A.; Li, J.; Kamachi, M. Nature (London) 1993, 364, 516–518. (13) Rosenoer, V. M., Oratz, M., Rothschild, M. A., Eds.; Albumin Structure, Function, and Uses: Pergamon: Oxford, U.K., 1977; p 397. (14) Il’ichev, Y. V.; Perry, J. L.; Simon, J. D. J. Phys. Chem. B 2002, 106, 452–459, and references therein. (15) Oyekan, A. O.; Thomas, W. O. A. J. Pharm. Pharmacol. 1984, 36, 831– 834. (16) Bree, F.; Urien, S.; Nguyen, P.; Riant, P.; Albengres, E.; Tillement, J. P. Eur. J. Drug Metab. Pharmacokinet. 1990, 15, 303–307. (17) Trnavska, Z.; Trnavsky, K.; Zlnay, D. Eur. J. Clin. Pharmacol. 1984, 26, 457–461.

Figure 1. (A) Schematic representation of molecular structures of the enol (E*), keto (K*), and keto rotamer (KR*) of 1′-hydroxy-2′acetonaphthone (HAN) upon electronic excitation of E to give K* through an intramolecular proton-transfer (IPT) reaction and a subsequent twisting motion to generate KR*. (B) X-ray structure of human serum albumin (HSA) protein adapted from ref 16 and molecular shape of heptakis2,6-di-O-methyl-beta-cyclodextrin (DM-β-CD).

to transport a wide variety of fatty acids, metal ions, steroid hormones, vitamins, and numerous pharmaceuticals, and it contributes significantly to colloid osmotic blood pressure.18-20 X-ray crystallographic data of HSA revealed that the protein, a 585 amino acid residues monomer, contains 3 homologous R-helical domains (I-III) and a single tryptophan (Trp 214).18,19,21 Each domain contains 10 helices and is divided into antiparallel 6-helix and subdomains (A and B). The ligands bind to HSA in regions located in the hydrophobic cavities of subdomains IIA (binding site I) and IIIA (binding site II). Binding to site I is dominated by the strong hydrophobic interactions with neutral heterocyclic compounds, whereas binding to site II involves ion (dipole)-dipole, van der Waals, and/or H-bonding interactions with the polar cationic groups of HSA (Figure 1). (18) He, X. M.; Carter, D. C. Nature 1992, 358, 209–215. (19) Carter, D. C.; Ho, J. X. AdV. Protein Chem. 1994, 45, 153–203. (20) Peters, J. T. All About Albumin: Biochemistry, Genetics, and Medical Applications. Academic Press: San Diego, CA, 1996; p 414. (21) Wardell, M.; Wang, Z.; Ho, J. X.; Robert, J.; Ruker, F.; Ruble, J.; Carter, D. C. Biochem. Biophys. Res. Commun. 2002, 291, 813–819.

10.1021/la801256h CCC: $40.75  2008 American Chemical Society Published on Web 08/15/2008

Relaxation of a Proton-Transfer Dye

1′-Hydroxy-2′-acetonaphthone (HAN) is an aromatic molecule with two groups capable of forming an intramolecular H bond (Figure 1). This bond is comparable to that in methyl salicylate,22,23 but with a larger π-electron molecular system. In previous reports, we have studied the photophysics of HAN using laser fluorescence spectroscopy under jet molecular beam conditions and its quantum chemistry in both the S0 and S1 states and in solutions of different cyclodextrins.1,11,24-26 The results show that the most stable conformer of HAN (enol structure, E) is the one having an intramolecular H bond between the OH and COCH3 groups and that the electronic excitation of E induces through or over a small energy barrier an excited-state intramolecular proton-transfer (ESIPT) reaction leading to a ketotype tautomer (K*) (Figure 1). This structure subsequently may lead to a rotamer (KR*) through a twisting motion of the C-C bond, linking the now protonated acetyl group and the naphthalene frame. The formation of KR* and its lifetime can be controlled by choosing an appropriate size of the host.1,11,26 Femtosecond experiments in the gas phase performed by the Cheng group27 found that the time constant for shifting the proton within E* to equilibrate rapidly with the generated K* structure is on the picosecond time scale. However, femtosecond pump-probe experiments showed that the ESIPT of HAN occurs in ∼30 fs.28 Previously, we reported on studies of HAN using cyclodextrins (R-, β-, and γ-CD) as host media.26,29 The addition of CDs to a water solution of HAN results in an intensity enhancement and shift of the emission band of keto phototautomers. Both effects reflect the formation and emission behavior of inclusion complexes. The stoichiometry of the complexes depends on the nature and size of the cage. For β- and γ-CD, the complex has 1:1 stoichiometry, whereas for R-CD the stoichiometry is 1:2 HAN/R-CD. In continuation of our previous efforts, we report here on studies of the excited-state dynamics of HAN in solution, in the presence of dimethyl-β-cyclodextrin (DM-β-CD), and HSA protein (Figure 1). The results reflect a large change in the photodynamics of HAN upon encapsulation by these systems. We explain it in terms of restriction of motion of the dye and the hydrophobic effect of the nanocages.

