Dynamical and Structural Changes of an Anesthetic Analogue in

Oct 8, 2008 - Carlos III, s/n, 45071 Toledo, Spain, and Departamento de. Quımica Fısica, Facultad de Ciencias, AVda Camilo José Cela 13071, Ciudad ...
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J. Phys. Chem. B 2008, 112, 13641–13647

13641

Dynamical and Structural Changes of an Anesthetic Analogue in Chemical and Biological Nanocavities ´ ngel Organero,† Boiko Cohen,† Cristina Martin,† Lucia Santos,‡ and Laura Tormo,† Juan A Abderrazzak Douhal*,† Departamento de Quı´mica Fı´sica, Seccio´n de Quı´micas, Facultad de Ciencias del Medio Ambiente, UniVersidad de Castilla-La Mancha, AVda. Carlos III, s/n, 45071 Toledo, Spain, and Departamento de Quı´mica Fı´sica, Facultad de Ciencias, AVda Camilo Jose´ Cela 13071, Ciudad Real, Spain ReceiVed: April 9, 2008; ReVised Manuscript ReceiVed: August 26, 2008

We report on photophysical studies of the interaction between an anesthetic analogue, methyl 2-amino-4,5dimethoxybenzoate (ADMB), with the human serum albumin (HSA) protein and the normal micelle of n-octylβ-D-glucopyranoside (OG) in water solutions. We used steady-state and picosecond time-resolved emission spectroscopy to follow the dynamical and structural changes due to their hydrophobicity and confinement on the photophysical behavior of ADMB. The formed 1:1 complex with the protein is robust with an equilibrium constant of 9.6 × 104 M-1 at 293 K. The fluorescence lifetimes of the 1:1 entity become longer (up to ∼10 ns), and the emission transients show complex behavior due to the heterogeneity of the media. Rotational time (45 ns) from picosecond anisotropy measurements clearly indicates strong confinement in the robust ADMB:HSA complex. For the ADMB:OG one, the anisotropy decays give time constants of 50 and 980 ps, assigned to free and restricted rotors within the micelle, respectively. The process of energy transfer from the excited tryptophan 214 (Trp214) of HSA to the trapped ADMB occurs with an efficiency of 50%, and the calculated distance between both chromophores is 19 Å. We believe that these results are important for a better understanding of processes occurring in encapsulated drugs and thus should be relevant to nanopharmacodynamics. 1. Introduction Knowledge of the nature of the interactions between drugs and proteins and the time scale of the involved processes are crucial for the understanding of the biochemical consequences of the drugs inside the human body1-5 and their importance in pharmacology and pharmacodynamics.6,7 Human serum albumin (HSA) protein is one of the most abundant proteins in the circulatory system, forming about 60% of the mass of plasma protein, with a typical concentration of 5 g/100 mL in the bloodstream.7 It is the most important delivery system for a wide variety of fatty acids, metal ions, steroid hormones, vitamins, and drugs such as tranquilizers and general anesthetics. Crystallographic studies of HSA indicate that at pH 7 the protein adopts a heart-shaped three-dimensional structure with three homologous R-helical domains I-III (Figure 1).8-10 Each domain contains 10 helices, and it is divided into antiparallel six-helix and subdomains (A and B). The ligands bind to HSA in regions located in hydrophobic cavities of subdomain IIA (binding site I) and IIIA (binding site II). Binding in site I is dominated by the strong hydrophobic interactions with neutral heterocyclic compounds, while binding to site II involves ion (dipole)-dipole, van der Waals, and/or H-bonding interactions with the polar cationic groups of HSA.11-13 The effect of HSA on the structure and stability of caged drugs (in real time) is crucial for understanding its physiological functions and for the knowledge of delivery and efficiency of drugs into the human body. Several techniques have been used to analyze the * To whom correspondence should be addressed: Tel +34-925265717, Fax +34-925268840, e-mail [email protected]. † Universidad de Castilla-La Mancha. ‡ Facultad de Ciencias.

