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Aggregation Number for Sodium Deoxycholate from Steady-State and Time-Resolved Fluorescence A. Jover, F. Meijide, E. Rodrı´guez Nu´n˜ez, and J. Va´zquez Tato* Universidad de Santiago, Campus de Lugo, Facultad de Ciencias, Departamentos de Quı´mica Fı´sica y Fı´sica Aplicada, 27002 Lugo, Spain
M. Mosquera Universidad de Santiago, Facultad de Quı´mica, Departamento de Quı´mica Fı´sica, 15706 Santiago de Compostela, Spain Received March 25, 1996. In Final Form: October 15, 1996X Steady-state fluorescence and time-resolved fluorescence have been used to study sodium deoxycholate aggregates at basic pH. Pyrene was used as a probe, and dimethylaniline, as a quencher. It is concluded that dynamic quenching with partially micellized quenchers and not static quenching with the quencher totally micellized, as has been often quoted in the literature, must be considered to interpret the experimental results. Fluorescence lifetimes for pyrene in the absence of quencher and the kinetic constants involved in quenching by dimethylaniline are given and compared to those for similar systems. The derived aggregation number, equal to 8 ( 2, is close to the ones determined by other authors from freezing point depression and static light scattering. The possible distortion of these small aggregates by probes is also discussed.
Introduction
ln(I0/I) ) [Qt]/[M]
(1)
1-8 and paramagnetic9 probes are often used
Fluorescent to study the structure of bile salt aggregates. Steadystate fluorescence is the most common technique, since it is inexpensive and experimentally simple10 and allows the determination of both the aggregation number and probe microenvironment. Among the polycyclic aromatic hydrocarbon probes, pyrene is the most common one used to study the associative behavior of bile salts.3-7 For these systems, there is no doubt that added hydrophobic probes are located in the interior of the aggregate1,4 and wellprotected from the environment (in particular from oxygen and water).6,8 These experimental results lead to proposed models for bile salt aggregates in which monomers are packed in a back-to-back way with the hydrophobic surface of the steroid group toward the aggregate interior and the hydrophilic surfaces toward the solvent.9,11 To determine the aggregation number from steady-state fluorescence measurements, the addition of a quencher is also required. Experimental results are usually analyzed through eq 1, derived by Turro and Yekta12 X Abstract published in Advance ACS Abstracts, December 15, 1996.
(1) Chen, M.; Gra¨tzel, M.; Thomas, J. K. J. Am. Chem. Soc. 1975, 97, 2052. (2) Fisher, L.; Oakenfull, D. Aust. J. Chem. 1979, 32, 31. (3) Hashimoto, S.; Thomas, J. K. J. Colloid Interface Sci. 1984, 102, 152. (4) Zana, R.; Gu¨veli, D. J. Phys. Chem. 1985, 89, 1687. (5) (a) Ueno, M.; Kimoto, Y.; Ikeda, Y.; Momose, H.; Zana, R. J. Colloid Interface Sci. 1987, 117, 179. (b) Matsuzaki, K.; Yokoyama, I.; Komatsu, H.; Handa, T.; Miyajima, K. Biochim. Biophys. Acta 1989, 980, 371. (c) Meyerhoffer, S. M.; McGown, L. B. Anal. Chem. 1991, 63, 2082. (6) Vethamuthu, M. S.; Almgren, M.; Mukhtar, E.; Bahadur, P. Langmuir 1992, 8, 2396. (7) Li, G.; McGown, L. B. J. Phys. Chem. 1994, 98, 13711. (8) Jover, A.; Meijide, F.; Rodrı´guez Nu´n˜ez, E.; Va´zquez Tato, J.; Mosquera, M.; Rodrı´guez Prieto, F. Langmuir 1996, 12, 1789. (9) Kawamura, H.; Murata, Y.; Yamaguchi, T.; Igimi, H.; Tanaka, M.; Sugihara, G.; Kratohvil, J. P. J. Phys. Chem. 1989, 93, 3321. (10) (a) Goodling, K.; Johnson, K.; Lefkowitz, L.; Williams, B. W. J. Chem. Educ. 1994, 71, A8. (b) Rodrı´guez Prieto, M. F.; Rios, M. C.; Mosquera, M.; Rios, A. M.; Mejuto, J. C. J. Chem. Educ. 1995, 72, 662. (11) Coello, A.; Meijide, F.; Rodrı´guez Nu´n˜ez, E.; Va´zquez Tato, J. J. Pharm. Sci. 1996, 85, 9. (12) Turro, N. J.; Yekta, A. J. Am. Chem. Soc. 1978, 100, 5951.