2. Experimental Details HAN (Sigma-Aldrich, 99%), HSA protein (Fluka-Sigma-Aldrich, 99%), and DM-β-CD (Across Organics, 99%) were used as received. Sodium phosphate buffer 0.1 M at pH 7.0 was used in the preparation of the samples. Steady-state absorption and emission spectra were recorded on Varian (Cary E1) and Perkin-Elmer (LS 50B) spectrophotometers, respectively. The HAN concentration was kept around 10-5 M when studying the interaction with DM-β-CD and was varied up to 1.6 × 10-4 M in experiments with HSA. The emission lifetimes were measured using a previously described timecorrelated single-photon-counting picosecond spectrophotometer (FluoTime 200).26 The sample was excited by a 40 ps pulsed (20 MHz) laser centered at 371 or 393 nm, and the emission signal was collected at the magic angle (54.7°). The instrument response function (IRF) was typically 65 ps. The emission decays were convoluted to (22) Smith, K. K.; Kaufmann, K. J. J. Phys. Chem. 1981, 85, 2895–2897. (23) Felker, P. M.; Lambert, W. R.; Zewail, A. H. J. Chem. Phys. 1982, 77, 1603–1605. (24) Douhal, A.; Lahmani, F.; Zewail, A. H. Chem. Phys. 1996, 207, 477–498. (25) Organero, J. A.; Moreno, M.; Santos, L.; Lluch, J. M.; Douhal, A. J. Phys. Chem. A 2000, 104, 8424–8431. (26) Organero, J. A.; Tormo, L.; Douhal, A. Chem. Phys. Lett. 2002, 363, 409–414. (27) Lu, C.; Hsieh, R. M. R.; Lee, I. R.; Cheng, P. Y. Chem. Phys. Lett. 1999, 310, 103–110. (28) Lochbrunner, S.; Schultz, T.; Schmitt, M.; Shaffer, J. P.; Zgierski, M. Z.; Stolow, A. J. Chem. Phys. 2001, 114, 2519–2522. (29) Organero, J. A.; Douhal, A. Chem. Phys. Lett. 2003, 373, 426–431.

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Figure 2. UV-visible absorption (Abs) and emission (Emi) spectra of 10-5 M HAN in a phosphate buffer solution at pH 7 (---) and in the presence of 14 µM HSA (-). The excitation wavelength was 371 nm.

Figure 3. Emission spectra of 14 µM HSA in water (pH ∼7) and in the presence of different amounts of HAN {(0-2.2) × 10-5 M}. The excitation wavelength was 280 nm. The inset is an enlargement of the HAN emission band. The spectra were corrected for the inner filter effect of increasing HAN concentration. See Supporting Information for the absorption spectra at different HAN concentrations.

the IRF and fitted to a multiexponential function using the Fluofit package. The time-resolved anisotropy was constructed using the expression r(t) ) (I| - GI⊥)/(I| + 2GI⊥), where G is the ratio between the fluorescence intensity at parallel (I|) and perpendicular (I⊥) polarizations of the emission with respect to the excitation beam. The value of G was measured at a gating window in which the fluorescence is almost completely depolarized (tail-matching technique). The quality of the fits was characterized in terms of the residual distribution and reduced χ2 values. All measurements were made at 293 ( 1 K.