Figure 1. (A) Schematic representation of conformers I and II of 2-amino-4,5-dimethoxymethyl benzoate (ADMB). (B) X-ray structure from ref 8 of human serum albumin (HSA). (C) Molecular structure of the n-octyl-β-D-glucopyranoside (OG) micelle.

interaction between HSA and different probes and to identify the specificity of binding sites of the protein.14-21 The difficulty to study the interactions between drugs and biological systems comes from the complexity of the latter ones. Thus, simple systems such as micelles that mimic biological media and cell membrane structures have been used.22-24 The normal micelle, alkylglucoside n-octyl-β-D-glucopyranoside (OG), and chosen

10.1021/jp803083y CCC: $40.75  2008 American Chemical Society Published on Web 10/08/2008

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here, is a nonionic detergent that consists of an octane group attached to a glucose unit (Figure 1). Its structural and thermodynamic properties, as well as the spectroscopic studies of different probes embedded within, have been widely studied.2,23,25,26 The probe selected here, 2-amino-4,5-dimethoxymethyl benzoate (ADMB), is an anesthetic analogue to procaine and tetracaine (p-aminobenzoate derivates), with two functional groups (amino and carboxylate) that are able to form an intramolecular H-bond (Figure 1). This bond is comparable to that found in methyl salicylate, where the hydroxyl group has been substituted by the amino one. The spectral and dynamical properties of ADMB in liquid media, as well as the confinement effect on its emission in cyclodextrins, have been reported previously.27,28 The results showed that upon encapsulation the excited-state lifetime becomes longer. Since the guest molecule may adopt different conformations inside the nanocavity and different H-bonding interactions with water molecules, the emission decays are rather complex.27,28 In continuation to our previous studies in this field,2,4,15,22-24 we report here on the effects of the cavity environment of HSA protein and OG on the structures and photodynamics of ADMB. Steady-state and picosecond-time-resolved experiments were used to elucidate the nature of the formed complexes and their photodynamics. The results reveal important information for a better understanding of the interactions of structurally comparable drugs in chemical and biological nanocavities. 2. Experimental Part ADMB was synthesized by a standard methyl esterification procedure of the parent acid compound, and the purity of the sample was checked taking into account the emission data in noninteracting solvents like methylcyclohexane, where only one structure exists at both ground and electronically first excited states. HSA protein (Fluka-Sigma-Aldrich, 99%) and OG (Across Organics, 98%) were used as received. Sodium phosphate buffer (0.1 M) giving pH 7.0 was used in the measurements. Steady-state absorption and emission spectra were recorded on Varian (Cary E1) and Perkin-Elmer (LS 50B) spectrophotometers, respectively. Emission lifetimes were measured by using a time-correlated single-photon counting picosecond spectrophotometer (FluoTime 200) previously described.29 The sample was excited by a 40 ps pulsed (20 MHz) laser centered at 371 or at 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 the IRF and fitted to a multiexponential function using the Fluofit package. The time-dependent 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 fit of the data was characterized in terms of residual distribution and reduced χ2 values. All measurements were done at 293 ( 1 K. 3. Results and Discussion 3.1. Steady-State Studies. As said above, we have previously reported on the photophysics of ADMB in water and in presence of cyclodextrin, and we have discussed the involved transitions and relaxation pathways of ADMB in these media.27,28 Here we show and discuss the results of experiments studying the interactions of ADMB with HSA protein in water using UV-vis

Figure 2. (A) UV-vis absorption (Abs) and emission (Em) spectra of ADMB (5 × 10-5 M) in a phosphate buffer solution at pH ) 7 (---) and in presence of 20 µM of HSA (s). (B) Emission spectra of ADMB in water solution (---) and upon addition of 30 mM OG micelle (s). In both cases, the excitation wavelength was 340 nm.