In this equation [M] is the concentration of micelles given by eq 2
[M] )
[surfactant] - cmc N
(2)
I0 and I are the fluorescence intensities at zero and [Qt] concentrations of the quencher, and N is the aggregation number of the aggregate. Equation 1 is derived under the following hypotheses: (1) the quencher and the fluorescent probe are entirely in the micellar phase, (2) the distributions of the quencher and the probe obey Poisson statistics, and (3) the probe only fluoresces in the absence of the quencher. Following Kalyanasundaram,13 other possibilities, however, do exist: (a) static quenching where the quencher is totally micellized, (b) static quenching where the quencher is partially micellized, (c) dynamic quenching with totally micellized, immobile quenchers, and (d) dynamic quenching with partially micellized quenchers (quenchers which partition between the micellar and aqueous phases). The Turro and Yekta model corresponds to case a. It implies that fluorescence decay curves should be monoexponentials with measured lifetimes independent of added quencher concentration. Therefore, for a given system, it is possible to test the validity of the Turro and Yekta equation by carrying out time-resolved fluorescence studies of the probe in the presence of different quencher concentrations. It is important to notice here that, in determining bile salt aggregation numbers, eq 1 has been used in the literature4,5a without testing its applicability. Here we present results from steady-state and time-resolved fluorescence studies for sodium deoxycholate (NaDC) and show that in fact case d is followed. NaDC was chosen for comparison purposes, pyrene as the fluorescent probe, and dimethylaniline (DMA) as the quencher. (13) Kalyanasundaram, K. Photochemistry in Microheterogeneous Systems; Academic Press, Orlando, FL, 1987.
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Jover et al. Table 1. Values for Parameters A, B, and C of Eq 3a [NaDC]/mM
[DMA]/mM
10-6A/s-1
B
10-8C/s-1
33.88 33.88 33.88 33.88 16.94 16.94
0.195 0.779 1.558 2.338 0.195 1.948
2.86 3.52 4.37 5.25 2.89 6.06
0.855 0.909 0.973 1.128 0.699 1.597
4.48 4.40 3.96 3.33 2.69 3.35
a They are related through eqs 4-6 to k (rate constant for the 0 unquenched probe decay), k- (kinetic constant for the exit of a quencher molecule from aggregates), kq (first-order quenching constant), and n j (mean occupancy number).
of this parameter on the resulting kinetic constants and aggregation number (see below). Modifying the cmc value from 6 to 14 mM, an influence lower than 5% was observed for the kinetic constants and a negligible one for the aggregation number.
Results and Discussion Figure 1a shows that the fluorescence intensity of pyrene in the absence of DMA follows a single exponential decay, the characteristic lifetime being τ0 ) 384 ( 1 ns, in agreement with previous measurements.8 The high value for τ0, close to the ones obtained in apolar solvents and deoxygenated solutions, means that quenching by oxygen does not exist, as has been commented on elsewhere,6,8 since solutions were not deoxygenated at all. Figure 1b shows a typical fluorescence decay in the presence of DMA; it is clear that in this case two exponentials are necessary to fit experimental results. Following Kalyanasundaram,13 eq 3 was used to fit experimental curves.
[]
ln
Figure 1. Fluorescence decay of pyrene (a) in the absence of quencher at [NaDC] ) 33.9 mM and (b) in the presence of [DMA] ) 1.95 mM and [NaDC] ) 16.9 mM.