3. Results and Discussion 3.1. Steady-State Observation. Figure 2 shows the UV-visible absorption and emission spectra of HAN in neutral water and in the presence of 14 µM HSA. Upon addition of the protein, the absorption band shows a small variation. However, the corresponding emission band exhibits a significant enhancement in intensity and shifts from ∼480 to 465 nm. This change indicates the effect of the surrounding environment on HAN, provided by the internal cavities of HSA. Thus, the emission spectrum of HAN in the presence of HSA is similar to that recorded using a restricted, hydrophobic pocket such as that provided by β-CD or DM-β-CD (vide infra), where the position of the maximum is at 460 nm.26 Therefore, emission within HSA is from that of a caged HAN structure within a hydrophobic binding site. Figure 3 shows the change in the fluorescence spectra of HAN at different concentrations in neutral buffer solution containing a fixed concentration of HSA and excited at 280 nm. (The absorption spectra of these solutions are given in the Supporting Information). The emission band with a peak at 337 nm is due

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Figure 5. Fluorescence excitation spectra of HSA protein in water (pH ∼7) ( · · · ) observed at 330 nm. The dashed and solid lines correspond to the normalized fluorescence excitation spectra of HAN in water (pH ∼7) without and in presence of 14 µM HSA protein, respectively (λem ) 465 nm).

the maximum in the fluorescence intensity occurs at fHAN ) 0.5 HAN; therefore, the complex has 1:1 stoichiometry. To determine the binding constant between both entities, we used eq 1,30 which assumes an equilibrium between free HAN, HSA, and the 1:1 complex ∆Ii )

{ [( ∆IS

) (

1 + [HAN] + [HSA] Keq

[HAN] + [HSA] +

1 Keq

)

2

- (4[HAN][HSA])

]}

2[HAN]

Figure 4. (A) Emission spectra of 14 µM HSA protein in water (pH ∼7) and in the presence of different amounts of HAN {(0-1.6) × 10-4 M}. The excitation wavelength was 365 nm. (B) Change in the emission intensity (I - I0) at 460 nm of the HSA protein (14 µM) solution with the concentration of HAN (λex ) 365 nm). The solid line shows the best fit assuming a 1:1 HAN/HSA complex using a model described in ref 30. The inset shows a Job’s plot of HAN/HSA emission at 460 nm upon increasing the molar fraction of HAN (fHAN) and upon excitation at 370 nm.

to the tryptophan (Trp214) residue of the protein. The addition of HAN leads to a concentration-dependent quenching of the Trp214 emission with the appearance and concomitant increase in the HAN emission at ∼470 nm. The emission spectra of the protein at 337 nm shift to ∼308 nm with increasing HAN concentration. These spectra were corrected by taking into account the inner filter effect due to the increasing absorption of HAN in this region. Thus, the observed change in Figure 3 is due to a variation in the intrinsic fluorescence of Trp214 caused by the presence of HAN within HSA. The remaining UV emission is a result of a change in the local environment of Trp214 and/or of the tyrosines of HSA. However, the intensity decrease in this emission and the simultaneous increase in that of HAN suggest an energy-transfer process from Trp214 (donor) to encapsulated HAN (acceptor). The inset of Figure 3 reveals an isoemissive point at ∼445 nm, indicating a correlation between these emissions and therefore supporting the energy-transfer explanation. Figure 4A shows the emission spectra of the above solutions, but upon excitation in the S0-S1 band of HAN. It is clear that the emission of the interaction of HAN with the protein is larger than that of the free species. The intensity maximum of the emission spectra at the saturation of the complex is located at ∼460 nm, whereas that of free HAN in water is at ∼490 nm. Again, the shift indicates a change in the photodynamics of HAN upon encapsulation by HSA. To obtain the stoichiometry of the complex, we used the Job’s plot technique and plotted the change in the fluorescence intensity of the complex versus the molar fraction of HAN (fHAN) in the solutions (inset of Figure 4B). The plot clearly shows that