absorption and emission spectroscopy. Figure 2A shows the UV-vis absorption and emission spectra of ADMB (5 × 10-5 M) in a phosphate buffer solution (pH ) 7) and in the presence of 20 µM HSA. The absorption spectra of the probe show no significant changes upon addition of HSA. The observed change in the intensity is due to the protein absorption. However, the increase in emission intensity of ADMB indicates its interaction with the HSA protein. This interaction also leads to a shift of the maximum of the emission spectrum from ∼437 to 414 nm, and it is explained in terms of binding of ADMB to the protein. This binding induces changes in the molecular structure and in nonradiative deactivation processes of the interacting ADMB. Previously, we have reported on similar effect due to the encapsulation by cyclodextrins.27,28 The result was explained in terms of confinement and hydrophobicity effect of the nanocages, and we use the same argument for explaining the observed changes in presence of HSA. A similar result was also observed upon addition of 30 mM OG micelle to an aqueous solution of ADMB (Figure 2B). In this case, the emission intensity increases and its maximum shifts from 437 to 434 nm. This smaller shift (∼3 nm) reflects a different environment from that of the protein cavity, and it is explained in terms of a relatively large flexibility of OG tails forming the cavity when compared to that of HSA pockets (vide anisotropy part). Previously, we have also observed a similar trend using a proton transfer probe.2 Figure 3 shows the changes in the UV-vis absorption and fluorescence spectra of HSA in the presence of different concentrations of ADMB and upon excitation at two different wavelengths (280 and 340 nm). In the UV-vis absorption spectra, the increase in the intensity at 265 and 333 nm bands upon addition of ADMB is due to its absorption. However, in the emission spectra, addition of ADMB induces a concentra-

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Figure 4. Change in the emission intensity (I - I0) at 420 nm of HSA in water at pH ) 7 versus the concentration of ADMB. The solid line shows the best fit obtained for a 1:1 equilibrium ADMB:HSA using a model described in ref 30. The inset shows a Job’s plot of ADMB/ HSA emission at 420 nm in water at pH ) 7 and upon increasing the molar fraction of ADMB (fADMB). The excitation wavelength was 340 nm.

Figure 5. Fluorescence excitation spectra of ADMB (10-5 M) in aqueous solution (b) and in presence of HSA (10 µM) (---) and OG micelle (30 mM) (O) observed at 420 nm and of HSA protein (10 µM) (s) observed at 330 nm in a phosphate buffer solution (pH ) 7). The optical density of the solutions at the maxima was lower than 0.4 to ensure a linear response of the apparatus. The spectra are normalized at the S0-S1 transition band. Figure 3. (A) UV-vis absorption and (B, C) emission spectra of HSA (10 µM) upon addition of different concentrations of ADMB ((0.00, 0.25, 0.66, 1.00, 1.50, 2.00, 2.33, 2.83, 3.20) × 10-5 M). The excitation wavelengths were 280 and 340 nm for (B) and (C), respectively.

tion-dependent quenching of Trp214 emission, and a blue shift from 333 to 328 nm is observed (Figure 3B). The recorded spectra were corrected for the inner filter effect due to the increasing absorption of ADMB in this region and for its relative absorption at the excitation wavelength (280 nm). As a result, the observed change in the emission spectra presented in Figure 3B is due to a modification in the intrinsic fluorescence of Trp214 caused by the presence of ADMB within HSA. The remaining emission in the UV arises from local environment changes around the Trp214 and/or around the tyrosines of HSA. Nonetheless, the intensity decrease in this emission and the concurrent increase in that of the probe indicate an energytransfer process from Trp214 (donor) to encapsulated ADMB (acceptor). The existence of an isoemissive point at ∼375 nm in Figure 3B points toward correlation between these emissions and therefore further supports the energy-transfer explanation. The inset of Figure 4 depicts the change in the fluorescence intensity of the complex versus the molar fraction of ADMB (fADMB) in the studied solutions (Job’s plot). It provides information regarding the stoichiometry of the formed complex. The plot clearly shows that the maximum in the fluorescence intensity occurs at fADMB ) 0.5 of ADMB, and therefore the

