J. Phys. Chem. B 2008, 112, 3575-3580
3575
Femtosecond Solvation Dynamics in Different Regions of a Bile Salt Aggregate: Excitation Wavelength Dependence Aniruddha Adhikari, Shantanu Dey, Ujjwal Mandal, Dibyendu Kumar Das, Subhadip Ghosh, and Kankan Bhattacharyya* Physical Chemistry Department, Indian Association for the CultiVation of Science, JadaVpur, Kolkata 700 032, India ReceiVed: NoVember 6, 2007; In Final Form: December 13, 2007
Solvation dynamics of coumarin 480 (C480) in the secondary aggregate of a bile salt (sodium deoxycholate, NaDC) is studied using femtosecond up-conversion. The secondary aggregate resembles a long (∼40 Å) hollow cylinder with a central water-filled tunnel. Different regions of the aggregate are probed by variation of the excitation wavelength (λex) from 375 to 435 nm. The emission maximum of C480 displays an 8 nm red shift as the λex increases from 345 to 435 nm. The 8 nm red edge excitation shift (REES) suggests that the probe (C480) is distributed over regions of varied polarity. Excitation at a short wavelength (375 nm) preferentially selects the probe molecule in the buried locations and exhibits slow dynamics with a major (84%) slow component (3500 ps) and a small (16%) contribution of the ultrafast component (2.5 ps). Excitation at λex ) 435 nm (red end) corresponds to the exposed sites where solvation dynamics is very fast with a major (73%) ultrafast component (e2.5 ps) and relatively minor (27%) slow (2000 ps) component. In sharp contrast to solvation dynamics, the anisotropy decay becomes slower as λex increases from 375 to 435 nm. It is proposed that the buried locations (λex ) 375 nm) offer lower friction because of the rigid sheetlike structure of the bile salt.
1. Introduction Bile salts are bioactive natural surfactant molecules that are synthesized in the liver and stored in gall bladder. Micellization of bile salts and their interaction with biological membranes play an important role in biliary secretion and solubilization of cholesterol, lipid, bilirubin, fat-soluble vitamins and many other species in living organisms.1 A bile salt exhibits facial amphiphilicity. The nonplanar bile salt, sodium deoxycholate (NaDC, Scheme 1a) consists of a convex hydrophobic surface (the steroid ring) and a concave hydrophilic surface (hydroxyl groups and carboxylate ions). In a slightly alkaline medium (pH > 7.5), NaDC displays two critical micellar concentrations (CMC1 and CMC2) at 10 and 60 mM, respectively. Above CMC1, primary aggregates are formed that consist of very few (2-10) monomers.1a In the primary aggregate, the polar face of NaDC (hydroxyl group and ions) points outward (Scheme 1b). The secondary aggregate (formed at a concentration >60 mM) resembles an elongated hollow cylinder with a central water filled tunnel (Scheme 1b).1-2 For NaDC, the cylinder is about 40 Å long and radius of the hollow core is ∼8 Å.2 The microenvironment and binding dynamics of many fluorescent probes in different sites of a bile salt aggregate has been studied extensively.3-4 Solvation dynamics in the secondary aggregate has been studied earlier using picosecond techniques.4 The ultrafast component of solvation dynamics in a bile salt aggregate has not been studied before. Further, there has been no effort to study solvation dynamics in different regions of a bile salt aggregate. In this work, we report on a femtosecond study of solvation dynamics in a secondary aggregate of NaDC. * To whom correspondence should be addressed. E-mail: pckb@ mahendra.iacs.res.in. Fax: (91)-33-2473-2805.
To probe different regions of the bile salt aggregate, we take recourse to excitation wavelength dependence. Several groups have reported earlier that variation of excitation wavelength selects probes in different regions of a heterogeneous organized assembly.5 Excitation at a short wavelength (blue end) selects probe in a less polar and hydrophobic (“buried”) region, and a long wavelength (red end) excites probe in a polar and exposed region. As a result, with increase in the excitation wavelength (λex) the emission maximum exhibits a gradual red shift. This phenomenon is known as red edge excitation shift (REES).5 The effect of variation of λex on steady-state emission spectrum has received attention for a long time. Recently, λex dependence has been applied to study dynamics in different regions of many organized assemblies. For instance, λex variation has revealed the differences in solvation dynamics in different regions of an ionic liquid,6a reverse micelle,6b triblock copolymer micelle,6c gel,6d lipid vesicles,6e and polymer-surfactant aggregate.6f Fluorescence resonance energy transfer in different regions of a reverse micelle,7a and polymer hydrogel7b has also been studied using λex variation. In this work, we apply this method to study solvation dynamics in different regions of a bile salt aggregate. We used coumarin 480 (C480, Scheme 1c) as a fluorescent probe. It may be recalled that many recent computer simulations also predict that the dynamics may vary markedly from one region to another in the case of a protein,8 micelle,9 surfaces,10 many hydrophobic cavities,11 and most recently in ionic liquids.12 Several recent experimental works also indicate variation of dynamics in different regions of micelles and other organized assemblies.13-16 Because of the importance of regioselectivity in many biological processes, in this work we focus on solvation dynamics in different sites of a bile salt (NaDC) aggregate.
