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Unusual Pyrene Excimer Formation during Sodium Deoxycholate Gelation† A. Jover, F. Meijide, E. Rodrı´guez Nu´n˜ez, and J. Va´zquez Tato* Departamentos de Quı´mica Fı´sica y Fı´sica Aplicada, Universidad de Santiago, Campus de Lugo, Facultad de Ciencias, 27002 Lugo, Spain
M. Mosquera and F. Rodrı´guez Prieto Departamento de Quı´mica Fı´sica, Universidad de Santiago, Facultad de Quı´mica, 15706 Santiago de Compostela, Spain Received July 28, 1995. In Final Form: January 22, 1996X Steady-state and time-resolved fluorescence have been used to study the gelation of sodium deoxycholate at pH values close to neutrality. Pyrene was used as a probe. The results suggest the formation of pyrene excimer as a result of the aggregation process: when two clusters carrying probes form a larger one, pyrene molecules can interact in both the ground and excited states. Fluorescence lifetimes for monomer and excimer pyrene are given and favorably compared to similar systems. A structure for the aggregates is also suggested.
Introduction The dihydroxy bile salt sodium deoxycholate (Figure 1, NaDC1 ) has been the subject of numerous studies2 and it is well-known that it forms aggregates in aqueous solutions. Compared with NaC, its homologous trihydroxy bile salt, NaDC shows three main differences. First, the rate of average aggregation number growth for NaDC with the addition of inert salts as NaCl,3-4 is greater than for NaC aggregates.5 Second, the fraction of bound counterions of NaC increases with concentration5,6 with an average value of 0.077 at concentrations below 0.25 mol kg-1;5 however, for NaDC this fraction is 0.32 (i.e., one counterion per three monomers) and remains independent of its concentration. Finally, at pH values close to neutrality, NaDC forms gels,7-12 a characteristic uncommon to most other bile salts. Although many authors have studied the aggregation behavior of NaDC at high pH values, less attention has been paid to the study of the gelation of NaDC, first noticed by Sobotka et al.7 Blow and Rich8,9 propose a helix structure for the aggregates
Figure 1. Molecular structure of NaDC.
† Part of this paper was presented at the 13th International IUPAC Conference on Chemical Thermodynamics, ClermontFerrand (France), 17-22 July, 1994. X Abstract published in Advance ACS Abstracts, March 15, 1996.
at pH values close to neutrality, Small13 gives an aggregation number of 552 at pH 7.3, and, finally, Sugihara et al. have carried out studies related to its behavior in capillary flow,11 the sol-gel transition under high pressures,12 and the formation of polymer-like aggregates at pH 7.8.14 Sugihara et al.11 accept that the gelation process implies the formation of secondary micelles from the primary ones.13 Changes during sol-gel processes have been examined by fluorescence techniques by using pyrene as a probe.15 They have also been used to study the aggregation behavior of several bile salt-aqueous solutions systems16-24 leading to important structural conclusions. Therefore, it is appropriate to apply fluorescence techniques to the gelation process of NaDC. In this paper, we present our results obtained from steady-state and lifetime fluorescence measurements using pyrene as a probe.