complex has 1:1 stoichiometry. The binding constant was calculated using the increase in the emission intensity at 420 nm upon addition of ADMB to a solution having a fixed concentration of HSA (10 µM) and upon excitation at 340 nm (Figure 3C). A nonlinear fit model30 to the experimental data assuming a 1:1 stoichiometry complex gives an equilibrium constant of (9.4 ( 0.4) × 104 M-1 at 293 K (Figure 4). Endogenous and exogenous ligands reversibly bind to HSA with constants in the range of 104-108 M-1.7,10 Thus, the value for equilibrium constant between HSA and ADMB and the complex indicates a high affinity of this anesthetic analogue to the HSA protein. Figure 5 shows a comparison of the fluorescence excitation spectra of HSA (10 µM), ADMB (10-5 M) in buffer solution and upon addition of HSA (10 µM) and OG micelle (30 mM). We checked that within the used optical density (lower than 0.4 at the maximum of absorption) of the samples the response of the used spectrophotometer is linear. Within both cavities (HSA and micelle), the excitation spectrum of ADMB shows a red shift of the S0-S1 transition (from ∼326 to ∼343 nm). In pure water, ADMB molecule is exposed to the solvent interactions that break its intramolecular H-bond (IHB), leading to a blue-shifted absorption spectrum. Upon encapsulation, the IHB is formed, which leads to an increase in its electronic conjugation and therefore to a long wavelength shift in the absorption

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Figure 6. Emission decays of ADMB (5 × 10-5 M) in the presence of 30 mM OG micelle and 40 µM HSA protein observed at 425 nm and excited at 393 nm. The solid curves are from the exponential fit of the experimental data.

TABLE 1: Fluorescence Lifetimes (τi) and Normalized (to 1) Pre-exponential Factors (ai) from the Best Multiexponential Fits of ADMB Emission in Buffer Aqueous Solutions (pH ) 7) and in the Presence of HSA Protein and OG Micelle upon Excitation at 393 nm τi/ns (ai) medium

Em ) 425 nm

water, pH ) 7 0.06 (0.93) 0.19 (0.07) HSA/water, pH ) 7 0.06 (0.44) 0.2 (0.06) 2.2 (0.20) 9.7 (0.30) OG micelle/water 0.05 (0.32) 0.2 (0.17) 1.3 (0.21) 3.1 (0.30)

Em ) 480 nm 0.06 (0.93) 0.19 (0.07) 0.05 (0.47) 0.2 (0.17) 2.2 (0.17) 9.7 (0.19) 0.05 (0.33) 0.2 (0.17) 1.3 (0.20) 3.3 (0.30)

spectrum of ADMB. Moreover, upon addition of HSA the ratio between the intensity of the two bands observed in the excitation spectra changes from 1.05 to 1.21. The difference suggests also the occurrence of an energy transfer process from Trp214 of the protein to the ADMB probe. 3.2. Time-Resolved Studies. To get information on the photodynamics of ADMB (5 × 10-5 M) within HSA (40 µM) and OG (30 mM), we recorded their respective emission decays (Figure 6). The decay of HSA in water (not shown) was fitted first using a three-exponential function giving times of 166 ps (51%), 1.04 ns (32%), and 5 ns (17%),15 indicating multiple protein conformations interacting differently with the environment.31-33 Previously, we have shown that the emission decay of ADMB in water gives two components of 60 ps (93%) and 190 ps (7%).27 The first one is assigned to conformer II while the second one is attributed to conformer I (Figure 1). The optimized structures by ab initio calculations reveal that the internal H-bond in conformer II is weaker than in I by 7.2 kcal/mol.27 The emission decay times of ADMB interacting with HSA become longer due to the influence of the hydrophobic environment and caging effect of the protein on its photodynamics (Figure 6). The emission decays fit to a four-exponential function with time constants of 60 ( 7 ps, 200 ( 25 ps, 2.2 ( 0.1 ns, and 9.7 ( 0.7 ns (Table 1). Taking into account the results for ADMB in water (60 and 190 ps), the 60 and 200 ps components in the presence of HSA, are assigned to free ADMBsconformers II and I in buffer solution, respectively. The 2.2 and 9.7 ns components are assigned to the formed complexes with HSA. It is worth noting that the contribution of the 60 ps component decreases from 93% to 44% upon addition of 40 µM HSA to 5 × 10-5 M solution of ADMB, while that of the 200 ps one remains unaffected (∼7%). This behavior might be the result of preferential complexation of HSA with conformer II rather