(1)

where [HAN] and [HSA] represent their initial concentrations, respectively. Keq is the equilibrium constant, ∆Ii is the value of the fluorescence intensity change of the complex upon addition of a certain amount of HAN to a solution of HSA, and ∆IS is its value at saturation. The observed change was plotted against the HAN concentration to determine Keq of the 1:1 complex according to eq 1. The best fit gives Keq ) (2.6 ( 0.6) × 105 M-1 (Figure 4B). The excitation spectrum of HAN in the presence of HSA (Figure 5) shows that upon encapsulation its S0 f S1 transition shifts to longer wavelengths by ∼15 nm and the band becomes broader. The relative red shift is explained by an increase in the electronic conjugation in the dye induced by an increase in the intramolecular H-bond (IHB) strength between its OH and carbonyl parts when encapsulated by the hydrophobic pocket of the protein. In water, the formation of intermolecular H bonds between these groups and the water molecules weakens the IHB and therefore lower the π-electron conjugation, provoking a blue shift (to short wavelengths) of the absorption spectrum. Notice that as a result of weakening of the IHB in water, open enol forms of HAN lacking the IHB may be formed, thus giving an absorption band shifted to shorter wavelengths. In fact, upon substitution of the OH group by an OMe group in HAN, the maximum in the absorption spectrum in methycyclohexane shifts from 365 to 330 nm. This result clearly shows that an increase in the electronic conjugation by formation of the IHB in HAN leads to a red shift in its absorption band. Previously, we observed a red shift in the excitation spectrum of HAN when the cyclodextrin cavity size increased. The result was explained in terms of a larger relaxation of the guest inside a wider host cavity, allowing a larger amount of electronic conjugation between the H-bond chelate ring and the naphthalene frame of HAN.26 Furthermore, it has been shown that anthracene (a nonflexible molecule) within a confining medium (zeolite) undergoes a change in its 0-0 electronic (30) Croce, K.; Freedman, S. J.; Furie, B. C.; Furie, B. Biochemistry 1998, 37, 16472–16480.

Relaxation of a Proton-Transfer Dye

Figure 6. Emission spectra of HAN (∼10-5 M) in water (pH ∼7) upon addition of different amounts of DM-β-CD {(0-8) × 10-3 M} and excitation at 365 nm. (Inset) Fluorescence intensity variation of HAN with DM-β-CD concentration observed at 465 nm. The solid line is from the best fit using eq 1, assuming a 1:1 complex.

transition.31 In a stronger confining zeolite, the 0-0 transition shifts to the red side of the absorption spectrum, a result that is explained in terms of a decrease of the HOMO-LUMO band gap of the confined dye.31 Thus, ion addition to the above causes the shift in the HAN absorption spectrum to the red side upon encapsulation by the protein, and the confinement effect may participate in lowering the electronic band gap. The broadening of the spectrum reflects the heterogeneity of the sample formed by free and encapsulated dye in slightly different conformations (having a different extent of docking) inside the hydrophobic pocket of HSA. Moreover, in the presence of HSA the ratio between the intensity of the bands at 365 and 280 nm decreases (from 1 to 0.7). The change is due to the Trp214 absorption and therefore is another signature of an energy-transfer process between the excited Trp214 and caged HAN. As mentioned above, the hydrophobic interactions of a caged dye with CD hosts may lead to a large change in their photophysics and photochemistry. Depending on the size of the guest, the CD cavity may decrease the rate constant of some nonradiative processes such as twisting motions. Therefore, we studied HAN in an aqueous solution of DM-β-CD to compare its behavior with that observed within the HSA protein. The addition of DMβ-CD induces a clear increase in the emission intensity with a blue shift of the phototautomers’ band from 490 to 460 nm (Figure 6), indicating the formation of an inclusion complex. We find a binding constant Keq ) (3.6 ( 0.2) × 103 M-1 for a 1:1 complex. (We did not obtain an accurate fit when assuming a 1:2 complex.) In a previous study, using the same fitting procedure, we obtained Keq ) (1.4 ( 0.2) × 103 M-1 and (0.9 ( 0.1) × 103 M-1 for β- and γ-CD, respectively.26 The value of the inclusion equilibrium constant increases in the order DM-β-CD > β-CD > γ-CD, showing that both the hydrophobicity and size of the CD play a role in the formation and stability of the formed inclusion complexes. However, the stability depends on the interplay between van der Waals and hydrophobic forces involved in the guest-host interactions.1,32 In the HAN/DM-β-CD complex, the methyl groups at the other gate act as a hydrophobic protector screen of the guest from water molecules, and the restriction to the possible twisting motion of the guest is more significant in DM-β-CD than in β- and γ-CD entities. These latter cavities have similar height (∼8 Å); therefore, part of the (31) Ma´rquez, F; Garcia, H; Palomares, E; Fernandez, L; Corma, A. J. Am. Chem. Soc. 2000, 122, 6520–6521. (32) Rekharsky, M. V.; Inoue, Y. Chem. ReV. 1998, 98, 1875–1917.