10.1021/jp7106445 CCC: $40.75 © 2008 American Chemical Society Published on Web 02/27/2008
3576 J. Phys. Chem. B, Vol. 112, No. 11, 2008 SCHEME 1: Schematic Representation of (a) Bile Salt, NaDC, (b) Secondary Aggregate of NaDC, and (c) Coumarin 480 (C480)
Adhikari et al. setup consists of an Ortec 9327 CFD and a Tennelec TC 863 TAC. The data is collected with a PCA3 card (Oxford) as a multichannel analyzer. The typical fwhm of the system response using a liquid scatterer is about 90 ps. The fluorescence decays were deconvoluted using IBH DAS6 software. To fit the femtosecond transients, we first determined the long picosecond components by deconvolution of the picosecond decays. Then the long picosecond components were kept fixed to fit the femtosecond data. The time-resolved emission spectra (TRES) were constructed using the parameters of best fit to the fluorescence decays and the steady-state emission spectrum following the procedure described by Maroncelli and Fleming.17a The solvation dynamics is described by the decay of the solvent correlation function C(t), which is defined as
C(t) )
ν(t) - ν(∞) ν(0) - ν(∞)
(1)
where ν(0), ν(t), and ν(∞) are the peak frequencies at time 0, t, and ∞, respectively. Note, a portion of solvation dynamics is missed (at λex ) 405 and 435 nm) even in our femtosecond set up of time resolution 350 fs. The amount of solvation missed is calculated using the Fee-Maroncelli procedure.17b The emission frequency at time zero, νpem(0), may be calculated using the absorption frequency (νpabs) in a polar medium (i.e., C480 in NaDC) as17b np νpem(0) ) νpabs - (νnp abs - νem)
(2)
np where νnp em and νabs denote the steady-state frequencies of emission and absorption, respectively, of the probe (C480) in a nonpolar solvent (i.e., cyclohexane). To study fluorescence anisotropy decay, the analyzer was rotated at regular intervals to get perpendicular (I⊥) and parallel (I|) components. Then the anisotropy function, r(t) was calculated using the formula
2. Experimental Section Laser grade coumarin 480 (C480, Exciton) and bile salt sodium deoxycholate (NaDC, Aldrich) were used as received. The steady state absorption and emission spectra were recorded in a Shimadzu UV-2401 spectrophotometer and a Spex FluoroMax-3 spectrofluorimeter, respectively. In our femtosecond upconversion setup (FOG 100, CDP), the sample was excited at 375, 405, and 435 nm, respectively. Briefly, the sample was excited using the second harmonic of a mode-locked Ti-sapphire laser with an 80 MHz repetition rate (Tsunami, Spectra Physics), pumped by 5 W Millennia (Spectra Physics). The fundamental beam was frequency doubled in a nonlinear crystal (1 mm BBO, θ ) 25°, φ ) 90°). The fluorescence emitted from the sample was upconverted in a nonlinear crystal (0.5 mm BBO, θ ) 38°, φ ) 90°) using a gate pulse of the fundamental beam. The upconverted light is dispersed in a monochromator and detected using photoncounting electronics. A cross-correlation function obtained using the Raman scattering from ethanol displayed a full width at halfmaximum (fwhm) of 350 fs. The femtosecond fluorescence decays were fitted using a Gaussian shape for the exciting pulse. To fit the femtosecond data, one needs to know the long decay components. These were detected using a picosecond set up in which the samples were excited at 375, 405, and 435 nm using picosecond laser diodes (IBH Nanoleds) in an IBH Fluorocube apparatus. The emission was collected at a magic angle polarization using a Hamamatsu microchannel plate photomultiplier (5000U-09). The time correlated single photon-counting
r(t) )
I|(t) - GI⊥(t) I|(t) + 2GI⊥(t)
(3)
The G value of the picosecond set up was determined using a probe whose rotational relaxation is very fast, for example, coumarin 480 in methanol. The G value was found to be 1.5. 3. Results and Discussion 3.1. Steady-State Absorption and Emission Spectra: λex Dependence. All the experiments reported in this work are carried out in 105 mM NaDC. This is well above the CMC2 of NaDC and hence corresponds to a secondary aggregate. In a 105 mM NaDC solution, the absorption maximum of C480 is found to be at 391 nm (Figure 1). This is blue-shifted by 5 nm from the reported absorption maximum (396 nm)18 of C480 in water. Figure 2 shows the emission spectrum of coumarin 480 in NaDC for various excitation wavelengths (λex). The emission maxima of C480 at different λex in the secondary aggregate of NaDC are listed in Table 1. Figure 3 shows the λex dependence of emission maximum of C480 in NaDC. It is readily seen that emission maximum of C480 displays a red shift (REES) of 8 nm from 468 nm at λex ) 345nm to 476 nm at λex ) 435 nm. The observed REES suggests a distribution of the fluorophore (C480) in different regions of the bile salt. Evidently, excitation at a short wavelength (the blue end, 345 nm), preferentially selects the probes residing at the less polar core region and hence, gives rise to a blue-shifted emission spectrum. At a long λex (435 nm, red end), probe (C480) molecules in the hydrophilic
Solvation Dynamics in Different Regions of Bile Salt Aggregate
J. Phys. Chem. B, Vol. 112, No. 11, 2008 3577
Figure 1. Absorption spectrum of C480 in 105 mM NaDC. The excitation wavelengths are marked (×) at 375, 405 and 435 nm.