Tormo et al. than with conformer I. The observation of two lifetimes for the complexes is explained in terms of different heterogeneous environments. The longest time constant (9.7 ns) is comparable to those observed in aprotic solvents (6-9 ns), and therefore, the corresponding structure could be assigned to conformer I embedded into a hydrophobic pocket of the protein leading to encapsulated conformer I. The 2.2 ns component represents the contribution of conformer II bound to the protein, probably to its surface via intermolecular H-bonds. Both kinds of interactions result in 1:1 complex formation as indicated by the change in the ADMB emission (Figures 3C and 4). For 480 nm emission, the contribution of the 9.7 ns component (encapsulated conformer I) decreases to 19%. This change, which is in agreement with the steady-state emission spectra, indicates that the formed complexes emit at the blue part of the spectrum and that the confinement governs the relaxation routes of the caged species. Furthermore, at longer wavelengths of observation (480 nm) the contribution of the 2.2 ns component is relatively higher than that of the 9.7 ns one, suggesting that the longest component is due to a confined conformer I structure while the shortest one is due to the conformer II probably H-bonded to the protein surface. In fact, in buffer solution, as a result of the formation of intermolecular hydrogen bonds with water, the emission spectrum of ADMB shifts to longer wavelengths (437 nm) when compared to that in HSA protein (414 nm) (Figure 2A). Notice that upon interaction of ADMB with the hydrophobic protein pocket, conformer II, the most stable one in water, might undergo a rotation of the ester group to yield encapsulated conformer I. Supporting the assignment of the long-lived component to a confined conformer I and that of the short-lived one to conformer II probably is the increase in the pre-exponential ratio value of the relative contributions (related to the populations at the excited states) of conformers I and II upon addition of HSA to water solution (Table 1). For example at 425 nm, it goes from 0.07 in pure water to 0.14 and 1.5 in the protein for free and bound ADMB, respectively. Upon addition of 30 mM OG micelle, a four-exponential fit to the fluorescence decay was obtained at 425 nm with time constants of 50 ( 7 ps, 200 ( 30 ps, 1.3 ( 0.3 ns, and 3.1 ( 0.5 ns (Table 1 and Figure 6). Because of the heterogeneity of micellar systems (as seen with the protein complexes),2,23 it is expected to observe such multiexponential behavior for the emission decay of ADMB within the micelle. The contribution of 50 ps component decreases relatively to that of the 200 ps one, similar to that observed in the presence of HSA. With comparison to the results in water, we find an increase in the pre-exponential factor of the longer picosecond component (from 7% to 17% in the presence of OG), which corresponds to a relative decrease of the short picosecond contribution as a result of the interaction with the micelle. This can be explained in terms of the existence of free forms of conformer I of the probe or of specific interaction of the ADMB molecule with the glucose units at the micellar surface. The two additional nanosecond components are due to the heterogeneity of the micelar surrounding as it has been observed in other studies.27,28 The 3 ns component corresponds to the lifetime of ADMB inside the core of the micelle, where the hydrophobic pocket prevents the interactions between the guest and water molecules. The 1.3 ns lifetime corresponds to ADMB molecules interacting with the polar headgroups at the surface into the micelle. This latter type of ADMB molecules are more exposed to water, which can break the intramolecular H-bond of the probe, and favors the twisting motion of the ester group, thus leading to the formation of conformers II and giving rise to shorter emission

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Figure 7. Anisotropy decays of ADMB (5 × 10-5 M) in the presence of 30 mM OG micelle and 50 µM HSA protein monitored at 430 nm and excited at 393 nm. The solid curves are from the fit of the experimental data. The inset shows the anisotropy decay of ADMB: HSA complex in a larger time window.