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Figure 7. (A) Emission decays of (a) HAN in water (pH ∼7) and in the presence of (b) 8.4 mM DM-β-CD and (c) 14 µM HSA observed at 460 nm and excited at 371 nm. (Inset) Normalized 1 ns gated emission spectra of HAN in the previous solutions. (B) Variation of the preexponential factor (ai) corresponding to the time constants (130 ps (O), 600 ps (•), and 2 ns (∆)) from the multiexponential fit of HAN emission decay at 460 nm as a function of the molar fraction of HSA. Table 1. Lifetime (τi) and Preexponential Factor (ai) Values of the Emission Decays of HAN in Water (pH ∼7) and in the Presence of 14 µM HSA Protein (As Indicated by the Molar Fraction, fHSA) Observed at 460 and 560 nm fHSA

[HAN] µM

0 0.1 0.5 0.8

130 126 14 4

Em ) 460 nm τi/ns (ai %)

Em ) 560 nm τi/ns (ai %)

0.09(99) 4(1) 0.09(99) 4(1) 0.13(66) 0.63(29) 2(5) 0.12(84) 0.63(12) 2(4) 0.13(38) 0.63(52) 2(10) 0.13(58) 0.63(35) 2(7) 0.14(29) 0.63(59) 2(12) 0.14(47) 0.64(42) 2(11)

dye (∼10 Å) is outside of the nanocage. However, because of the methyl groups at the second rim, the height of the DM-β-CD cavity is longer (∼11 Å), protects HAN from water, and makes hydrophobic interaction stronger, thus producing a larger K value in this pocket. Similar observations have been reported for other caged molecules.5 3.2. Time-Resolved Observation. To get information on the picosecond relaxation dynamics, following the ultrafast protontransfer reaction, we recorded the emission decays of HAN in water and in the presence of DM-βCD and HSA (Figure 7). In neutral water, the emission decay at 480 nm fits to a biexponential function with time constants of ∼100 ( 10 ps (99%) and 4 ( 1 ns (1%), as previously reported.26 The picosecond component is assigned to the K* structure, whereas the nanosecond component is assigned to its twisted structure (KR*). In the presence of 0.5 mol fraction HSA, the emission decay at 460 nm fits to a three-exponential function, giving time constants of 130 ( 20 ps (38%), 630 ( 50 ps (52%), and 2.0 ( 0.5 ns (10%). The shortest component corresponds to that found in water (∼100 ps). The increase in this time to 130 ps is due to an increase in the viscosity of the medium upon addition of HSA. The intermediate (∼630 ps) and longest (∼2 ns) components are assigned to the encapsulated K* and KR* structures. Because of the protection of K* by the cage of the protein, its lifetime becomes longer. However, the contribution to the signal of the KR* component increases from 1% in water to 10% in the presence of 0.5 mol fraction HSA at 460 nm. In a parallel way, that of the shortest component decreases from 99 to 38%. This behavior clearly reveals the presence of at least two emitters of HAN bound to HSA (630 ps and 2 ns components), in addition to free HAN in solution. Note that the value of the preexponential factor of these components depends on the relative HSA concentration to that of HAN and on the emission wavelength (Table 1, Figure 7B). For example, the contribution of the 630 ps component in the presence of 0.5 mol fraction HSA decreases at longer wavelength and shows the following changes: 52%