(1) The following acronyms are used: NaC, sodium cholate; NaDC, sodium deoxycholate; NaTC, sodium taurocholate; NaGC, sodium glycocholate; NaTDC, sodium taurodeoxycholate; NaTCDC, sodium taurochenodeoxycholate; SDS, sodium dodecyl sulfate. (2) Coello, A.; Meijide, F.; Rodrı´guez Nu´n˜ez, E.; Va´zquez Tato, J. J. Pharm. Sci. 1996, 85, 9, and references there in. (3) Birdi, K. S. Finn. Chem. Lett. 1982, 142. (4) Kratohvil, J. P.; Hsu, W. P.; Kwok, D. I. Langmuir, 1986, 2, 256. Esposito, G.; Giglio, E.; Pavel, V.; Zanobi, A. J. Phys. Chem. 1987, 91, 356. (5) Coello, A.; Meijide, F.; Rodrı´guez Nu´n˜ez, E.; Va´zquez Tato, J. J. Phys. Chem. 1993, 97, 10186. (6) (a) Lindman, B.; Kamenka, K.; Brun B. J. Colloid Interface Sci. 1976, 56, 328. (b) Lindman, B.; Kamenka, N.; Fabre, H.; Ulmius, J.; Wieloch, T. J. Colloid Interface Sci. 1980, 73, 556. (c) Lindman, B. Hepatology 1984, 4, 103S. (d) Lindman, B.; Puyal, M. C.; Kamenka, N.; Rynden, R.; Stilbs, P. J. Phys. Chem. 1984, 88, 5048. (e) Gustavsson, H.; Lindman, B. J. Am. Chem. Soc. 1975, 97, 3923. (7) Sobotka, H.; Czeczowiczka, N. J. Colloid Sci. 1958, 13, 188. (8) Rich, A.; Blow, D. M. Nature 1958, 182, 423. (9) Blow, D. M.; Rich, A. J. Am. Chem. Soc. 1960, 82, 3566. (10) Sugihara, G.; Tanaka, M. Bull. Chem. Soc. Jpn. 1976, 49, 3457. (11) Sugihara, G.; Tanaka, M.; Matuura, R. Bull. Chem. Soc. Jpn. 1977, 50, 2542. (12) Sugihara, G.; T. Ueda, Kaneshina, S.; Tanaka, M. Bull. Chem. Soc. Jpn. 1977, 50, 604.
(13) Small, D. M. Adv. Chem. Ser. 1968, No. 84, 31. (14) Sugihara, G.; Yamakawa, K.; Murata, Y.; M. Tanaka, M. J. Phys. Chem. 1982, 86, 2784. (15) Matsui, K.; Tominaga, M.; Arai, Y.; Satoh, H.; Kyoto, M. J. NonCrystalline Solids 1994, 169, 295 and references therein. (16) Paul, R.; Mathew, M. K.; Narayanan, R.; Balaram, P. Chem. Phys. Lipids 1979, 25, 345. (17) De Vendittis, E.; Palumbo, G.; Parlato, G.; Bocchini, V. Anal. Biochem. 1981, 115, 278. (18) Fisher, L.; Oakenfull, D. Aust. J. Chem. 1979, 32, 31. (19) Zana, R.; Gu¨veli, D. J. Phys. Chem. 1985, 89, 1687. (20) Kawamura, H.; Manabe, M.; Narikiyo, T.; Igimi, H.; Murata, Y.; Sugihara, G.; Tanaka, M. J. Solution Chem. 1987, 16, 433. (21) Vethamuthu, M. S.; Almgren, M.; Mukhtar, E.; Bahadur, P. Langmuir 1992, 8, 2396. (22) Chen, M.; Gra¨tzel, M.; Thomas, J. K. J. Am. Chem. Soc. 1975, 97, 2052. (23) Hashimoto, S.; Thomas, J. K. J. Colloid Interface Sci. 1984, 102, 152. (24) (a) Meyerhoffer, S. M.; McGown, L. B. Langmuir 1990, 6, 187. (b) Meyerhoffer, S. M.; McGown, L. B. Anal. Chem. 1991, 63, 2082. (c) Meyerhoffer, S. M.; McGown, L. B. J. Am. Chem. Soc. 1991, 113, 2146. (d) Li, G.; McGown, L. B. J. Phys. Chem. 1993, 97, 6745. (e) Li, G.; L. B. McGown, L. B. J. Phys. Chem. 1994, 98, 13711.