Figure 8. Spectral overlap between ADMB (5 × 10-5 M) absorption and HSA (20 µM) emission upon excitation at 280 nm in a phosphate buffer solution (pH ) 7). The dotted lines represent the absorption and emission spectra of HSA protein while the solid ones are for the absorption and emission spectra of ADMB excited at 340 nm.

lifetimes. The relative contribution (pre-exponential factor) of the lifetimes does not show significant changes with the emission wavelength (see results at 480 nm in Table 1). 3.3. Time-Resolved Anisotropy. To get information on the rotational relaxation time of the ADMB (5 × 10-5 M) and on the robustness of the formed complexes, we performed steady state (r) and time-resolved anisotropy (r(t)) measurements (Figure 7). In the presence of HSA (50 µM) and OG (30 mM), 〈r〉 gives values of 0.254 ( 0.01 and 0.104 ( 0.01, respectively. While in a buffer water solution (pH ∼ 7) 〈r〉 ) 0.004 ( 0.001. The high value of 〈r〉 upon addition of HSA is remarkable and suggests that a strong restriction to free reorientation dynamics is acting during the lifetime of the caged probe. This further confirms the formation of robust complexes between the protein and ADMB. The smaller value of r observed in OG (0.104) indicates a less rigid caging cavity of the micelles, in agreement with a previous work.2 Since the hydrophobic core of the glucopyranoside units is formed by flexible alkyl chains, it allows reorientation of ADMB within the micelle, contrary to the situation within the protein. The time-resolved anisotropy (r(t)) decay in pure water fits to a single-exponential function giving φ ) 93 ( 10 ps upon excitation at 371 nm (82 ps when exciting at 393 nm).27 Modeling ADMB as a prolate ellipsoid, we calculated a rotational relaxation time of 45 and 25 ps under stick- and slipboundary condition limits, respectively.27 This result agrees with the existence of strong H-bonding interaction between ADMB and surrounding water molecules affecting its rotational relaxation time. In the presence of HSA, the anisotropy decay of ADMB shows a single-exponential behavior, giving a rotational time constant of 45 ( 7 ns, which corresponds to the global motion of HSA. This result clearly indicates that the probe is strongly bound to the hydrophobic pocket of HSA. The reported values for the global Brownian rotation of this protein are between 22 and 45 ns.34,35 The spread in this time constant arises from the difficulty to determine this parameter with accuracy due to the short lifetime of intrinsic fluorescence from HSA, which is extensively used in this type of analysis. In many cases, it is not possible to use the appropriate time windows for an accurate measurement. The rotational correlation time of 41 ns35 determined by using long lifetime luminescence transition metal complexes as ligands is then considered one of the most accurate values. The value of the initial anisotropy (r0 ) 0.31) is close to the ideal one (0.40) and gives a small angle (θ ) 23°) between the transition moments of absorption and emission of the caged analogue. The observed change is due to production of a charge-

transfer process in the excited ADMB, leading to a structure different from the absorbing one. In the case of OG micelle, we got a biexponential anisotropy decay with rotational relaxation times (at 425 nm) φ1 ) 50 ( 8 ps (19%) and φ2 ) 980 ( 70 ps (81%). At 480 nm the amplitudes of φ1 and φ2 components change to 39% and 61%, respectively. This variation suggests the existence of two main rotors emitting in overlapping regions, the slowest one being at the short wavelengths side. The shortest time (50 ps) is not very different from the one corresponding to ADMB in water (80 ps), and therefore we assign it to free probe. Those found near the heads of the micelle or within the alkyl chains feel a relative restriction of these environments to free motion. The value of φ2 is not the corresponding one of overall rotational time of the micelle. This value was estimated 11 ns using the Stokes-Einstein relation: τM ) 4πηrh3/3kT, where η is the viscosity of the solution and rh ∼ 23 Å36 is the dynamic radius of the spherical particle (micelle). The 980 ps value corresponds then to the rotational time of the caged ADMB molecules within the core of the micelle. As in the case of ADMB:HSA, the r0 value of 0.31 indicates an angle of 23° between the transition moments of absorption and emission. Again, the electronic structures of the absorbing and emitting species are different. 3.4. Intermolecular Energy Transfer. Energy transfer from an initially excited-state donor (D) to an acceptor (A) with Fo¨rster mechanism occurs by resonance energy transfer. This process happens by resonance interaction between both chromophores, over distances considerably greater than interatomic ones, without conversion to thermal and without kinetic collision of D and A. The donor is the probe that initially absorbs the photon energy (Trp214 in this case), and the acceptor is the one to which the energy is subsequently transferred (ADMB molecule).37 The steady-state absorption and emission spectra obtained upon excitation of Trp214 at 280 nm and ADMB at 340 nm suggest the existence of an energy-transfer process from Trp214 to the bound ADMB (Figures 3B and 5). Figure 8 shows the spectral overlap between protein emission and ADMB absorption spectra. It indicates the possibility of energy transfer from the excited Trp214 residue to the caged probe. According to Fo¨rster’s theory,38 the rate of a nonradiative energy transfer process depends on (i) the relative orientation of the donor and acceptor dipoles, (ii) the extent of overlap of the emission spectrum of the donor with the absorption spectrum of the acceptor, and (iii) the distance between the donor and the acceptor. The efficiency (E) of energy transfer from D to A