Experimental Section NaDC was purchased from Sigma and was purified as previously described.8 Other chemicals were purchased from Merck. Water was Milli-Q grade. Steady-state fluorescence measurements were recorded on a Hitachi (model F-3010) spectrofluorometer, and fluorescence lifetimes were measured in an Edinburgh Instruments CD900 fluorescence lifetime spectrometer, using the time-correlated single-photon counting technique. Temperature was kept constant at 20 °C with a Haake thermostat, and the pH was 11 (adjusted with NaOH and measured with a pH-meter Radiometer pHM-82 with a combined electrode Radiometer GK4201C). This high pH value assures that NaDC and DMA are totally unprotonated. λexc was 337 nm for both dynamic and steady-state studies. λem was 390 nm for dynamic experiments, and for steady-state measurements the averaged (during 30 s) fluorescence intensities were recorded at the maximum first and third vibronic pyrene peaks. For the analysis of experimental results a value of 10 mM was used as the cmc for NaDC.11 Since a great range of cmc values are published in the literature,11 we have checked the influence
It ) - At + B(e-Ct - 1) I0
(3)
In this equation It and I0 are the fluorescence intensities at t and zero times and the parameters A, B, and C are defined below. Table 1 shows the obtained results for these parameters at different quencher concentrations. Only results for two NaDC concentrations are shown. The standard deviation in parameter C is rather high due to the low statistical weight of the corresponding term compared to the first one in eq 3. Two cases are compatible with eq 3: (i) The characteristic lifetime for pyrene is unaffected by the quencher concentration (case c of the Introduction). (ii) The characteristic lifetime diminishes with increasing quencher concentration (case d). Comparison between Figure 1a and b shows that the second case applies.13,14 Therefore in eq 3, the parameters A, B, and C are given by
A ) k0 + B)
j k-kqn C
n j kq2 C2
C ) kq + k-
(4)
(5) (6)
where n j is the mean occupancy number given by
n j ) K[Qt]/(1 + K[M])
(7)
kq is the first-order quenching constant, k- is the kinetic constant for the exit of a quencher molecule from aggregates, k0 is the rate constant for the unquenched probe decay, and K ) k+/k-, where k+ is the rate constant for the (14) Yekta, A.; Aikawa, M.; Turro, N. J. Chem. Phys. Lett. 1979, 63, 453.
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Aggregation Number for Sodium Deoxycholate
Langmuir, Vol. 13, No. 2, 1997 163 Table 2. Summary of the Kinetic Constants and Aggregation Number (See Eqs 3-7 in the Text) for NaDC and Related Systems surfactant
τ0/ns
NaDC NaTCa SDSa
384 415-455 323b
a
Figure 2. Plot of A vs [DMA] according to eq 4.
Figure 3. Fluorescence intensities ratio, I0/I, vs quencher concentration at [NaDC] ) 135.5 mM. The line is derived from eq 7 and the parameter values shown in Table 2.
entry of a quencher molecule into aggregates. According to eq 4, the plot of A (the reciprocal of the characteristic lifetime of pyrene in the presence of quencher) vs [DMA] should be linear, the ordinate being 1/τ0. Figure 2 shows two typical examples at two different NaDC concentrations. On the other hand, the corresponding equation for steady-state fluorescence experiments for the case of a dynamic quenching with partially micellized quenchers (case d) is given by
1
I ) I0
[Qt]
1 + τ0k+ 1 + K[M]
exp[-n j (kq/C)2]
[n j (kq/C)2]i
∑ i)0 i!(1 + Ciτ ) 0
(8)
In this equation [M] is calculated by eq 2 and k+, K, and n j are the fitting parameters. Equation 8 was solved iteratively with the initial assumption that the summation in eq 8 was close to 1, which corresponds to the physical situation kq >> k-. With the resulting values for the kinetic constants this is in fact the final conclusion. Figure 3 shows some experimental results for the steadystate fluorescence intensity vs quencher concentration. The curve is calculated according to eq 8 and the different kinetic constants presented in Table 2. The shown values
10-8 10-6 10-9 (K ) kq/s-1 k-/s-1 k+/M-1 s-1 k+/k-)/M-1 3.4 0.50 0.3
5.1 3.8 2.2
1.0 1.4 2.9
205 370 1300
n 8(2 18
Reference 3. b Reference 4.