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Experimental Section NaDC was purchased from Sigma and was purified following the method published for NaC.5 Recrystallization was from methanol/water. NaOH, NaH2PO4, and pyrene were from Merck and the water was Milli-Q grade. pH was measured on a Radiometer pHM-82 with a Radiometer combined electrode GK4201C. Temperature was kept constant with a Haake thermostat. Steady-state fluorescence spectra were recorded on a Perkin-Elmer Model 650-40 and a Spex Fluorog 2 spectrofluorometer. The spectra were corrected for the wavelength dependence of the sensitivity of the system. Fluorescence lifetimes were measured in an Edinburgh Instruments CD900 fluorescence lifetime spectrometer, using the time-correlated single-photon counting technique. Running time for the accumulation of counts was always much shorter than the time required for gelation. Experiments were carried out in square quartz cells from Hellma. Cells were cleaned for 24 h with concentrated nitric acid to prevent catalysis by “seeding” of gel formed in a previous experiment.9 Solutions were prepared directly in the measuring cell by mixing filtered solutions of NaOH, NaH2PO4, NaDC, and NaDC + pyrene. Total volume was always 2 mL. NaDC concentrations used in the study were above the critical micellar concentration (cmc). cmc values ranging from 0.925 to 10 mM26 have been published.2 Reasons for this dispersity have been commented on elsewhere.5,27
Results and Discussion Figure 2a shows the emission and excitation spectra of pyrene at the beginning of a gelation experiment. The fluorescence emission spectrum of pyrene was recorded at different times during the gelation process; Figure 2b shows a typical experiment. The existence of a isosemission wavelength is observed at 450 nm. Below this wavelength the fluorescence spectra show the typical vibronic structure due to the pyrene monomer; the intensity decreases with time (see Figures 2b and 3). Above that wavelength, the fluorescence spectra show the structureless fluorescence emission due to the pyrene excimer (Figures 2b and 4a); its intensity increases with time (Figure 3). Once the gel is formed, the fluorescence emission intensities, for both the monomer and excimer, remain constant, indicating that the NaDC gel was completely formed (Figure 3). Since the concentration of pyrene is very low and the excimer formation requires the presence of two probe molecules in a given aggregate at the time of excitation,28 it can be concluded that the gel formation facilitates the interaction between two pyrene molecules. Figure 4b shows the excitation spectra recorded, once the gel is formed, at 382 nm, where only the pyrene monomer emits, and 480 nm, where the emission is mainly due to the excimer fluorescence. There is a clear displacement of the vibronic peaks of pyrene depending on emission wavelength, with a red shift of the excimer fluorescence excitation spectrum. I.e., the detected changes are only those corresponding to the emission intensities of both the monomer and excimer and the red shifted excitation spectrum corresponding to the excimer. These results can be interpreted by assuming that, in the gel, pyrene molecules exist in two different environments: as isolated monomers and near to a second pyrene molecule. In the later case, the proximity of the molecules implies an interaction between them in the ground state as is evident from the excitation spectrum, a fact that is well documented in the literature for other systems.29 In the excited state, this pair of molecules forms a “static excimer”.29 (25) Thomas, D. C.; Christian, S. D. J. Colloid Interface Sci. 1980, 78, 466. (26) Roda, A.; Hofmann, A. F.; Mysels, K. J. J. Biol. Chem. 1983, 258, 6362. (27) Kratohvil, J. P. Adv. Colloid Interface Sci. 1986, 26, 131.
Figure 2. (a) Emission and excitation spectra of pyrene before gelation: (a) λexc ) 337 nm; (b) λem ) 382 nm. Experimental conditions were as follows: [NaDC] ) 0.0362 mol dm-3, [pyrene] ) 1.24 × 10-6 mol dm-3, [NaH2PO4] ) 0.02 mol dm-3, pH ) 6.80, T ) 20.0 °C. (b) Variation of fluorescence spectrum of pyrene with time during gelation. Spectra were recorded every 5 min. λexc ) 338 nm.