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is related to the ADMB-Trp214 distance (R) according to the following equation:39

E ) 1 - (Φ/Φ0) ) 1 - (F/F0)

(1)

where Φ0 and Φ (or F0 and F) are the fluorescence quantum

E ) R06 /(R06 + R6)

(1a)

yields (or emission intensity) of the donor in absence and presence of an equal amount of acceptor, respectively. R0 is the Fo¨rster critical distance between A and D, at which 50% of the excitation energy is transferred to A. R0 can be obtained from D emission and A absorption spectra using the equation where ΦD ) 0.11 is the fluorescence quantum yield of

R0 ) 0.211[κ2nD-4ΦDJ(λ)]1/6

(2)

tryptophan,40 nD ) 1.333 is the refractive index of the solution, and κ2 is the orientation factor, commonly taken as 2/3, assuming a random orientation for both the donor and acceptor.41 The spectral overlap J between D emission spectrum and A absorption one is calculated by the following equation:

∫0

∞

J)

F(λ)ε(λ)λ4 dλ

∫0∞ F(λ) dλ

(3)

where F(λ) accounts for the fluorescence emission intensity of D at a wavelength λ and it is dimensionless, and ε(λ) is the molar absorption coefficient of A. From the spectra shown in Figure 8 and using eq 3, we calculated J ) 4.63 × 10-15 M-1 cm3. From eq 1 we obtained E from the F and F0 values for each ADMB and HSA concentration (Table 1 in Supporting Information). In the system under study the probe and the protein are not covalently linked by a chemical bond, and thus the obtained values of E are corrected (Ecorr ) E/f) by taking into account the molar fraction (f) of the ADMB:HSA complex. The molar fraction is deduced from the equilibrium constant and the initial concentrations of both the guest and host. We calculated a mean value of Ecorr ) 50 ( 5% from several independent experiments. This value indicates significant energy transfer efficiency from Trp214 to the caged ADMB. Equation 2 yields R0 ) 20 ( 2 Å, and from the mean value of Ecorr and eq 1a, we got the ADMB-Trp214 distance, R ) 19 ( 2 Å. According to the Fo¨rster theory, the rate constant of energy transfer (kET) from Trp214 to ADMB is given by the equation where τHSA is the mean fluorescence lifetime of the Trp214 in

kET ) τHSA-1(R0 /R)6

(4)

HSA (τHSA ) 3.53 ns).31 Using this value, we got kET ) 3.0 × 108 s-1. In general, Fo¨rster energy transfer from excited donor to unexcited acceptor has rate constants in the range of 106-1011 s-1.40 The energy-transfer rate constant from Trp214 to ADMB is not very large, and comparable values of kET and Trp214-guest distances have been reported for probes of not very different geometries.12,31,40 The large equilibrium constant (9.6 × 104 M-1 at 293 K) and the rotational emission time of 45 ns clearly show