correspond to a global fitting of 47 experiments carried out within the following ranges of concentrations: [NaDC] ) 34-136 mM and [DMA] ) 0.195-4.8 4 mM. The values for the kinetic constants shown in Table 2, deduced from steady-state experiments, reproduce quite well the dynamic experiments (eqs 3-6). In fact, the aggregation number is a fitting parameter (through eqs 2 and 7) considered to be independent of NaDC concentration. This assumption is supported by recent determinations of the aggregation number at every single bile salt concentration. For both tri- and dihydroxy derivatives, it has been shown11,15 that the aggregation number remains constant with surfactant concentration. The results are consistent with rigorous thermodynamical conclusions for surfactant behavior in aqueous solution,16 since fractions of bound counterions are also constant with bile salt concentration and the aggregation number slowly increases in the presence of added inert electrolytes.15,17 Although the partition and the different kinetic constants are very similar for NaDC, NaTC (in 1 M NaCl)3 and SDS3 (see Table 2), it is not easy to derive clear conclusions, since the structure for bile salt aggregates is quite different than the one for classical alkyl surfactant micelles.4 For instance, the ratio I1/I3 of the intensities of the first and third vibronic peaks of pyrene solubilized within NaDC and SDS micelles (0.70 and >1, respectively) clearly shows that the interior of the former is much more apolar than the later.3,4,8 Similar comments apply to the τ0 values shown in Table 2.8 Furthermore, Vethamuthu et al.6 have estimated that the fractions of pyrene in contact with water for sodium cholate and NaDC are 4 and 0%, respectively. This is a clear difference with classical surfactant micelles, for which pyrene is located in the palisade layer. In fact, the palisade layer, in the sense given to it in classical surfactant micelles, does not exist in bile salt aggregates.4 The resulting aggregation number for NaDC aggregates 8 ( 2 is a little higher than previous published ones. Coello et al.11 from freezing point depression measurements have found a value of 5.82 ( 0.04 while, from Kratohvil et al.17 results, derived from a very careful static light scattering study at 0.15 and 0.6 Na+ concentrations, a value of 6 is obtained by extrapolation at zero NaCl concentration.18 That higher value could arise from the fact that bile salt aggregates can be modified by the introduction of guest molecules, except if they are flexible enough to adopt the rigid inside form of the host aggregate.9 This interpretation would be in agreement with Li and McGown,7 who, in studying the aggregation behavior of sodium taurocholate by using an array of polycyclic aromatic hydrocarbon probes (ranging from pyrene to ovalene), affirm that “larger probes may cause distortions in the aggregate shapes”. Therefore, when aggregates are small in size, invasive methods must be considered carefully. (15) Coello, A.; Meijide, F.; Rodrı´guez Nu´n˜ez, E.; Va´zquez Tato, J. J. Phys. Chem. 1993, 97, 10186. (16) Nagarajan, R. Langmuir 1994, 10, 2028. (17) Kratohvil, J. P.; Hsu, W. P.; Kwok, D. I. Langmuir 1986, 2, 256. (18) Because of the monodispersity for these systems commented on above, the difference between weight and number average aggregation numbers does not apply.
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In conclusion, the results derived from three quite different experimental techniques (freezing point depression,11 static light scattering,17 and steady-state and timeresolved fluorescences (present work)) clearly show that NaDC aggregates are small in size, the aggregation number at zero NaCl concentration being quite probably 6. Higher aggregation numbers, such as for instance the one deduced by Carey19 from previous studies equal to 15 ( 3, are too high. A complete revision of published aggregation numbers for NaDC and other bile salts can be seen in ref 11. (19) (a) Carey, M. C. In Bile Acids in Gastroenterology; Barbara, L., Dowling, R. H., Hofmann, A. F., Roda, E., Eds.; MTP: Lancaster, 1982; Chapter 2. (b) Carey, M. C. In Sterols and Bile Acids; Danielsson, H., Sjo¨vall, J., Eds.; Elsevier: Amsterdam, 1985; Chapter 13.
Jover et al.
Finally, the present results also suggest that previous published values for the aggregation number obtained from static fluorescence measurements4,5a for bile saltaqueous solution systems derived from eq 1 must be considered carefully. The high aggregation number ()18) published by Ueno et al.5a for NaC compared to the value deduced from freezing point measurements ()3.1)15 supports this conclusion. Acknowledgment. We thank the Xunta de Galicia for financial support (Project XUGA 26203B94). LA9602877