Figure 5 (see also Table 1) shows logarithm decay curves obtained at zero (Figure 5a) and 65 min (Figure 5b). At the beginning of the process, a perfect fit of the data to a single exponential is observed, but when the gel is being formed, two exponentials are necessary to fit the experimental results according to
I(t) ) ae-t/τ1 + be-t/τ2
(1)
I(t) is the fluorescence intensity at t time, τ1 and τ2 are characteristic decay times, and a and b are pre-exponential factors. These two exponentials are more clearly observed once the gel is completely formed (see the decay curve in Figure 5c recorded at the end of the gelation process). Table 1 gives the corresponding values for the characteristic decay times and the pre-exponential constants of this experiment. Similar results are obtained when different excitation wavelengths are used. For a given gelation process and within experimental error, the characteristic lifetime τ1 remains constant (all over the process), but clear tendencies for the pre-exponential factors and τ2 are observed. From the steady-state fluorescence spectra commented on above we can accept that τ1 and τ2 are the lifetimes corresponding to the excited pyrene monomer and pyrene excimer, respectively. Since (28) Kalyanasundaram, K. Photochemistry in Microhetherogeneous Systems; Academic Press: Orlando, FL, 1987. (29) Winnik, F. M. Chem. Rev. 1993, 93, 587.
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Figure 3. (a) Fluorescence intensity (in arbitrary units) vs time for pyrene monomer (recorded at 385 nm) and pyrene excimer (480 nm). Experimental conditions were as in Figure 2.
Figure 4. (a) Emission spectra of pyrene obtained with two excitation wavelengths, (a) 337 nm and (b) 345 nm, at the end of the gelation process. (b) Excitation spectrum of pyrene recorded at emission wavelengths of (a) 382 nm and (b) 488 nm. Experimental conditions were as in Figure 2 except for T ) 25.0 °C.
both pre-exponential factors are always positive, it may be concluded that the excimer is formed from two molecules which are previously associated in the ground state. Since a diminishes and b increases with time, the number of associated pyrene molecules also increases due to gelation, in agreement with steady-state fluorescence results. Spectra in Figure 2 allow determination of the ratio I1/I3 of the intensities of the first and third vibronic peaks of monomeric pyrene solubilized within NaDC aggregates. It is well-known that this ratio reflects the polarity of the microenvironment of the pyrene probe and can thus be used to detect changes of polarity upon time in the medium.28 The obtained value, equal to 0.70 ( 0.01,
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remains constant during the gel formation and is close to published values for NaDC but lower than those for other bile salts (Table 2). For comparison purposes Table 2 also gives published values of I1/I3 for SDS, water, and cyclohexane. In fact, the value of I1/I3 for NaDC, very close to the one for the very unpolar cyclohexane, is the lowest one for pyrene solubilized in ionic micelles in aqueous solution. It indicates that the micelle interior where pyrene is solubilized is strongly apolar and squeezed between the lipophilic sides of several steroid nuclei which shield it very effectively against water and oxygen (see below).19,22 Although there is not a quantitative measurement of rigidity, it may be concluded that the probe environment in bile salts is more rigid than that in classical surfactant micelles,19,22,23 a conclusion in agreement with those derived from NMR studies.16,30,31 It cannot a priori be assumed that pyrene in the interior of the aggregates is perfectly shielded from oxygen. In fact, classical micelles with long alkyl chain seem to give no protection whatsoever. For SDS micelles the oxygen bimolecular quenching constant is approximately the same as in water.32 Vethamuthu et al.21 have quantified the shielding effect against oxygen quenching for NaC and NaDC and showed that it is very strong for both bile salts. This justifies the observed value for τ1 (see Table 1) in our system although our solutions were not deoxygenated at all. In fact, τ1 values are in agreement with previous published values for NaC, NaTC, NaTCDC, and NaDC (see Table 2) measured in