a strong docking of ADMB with the protein. As ADMB is a neutral aromatic compound and on the basis of the behavior of complexation to site I in subdomain IIA of a large number of neutral heterocyclic molecules to HSA, we suggest that this anesthetic analogue is most probably bonded to this site.11-13 4. Conclusion Steady-state and picosecond-time-resolved fluorescence measurements reveal strong hydrophobic and confinement effects of HSA protein and OG normal micelle on the structures and dynamics of the caged ADMB. Within the protein, the emission lifetime increases by a factor of 50, giving 2.2 and 9.7 ns for interacting conformers I and II, respectively, and the trapped anesthetic analogue cannot move as shown by the anisotropy experiments. The formed 1:1 complex is robust with an equilibrium complex of 9.6 × 104 M-1 at 293 K. Within the micelle, the results show a flexibility of the core allowing motion (diffusion) of the probe. For ADMB:HSA complex, the efficiency of the energy transfer process from excited Trp214 to ADMB is significant (50%) with a rate constant of ∼3.0 × 108 s-1, and the calculated interchromophore distance is ∼19 Å. These observations might provide invaluable information in the design of nanocapsules for this kind of drug and relevant to pharmacodynamics. Acknowledgment. This work was supported by the MEC and JCCM through the projects CTQ-2005-00114/BQU and PCI08-0037-5868. B.C. thanks MEC for the Ramon y Cajal fellowship. Supporting Information Available: Table 1 giving the observed (Eobs) and corrected (Ecorr) efficiency for energy transfer from tryptophan of HSA (10 µM) to ADMB at different concentrations. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Zhong, D. P.; Pal, S. K.; Wang, C.; Zewail, A. H. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 11873–11878. (2) Zhong, D. P.; Douhal, A.; Zewail, A. H. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 14056–14061. (3) Mataga, N.; Chosrowjan, H.; Taniguchi, S. J. J. Photochem. Photobiol., C 2004, 5, 155–168. (4) Douhal, A.; Sanz, M.; Tormo, L. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 18807–18812. (5) Narayanan, S. S.; Pal, S. S. Langmuir 2007, 23, 6712–6718. (6) (a) Kragh-Hansen, U. Pharmacol. ReV. 1981, 33, 17–53. (b) Sakai, T.; Yamasaki, K.; Sako, T.; Kragh-Hansen, U.; Suenaga, A.; Otagiri, M. R. Pharm. Res. 2001, 18, 520–524. (7) Peters, T., Jr. Biochemistry Genetics and Medical Applications; Academic Press: San Diego, 1996; pp 76-132. (8) He, X.; Carter, D. C. Nature (London) 1992, 358, 209–215. (9) 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) Carter, D. C.; Ho, J. X. AdV. Protein Chem. 1994, 45, 153–203. (11) Rosenoer, V. M.; Oratz, M.; Rothschild, M. A. Albumin Structure, Function, and Uses; Pergamon: Oxford, 1977. (12) II’ichev, I. V.; Perry, J. L.; Simon, J. D. J. Phys. Chem. B 2002, 106, 452–459, and references therein. (13) (a) Oyekan, A. O.; Thomas, W. O. A. J. Pharm. Pharmacol. 1984, 36, 831–834. (b) Bree, F.; Urien, S.; Nguyen, P.; Albengres, E.; Tillement, J. P. A. Eur. J. Drug Metab. Pharmacokinet. 1990, 15, 303–307. (c) Trnavska´, Z.; Trnavsky´, K.; Zlnay, D. Eur. J. Clin. Pharmacol. 1984, 26, 457–461. (14) Roloinsky, O. J.; Martin, A.; Birch, D. J. S. J. Biomed. Opt. 2007, 12, 034013-1–034013-7. (15) El- Kermany, M.; Gil, M.; Douhal, A. J. Med. Chem. 2007, 50, 2896–2902. (16) Vaya´, I.; Jime´nez, C.; Miranda, M. A. J. Phys. Chem. B 2007, 111, 9363–9371. (17) Qui, W.; Zhang, L.; Okobiah, O.; Yang, Y.; Wang, L.; Zhong, D. P.; Zewail, A. H. J. Phys. Chem. B 2006, 110, 10540–10549.

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