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(460 nm), 43% (480 nm), and 35% (560 nm). A similar trend, but more moderated, was observed in the nanosecond component (Figure 7B). The preexponential factor of the shortest component decreases from 66 to 29% in the presence of 0.1 and 0.8 mol fractions HSA, respectively, whereas those of the 600 ps and 2 ns components change from 29 to 59% and from 5 to 12%, respectively (Table 1). These data clearly indicate that the middle and longest components are related to the encapsulated K* and KR* structures. Thus, the biological confinement governs the relaxation route (time and yield) of the keto tautomers. Using 126 µM of HAN and 14 µM of HSA (molar fraction of HSA, fHSA ) 0.1) and the obtained equilibrium constant (Keq ) 2.6 × 105 M-1), we found 88% free HAN. From the lifetimes and preexponential factors of the emission decays (Table 1), we calculated the contribution of free HAN (130 ps component) in the total emission signal of the mixture under the same concentrations (HAN and HSA) and obtained ∼40% at 560 nm. Note that the contribution does not reflect the real population of this entity in the S1 state because the emission spectra of the free and encapsulated forms are not fully overlapping, thus hindering the comparison with the population of free HAN as calculated from Keq. (10-5

We recorded the emission decays of HAN M) in a saturated solution of DM-βCD (8.5 mM). At 460 nm, we obtained a three-exponential fit of the decay with time constants of (115 ( 10) (42%), (430 ( 20) (49%), and (920 ( 50) ps (9%). The shortest component is from free HAN in solution, whereas the other two are due to encapsulated K and KR*. These components are longer than those previously obtained in β-CD solutions: 90 (34%), 200 (63%), and 742 ps (3%).26 However, they are shorter than those obtained in the presence of γ-CD: 100 ps (82%), 650 ps (9%), and 4.2 ns (9%).26 The result shows the effect of the methyl groups of the nanocapsule on the relaxation of the caged dye. Within β-CD, the dye is not deeply embedded, and part of it (hydroxyl and acetyl groups) is exposed to water, making the nonradiative processes due to intermolecular H bonds with water more efficient. The methoxy groups of DM-β-CD create a larger hydrophobic cavity, leading to longer emission lifetimes of caged K* and KR*. The inset of Figure 7 shows the 1 ns gated emission spectra of HAN in water and in the presence of HSA and DMβ-CD. In water, the emission band shifts to the red side (maximum at 490 nm) when compared to those of HAN/HSA and HAN/ DM-β-CD (maxima at 460 nm) complexes. The red shift in neat water is due to the more efficient relaxation of K* to yield KR*, a process that is not favorable in C because it involves the twisting motion of the protonated acetyl group.26 3.3. Intermolecular Energy Transfer. The obtained steadystate emission spectra upon excitation of Trp214 (280 nm) and HAN (365 nm) suggest the occurrence of energy transfer from Trp214 to caged HAN. Figure 8 shows the spectral overlap between the emission band of HSA and the absorption band of HAN. Fo¨rster theory for nonradiative energy transfer is used to determine the distance between the donor (D, Trp214) and the acceptor (A, caged HAN). The efficiency (E) of the transfer from D to A is related to the distance (R) separating both partners according to eq 2,33 (33) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd ed.; Kluwer Academic/Plenum Publishers: Boston, 1999; p 725.

Figure 8. Spectral overlap between HAN absorption (-) and HSA emission ( · · · ) in a phosphate buffer solution (pH 7). The two spectra are normalized at their respective maxima.

E)1E)

Φ F )1Φ0 F0 R06 R06 + R6

(2) (2′)

where Φ0 (F0) and Φ (F) are the fluorescence quantum yields (intensities) of D in the absence and presence of equal amounts of A, respectively. R0 is the Fo¨rster critical distance between them, at which 50% of the excitation energy is transferred to A, which can be obtained from D emission and A absorption spectra using eq 3,33

R06 ) 8.8 × 10-5κ2nD-4ΦD

∫0∞ ID(λ)εA(λ)λ4dλ

(3)