oxygen-free solutions. That value indicates that the pyrene microenvironment is very apolar, in agreement with steady-state fluorescence results and close to published values for fluorescence lifetime of pyrene in hydrocarbon solvents.33 For NaC and NaDC, Vethamuthu et al21 have estimated the fraction of pyrene in contact with water. The observed values are 4 and 0%, respectively, suggesting that pyrene is in the interior of the aggregate. This is a clear difference with classical surfactant micelles for which pyrene is located in the palisade layer. Such a difference has been related to the lower surface charge of bile salt micelles than classical ones, i.e., the palisade layer, in the sense given to it in classical surfactant micelles, does not exist in bile salt aggregates.19 This is in agreement with the values obtained for the fraction of bound counterions for NaC,5 sodium fusidate (an antibiotic with a similar structure to that of a bile salt),34 and other bile salts.2 For most of these compounds, and in particular for NaDC, a value of 3:1 is observed for the ratio of monomers:bound counterions, which has been related to a structure in which the monomers are packed in a back-to-back way13,35 with the orientation of monomers alternatively up and down, the lateral chain supporting the carboxylate group which defines it. Finally, our conclusion also agrees with the quenching of the excited state of pyrene by several cations observed by Chen et al.22 and Hashimoto and Thomas23 in NaTC micelles. Chen et al. have reported that positively charged quenchers such as Cu2+ and Tl+ are strongly adsorbed at the surface of NaTC micelles, which is also in agreement with the reduction of quenching by Iobserved on addition of Mg2+ while the addition of benzyl alcohol increases it. (30) Kay, L. E.; Prestegard, J. H. J. Am. Chem. Soc. 1987, 109, 3829. (31) Wong, T. C.; Wang, P. L.; Duh, D. M.; Hwang, L. P. J. Phys. Chem. 1989, 93, 1295. (32) Almgren, M.; Grieser, F.; Thomas, J. K. J. Am. Chem. Soc. 1980, 102, 3188. (33) Malliaris, A. Int. Rev. Phys. Chem. 1988, 7, 95. (34) Coello, A.; Meijide, F.; Rodrı´guez Nu´n˜ez, E.; Va´zquez Tato, J. J. Pharm. Sci. 1994, 83, 828. (35) Kawamura, H.; Murata, Y.; Yamaguchi, T.; Igimi, H.; Tanaka, M.; Sugihara, G.; Kratohvil, J. P. J. Phys. Chem. 1989, 93, 3321.
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Figure 5. Fluorescence decay of pyrene at (a) zero, (b) 65 min, and (c) infinite time: (a and b) λexc ) 337 nm, λem ) 400 nm; (c) λexc ) 325 nm, λem ) 450 nm. Other experimental conditions were as in Figure 4. Table 1. Experimental Values for Parameters in Equation 1 Derived from Experiment Described in Figure 2 2 min τ1/ns τ2/ns a b χ2
15 min
370
364
1525
1607
1.131
1.084
65 min
85 min
105 min
354 67 1188 164 1.101
351 80 994 161 1.095
400 119 355 420 1.052
A very important result is the constancy observed for both the I1/I3 ratio and τ1 during the gelation process. This means that the environment of pyrene monomers is
not modified during the gel formation; i.e., the interior aggregate structure remains unchangeable during the process and the probe does not probably leave the aggregate. The size or aggregation number of a given cluster can increase by the addition of a monomer or a second cluster. Models for these particle-cluster and cluster-cluster aggregation processes are well-known.36 When two clusters carrying probes form a larger aggregate, the pyrene molecules can interact in both the ground and excited states. Therefore, there will coexist (a) isolated pyrene molecules which upon excitation fluoresce with a typical lifetime of several hundred nanoseconds,37 and (b)
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Table 2. Monomeric Pyrene Fluorescence Lifetimes and Values of I1/I3 for Various Bile Salts Taken from Literature compound NaC NaTC
NaGC NaDC
NaTCDC NaTDC SDS
temp, °C 25 25 25 23 25 20.0 25 25 28 25 25 20 25 28
I1/I3 0.86 0.72 0.79 0.91 1.03 0.82 0.79 0.68c 0.80 0.66 0.70 ( 0.01 0.83 0.77 1.04 1.16
424 393 (415-455) ( 10 263a 350 ( 4b 406c 456 406 ( 10a 371 329a 323 150a
25 cyclohexane
τ1/ns
1.32 0.54 0.61 0.59
water 1.96
175 200 ( 20
reference 21 41 42 23 24b 24e 41 41 19 21 41 d 19 24b 19 23 24b 43 19 23 24b 21 23
a Non-deoxygenated solutions. b [NaTC] ) 30 mM. c Average value. d This paper.