Figure 2. Emission spectra of C480 in 105 mM NaDC when excited at (a) 375 nm, (b) 405 nm, (c) 435 nm, and (d) C480 in water (‚‚‚‚‚) (λex ) 375-435 nm).
Figure 4. Picosecond fluorescence decays of C480 in NaDC at λex ) 375 nm (A-C) and 435 nm (D-F). λem at (A) 540 nm, (B) 470 nm, (C) 410 nm, (D) 560 nm, (E) 470 nm, and (F) 445 nm.
Figure 3. Plot of emission maximum of C480 in 105 mM NADC as a function of excitation wavelength.
TABLE 1: λex Dependence of Steady-State Emission Maximum and Width (fwhm) of C480 in 105 mM NaDC and Water medium
λex (nm)
λmax em (nm)
fwhm (cm-1)
NaDC NaDC NaDC water
375 405 435 375-425
469 473 476 489
3250 2900 2600 2600
exposed sites are excited almost exclusively, and this leads to a red-shifted emission spectrum. 3.2. Time-Resolved Studies. 3.2.1. SolVation Dynamics of C480 in NaDC: λex Dependence. Figures 4 and 5 show the picosecond and femtosecond fluorescence transients of C480 in 105 mM NaDC. At the red end of the emission spectrum, a rise precedes the decay, and at the blue end a decay (with no rise) is observed. Such a wavelength dependence of fluorescence decays is a clear indication of solvation dynamics. For λex ) 375 nm (buried site), in NaDC the fluorescence decay at 410 nm (blue end) exhibits four decay components: 3, 126, 950, and 2700 ps. At 540 nm (red end), there are three rise
Figure 5. Femtosecond fluorescence transients of the C480 in NaDC at λex ) 375 nm (A-C) and 435 nm (D-F). λem at (A) 540 nm, (B) 470 nm, (C) 410 nm, (D) 560 nm, (E) 470 nm, and (F) 445 nm.
components, 3.5, 260, and 2230 ps, with a long decay component of 7130 ps. For excitation at the red end (λex ) 435 nm), that is, in the polar exposed site, the decay displays two rise components of 2.5 and 690 ps followed by a long decay component of 6800 ps at the red end (560 nm). The fluorescence transient at the
3578 J. Phys. Chem. B, Vol. 112, No. 11, 2008
Adhikari et al.
Figure 6. TRES of C480 in NaDC at λex ) 375 nm at time (A) 0 ps (9), (B) 700 ps (O), (C) 3000 ps (2), and (D) 12000 ps (3). Figure 8. Fluorescence anisotropy decays of C480 (λem) 460 nm) along with fitted curves in NaDC at (A) λex ) 375 nm (O), (B) λex ) 405 nm (2), and (C) λex ) 435 nm (0). The fitted curves are shown in the inset.
TABLE 3: λex Dependence of Anisotropy Decay of C480 in 105 mM NaDC λex
r0
τr1(a1) (ps)
τr2(a2) (ps)
aav (ps)
375 405 435
0.31 0.35 0.35
600 (0.68) 600 (0.39) 600 (0.32)
2800 (0.32) 2800 (0.61) 2800 (0.68)
1300 1950 2100
a
Figure 7. Complete decay of the solvent response function C(t) of C480 in NaDC for λex)375 nm (4), λex)405 nm (b), and λex)435 nm (0). The points denote the actual values of C(t), and the solid line denotes the best fit. Initial portions of the decays are shown in the inset.
TABLE 2: Decay Parameters of C(t) of C480 in NaDC at Different Excitation Wavelengths (λex) λex (nm)
ν (0) (cm-1)
∆νobsa (cm-1)
τ1 (a1) (ps)
τ2 (a2) (ps)
τ3 (a3) (ps)
c (ps)
375 405
22500 22100
1500 1100
3500 (0.84) 400 (0.15)
3500 (0.43)
2940 1570
435
21300
500
2.5 (0.16)