ID(λ) is the normalized fluorescence spectrum of D, and ε(λ) is the molar absorption coefficient of A. The fluorescence quantum yield (ΦD) of tryptophan in HSA is 0.14,34 and the refractive index (nD) of water is 1.333. The orientation factor (κ2) is taken to be 2/3, assuming random orientations for both donor and acceptor.35 This value is reasonable for the present system because the tryptophan chromophore rotates very rapidly in the protein (isotropic system), as revealed by the picosecond component in its emission anisotropy.36-38 Using eq 2, we obtained E from F and F0 values for each HAN and HSA concentration (Table 1 in Supporting Information). Because the dye and the protein are not covalently linked by a chemical bond, the obtained values of E from F and F0 are corrected (Ecorr ) E/f) by taking into account the molar fractions (f) of the HAN/HSA complex deduced from the equilibrium constant and the initial concentrations of both guest and host. From several experiments, we obtained the values of the corrected efficiency and the mean value Ecorr ) 71 ( 3%, which indicates a high energy-transfer efficiency from Trp214 to the caged HAN. Equation 3 yields R0 ) 20 ( 2 Å, and using the mean value of Ecorr and eq 2′ involving R0 and R, we obtained the HAN-Trp214 distance, R ) 17 ( 2 Å. Previous studies have suggested dyes and drugs with similar size and aromatic moieties comparable to that of HAN, bound to site I of subdomain IIA, and comparable distances for energy transfer have been calculated.5,14,34 Because HAN is a neutral aromatic compound and on the basis of the (34) Sytnik, A.; Litvinyuk, I. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 12959– 12963. (35) Vazquez-Ibar, J. L.; Weinglass, A. B.; Kaback, H. R. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 3487–3492. (36) Maliwal, B. P.; Lakowicz, J. R. Biophys. Chem. 1984, 19, 337–344. (37) Marzola, P.; Gratton, E. J. Phys. Chem. 1991, 95, 9488–9495. (38) Hansen, J. E.; Rosenthal, S. J.; Fleming, G. R. J. Phys. Chem. 1992, 96, 3034–3040.

Relaxation of a Proton-Transfer Dye

Langmuir, Vol. 24, No. 18, 2008 10357

Figure 9. Decays of emission anisotropy of HAN (a) in water at pH ∼7, (b) in the presence of 8.4 mM DM-β-CD, and (c) in 14 mM HSA collected at 460 nm and excited at 371 nm. The solid lines represent the best fits.

behavior of complexation to site I in subdomain IIA of a large number of neutral heterocyclic molecules to HSA, we suggest that this dye is most probably bonded to the same site.13-17 According to Fo¨rster theory, the rate constant of energy transfer (kET) from D (Trp214) to A (HAN) is given by

( )

kET ) τHSA-1

R0 R

secondary structure of the HSA protein upon aromatic molecule encapsulation. Because of the electronic change in the emitting structures (K* and KR*) when compared to the absorbing one (E), the corresponding transient moments have different orientations, making the initial value of r(0) ) 0.25 different from the ideal one (0.40). Within DM-β-CD (8.4 mM), the anisotropy decay of emission exhibits biexponential behavior (Figure 9) and shows the existence of 1:1 complex in agreement with the steady-state emission and lifetime data. The fit of r(t) decay collected at 460 nm gives φ1 ) (135 ( 10) (32%) and φ2 ) (660 ( 50) ps (68%). The value of φ1 and the corresponding contribution slightly change with the gated region: (115 ( 10) (28%) and (105 ( 10) ps (25%) when the emission is collected at 480 and 560 nm, respectively. The small variation suggests the existence of several rotors that contribute to this component: free dye and caged dye undergoing a fast change in anisotropy due to internal motion within the cage. We attribute φ2 (660 ps) to the global rotational relaxation time of the 1:1 complex. Previously,26 we found φ2 ) 540 and 840 ps for HAN/β-CD and HAN:γ-CD complexes, respectively. The difference reflects the size of the cage and the formed complex.

4. Conclusions

6

(4)