associated pyrene molecules close enough as to interact in the ground state and form excimers in the excited state. In our case, this process does not imply a matter transfer of the probes between micelles which is typical for classical surfactants at high fluorophore concentrations.28 Such an interpretation is also in agreement with (a) the results outlined above from Figures 2 and 3 which show an intensity increment with time due to the excimer, (b) the spectrum of Figure 4 in which the vibronic peaks suggest that pyrene molecules may exist in two different environments, (c) the fluorescence decay data, (d) the absence of oxygen quenching, and (e) the positive values observed for the pre-exponential factors in eq 1. (36) (a) Meakin, P. Phys. Scr. 1992, 46, 295. (b) Meakin, P. Croat. Chem. Acta 1992, 65, 237. (c) Meakin, P. The Fractal Approach to Heterogeneous Chemistry. Surfaces, Colloids, Polymers; Avnir, D., Ed.; Wiley: Chichester, 1989. (d) Kinetics of Aggregation and Gelation; Family, F., London, D. P., Eds.; North-Holland: Amsterdam, 1984.
Less information exists for the fluorescence lifetime of pyrene excimer due to the limitation of the solubility of pyrene in bile salt systems. The only reference is due to Hashimoto and Thomas23 who have observed that the fluorescence of the excimer in NaTC shows a faster rise time and a slower decay than those in SDS, relating this result to a lower fluidity of the former system. As is wellknown, such an efficiency of excimer formation has been used by several authors to investigate which statistical model follows the probability of finding singly and multiply occupied micelles.28 The excimer lifetime depends on several kinetic processes,38 being not possible to relate it to a particular one. However it is observed that its lifetime increases as gelation time increases (see Table 1); this can be related to a reversible formation of aggregates: if the fragmentation of a large cluster (carrying two interacting probes) to form smaller clusters takes place during the lifetime of the excimer (i.e., the fragmentation kinetic constant > τ2-1), the excimer lifetime will consequently be reduced. Because of the rigidity of NaDC aggregates commented on above, it is expected that the residence lifetime of pyrene in its host aggregate is much longer than in classical micelles (for which values of 10-3 to 10-4 s have been published).39 This allows the treatment of fluorophors as immobile solubilizates33 and suggests that the lifetime of pyrene excimer, at the end of the gelation process, could be compared with the excimer-type fluorescence of pyrene crystals40 which have been studied as a function of temperature. At 296 K a value of 108 ns has been reported, to which our value of 95 ( 17 ns compares favorably. Acknowledgment. We thank the DGICYT for financial support (Project PB90-0758). LA9506335 (37) Gra¨tzel, M.; Thomas, J. K. J. Am. Chem. Soc. 1973, 95, 6885. (38) Birks, J. B. Prog. React. Kinet. 1970, 5, 181. (39) Thomas, J. K.; Grieser, F.; Wong, M. Ber. Bunsenges. Phys. Chem. 1978, 82, 937. (40) (a) Birks, J. B.; Kazzaz, A. A.; King, T. A. Proc. R. Soc. London, Ser. A 1966, 291, 556. (b) Horiguchi, R.; Iwasaki, N.; Maruyama, Y. J. Phys. Chem. 1987, 91, 5135. (c) Seyfang, R.; Betz, E.; Port, H.; Schrof, W.; Wolf, J. C. J. Lumin. 1985, 34, 57. (41) Matsuzaki, K.; Yokoyama, I.; Komatsu, H.; Handa, T.; Miyajima, K. Biochim. Biophys. Acta 1989, 980, 371. (42) Ueno, M.; Kimoto, Y.; Ikeda, Y.; Momose, H.; Zana, R. J. Colloid Interface Sci. 1987, 117, 179. (43) Llanos, P.; Zana, R. J. Phys. Chem. 1980, 84, 3339.