where τHSA is the mean fluorescence lifetime of the tryptophan in HSA, τHSA ) 3.53 ns.39 Using eq 4, we obtained kET ) 7.5 × 108 s-1, a value that is within the range of reported values (106-1011 s-1) in the literature.34 3.4. Time-Resolved Anisotropy Observation. To get further information on the rotational photodynamics of HAN/HSA and HAN/DM-β-CD complexes, we performed time-resolved emission anisotropy (r(t)) measurements of HAN in neutral water in the presence of HSA (14 µM) and DM-β-CD (8 mM). Figure 9 shows representative r(t) decays at 460 nm and their exponential fits. In pure water, the decay fits to a single-exponential function yielding a rotational time of φ ) (70 ( 15) ps. Comparable rotational times have been obtained for dyes of analogous size.7,40-42 In the presence of HSA, the anisotropy decay of HAN significantly changes (Figure 9). It displays doubleexponential behavior, with rotational time constants φ1 ) (65 ( 15) ps (15%) and φ2 ) (6 ( 1) ns (85%) at 450 nm. The first component is due to free HAN in solution, and it increases its contribution on the red side of the spectrum (20 and 40% at 480 and 560 nm, respectively), whereas the contribution of the HAN/ HSA complex is smaller. The observed rotational time value of caged HAN is 5 times shorter than the global motion of HSA (∼20 ns),37 indicating a diffusive motion of the drug in the hydrophobic pockets of the protein. However, the value is large enough, suggesting a robust complex. At the moment, we cannot speculate on the induced change (if any) in the secondary structure of HSA due to the encapsulation of HAN. However, structural studies have shown minor43-46 to moderate47-49 changes in the (39) Kamal, J. K. A.; Behere, D. V. J. Biol. Inorg. Chem. 2002, 7, 273–283. (40) Balabai, N.; Linton, B.; Napper, A.; Priyadarshy, S.; Sukharevsky, A. P.; Waldeck, D. H. J. Phys. Chem. B 1998, 102, 9617–9624. (41) El-Kemary, M.; Organero, J. A.; Santos, L.; Douhal, A. J. Phys. Chem. B 2006, 110, 14128–14134. (42) Tormo, L.; Organero, J. A.; Douhal, A. J. Phys. Chem. B 2005, 109, 17848–17854. (43) Petitpas, I.; Bhattacharya, A. A.; Twine, S.; East, M.; Curry, S. J. Biol. Chem. 2001, 276, 22804–22809. (44) Fleury, F.; Ianoul, A.; Berjot, M.; Feofanov, A.; Alix, A. J. P.; Nabiev, I. FEBS Lett. 1997, 411, 215–220.

In this study, we reported on photophysical data related to the effect of biological (HSA protein) and chemical (DM-β-CD) nanocaging of a proton-transfer dye (HAN). Both steady-state and time-resolved measurements clearly indicate a strong docking of HAN within both cavities, giving a 1:1 complex and longer emission lifetimes of the formed caged tautomers. The complexation with the DM-β-CD cavity yields emission lifetimes (430 and 920 ps) that are longer than those found using the nonmethylated nanocage. The methoxy groups of the former increase the size of the hydrophobic cage, thus protecting and reducing the nonradiative rate constants of the encapsulated tautometic forms. For the protein complexes, we obtained 630 ps and 2 ns. Picosecond-resolved anisotropy experiments for both DM-β-CD and HSA entities reflect the robustness of the complexes. Energy transfer from excited Trp240 to an ∼17 Å distant, caged HAN occurs 71% of the time. The reported results may help in understanding the dynamic behavior of caged aromatic molecules and especially drugs, such as those in nanopharmacodynamics. Acknowledgment. This work was supported by the MEC and JCCM through projects CTQ-2005-00114/BQU and PCI08-00375868. B.C. thanks MEC for the Ramon y Cajal fellowship. Supporting Information Available: Change in the UV-visible absorption spectra of HAN and HSA protein in water at pH 7 upon addition of HAN. Observed (Eobs) and corrected (Ecorr) efficiency for energy transfer from the tryptophan of HSA (14 µM) to HAN at different concentrations. This material is available free of charge via the Internet at http://pubs.acs.org. LA801256H (45) Bhattacharya, A. A.; Grune, T.; Curry, S. J. Mol. Biol. 2000, 303, 721– 732. (46) Curry, S.; Mandelkow, H.; Brick, P.; Franks, N. Nat. Struct. Biol. 1998, 5, 827–835. (47) Ahmed-Ouameur, A.; Diamantoglou, S.; Sedaghat-Herati, M. R.; Nafisi, S.; Carpentier, R.; Tajmir-Riahi, H. A. Cell Biochem. Biophys. 2006, 45, 203– 213. (48) Li, Y.; He, W.; Tian, J.; Tang, J.; Hu, Z.; Chen, X. J. Mol. Struct. 2005, 743, 79–84. (49) Xie, M.-X.; Xu, X.-Y.; Wang, Y.-D. Biochim. Biophys. Acta 2005, 1724, 215–224.