2038
Langmuir 2000, 16, 2038-2041
Notes Aggregation Dynamics of Sodium Taurodeoxycholate and Sodium Deoxycholate
Chart 1
Y. Li,† J. F. Holzwarth,† and C. Bohne*,‡ Abteilung Physikalische Chemie, Fritz-Haber-Institut der Max-Planck-Gesellschaft, Faradayweg 4-6, D-14195 Berlin-Dahlem, Germany, and Department of Chemistry, University of Victoria, P.O. Box 3065, Victoria, B.C., Canada, V8W 3V6 Received March 30, 1999. In Final Form: October 20, 1999
Introduction Bile salts are derivatives of cholesterol that aggregate in aqueous solution, due to their planar polarity.1-6 The polarity of bile salts, such as sodium deoxycholate (NaDC) and sodium taurodeoxycholate (NaTDC) (Chart 1), is defined by the hydrophilic hydroxyl groups on the concave face and the hydrophobic methyl groups on the convex face of these molecules. Several models have been proposed to explain the progressive aggregation of bile salts. The primary/secondary1,3,7 and disklike8 models suggest that aggregation is initially driven by the interaction of the hydrophobic faces, whereas the helical model suggests that aggregation initially occurs due to the hydrogen bonding of the hydroxyl groups.9,10 Although this controversy has not been fully resolved, there is increasing evidence that the aggregation is driven by the interaction of the hydrophobic faces.7,8,11-18 For this reason the discussion of our results is based on the primary/secondary aggregation model, where at low concentrations the bile * Corresponding author. Phone: 250-721-7151. Fax: 250-7217147. E-mail:
[email protected]. Web page: http://www.foto.chem. uvic.ca/. † Fritz-Haber-Institut der Max-Planck-Gesellschaft. ‡ University of Victoria. (1) Small, D. M. In The Bile Salts; Nair, P. P., Kritchevsky, D., Eds.; Plenum Press: New York, 1971; Vol. 1, p 249. (2) Carey, M. C.; Small, D. M. Arch. Intern. Med. 1972, 130, 506. (3) O’Connor, C. J.; Wallace, R. G. Adv. Colloid Interface Sci. 1985, 22, 1. (4) Kratohvil, J. P. Adv. Colloid Interface Sci. 1986, 26, 131. (5) Hofmann, A. F.; Mysels, K. J. Colloids Surf. 1988, 30, 145. (6) Hofmann, A. F. In The Liver: Biology and Pathobiology, 3rd ed.; Arias, I. M., Schachter, A. D., Shafritz, D. A., Eds.; Raven Press Ltd.: New York, 1994; p 677. (7) Small, D. M.; Penkett, S. A.; Chapman, D. Biochim. Biophys. Acta 1969, 176, 178. (8) Kawamura, H.; Murata, Y.; Yamaguchi, T.; Igimi, H.; Tanaka, M.; Sugihara, G.; Kratohvil, J. P. J. Phys. Chem. 1989, 93, 3321. (9) Campanelli, A. R.; de Sanctis, S. C.; Chiessi, E.; D’Alagni, M.; Giglio, E.; Scaramuzza, L. J. Phys. Chem. 1989, 93, 1536. (10) Campanelli, A. R.; De Sanctis, S. C.; Giglio, E.; Pavel, N. V.; Quagliata, C. J. Inclusion Phenom. Mol. Recognit. Chem. 1989, 7, 391. (11) Chen, M.; Gra¨tzel, M.; Thomas, J. K. J. Am. Chem. Soc. 1975, 97 (7), 2052. (12) Zana, R.; Guveli, D. J. Phys. Chem. 1985, 89, 1687. (13) Meyerhoffer, S. M.; McGown, L. B. Anal. Chem. 1991, 63, 2082. (14) Li, G.; McGown, L. B. J. Phys. Chem. 1994, 98, 13711. (15) Funasaki, N.; Ueshiba, R.; Hada, S.; Neya, S. J. Phys. Chem. 1994, 98, 11541. (16) Funasaki, N.; Hada, S.; Neya, S. J. Phys. Chem. B 1999, 103, 169. (17) Coello, A.; Meijide, F.; Rodrı´guez Nu´n˜ez, E.; Va´zquez Tato, J. J. Pharm. Sci. 1996, 85, 9. (18) Ju, C.; Bohne, C. Photochem. Photobiol. 1996, 63, 60.
salts form primary aggregates due to the hydrophobic interaction of the convex faces and at higher bile salt concentrations the primary aggregates form larger secondary aggregates. Bile salts form aggregates with a more complex structure than that of simple micelles, such as those formed from alkyl sulfates. For example, guests are solubilized in a less polar and more rigid environment in bile salt aggregates,12-14,19,20 and hydrophobic molecules can induce the formation of aggregates.14,18 The dynamics of guest complexation was studied,11,19,21-23 and detailed quenching experiments showed that bile salt aggregates at high concentrations have at least two binding sites for guest molecules.22 The monomer exchange rate in the primary aggregates and the dynamics of secondary aggregation are different types of dynamics that are relevant to understand the mechanisms for bile salt aggregation. In the only report in which the dynamics of bile salt aggregation was studied,24 ultrasonic relaxation measurements established that ultrasonic absorptions occurred for a distribution of frequencies. These results suggest that the association of the bile salts into aggregates occurred for a range of concentrations and not at a critical micellar concentration. We report here on salt-jump experiments using the stopped flow technique and on iodine-laser temperature-jump experiments which indicate that the dynamics of secondary aggregation is a fast process. This study provides the first estimate for the relaxation lifetimes involved in the aggregation dynamics of bile salts. Experimental Section Sodium taurodeoxycholate (NaTDC, Sigma >97% or SigmaUltra >97%), sodium deoxycholate (NaDC, Sigma >97% or SigmaUltra >99%), 1,6-diphenyl-1,3,5-hexatriene (DPH, Fluka 99%), p-toluenesulfonate salt of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH, Molecular Probes), NaCl (Merck, >99.5%), and methanol (Merck, 99.8%) were used as received. Purified water (Milli-Q Ultrapure Water System) was employed for the bile salt solutions. (19) Hashimoto, S.; Thomas, J. K. J. Colloid Interface Sci. 1984, 102, 152. (20) Nithipatikom, K.; McGown, L. B. Photochem. Photobiol. 1988, 47, 797. (21) Seret, A.; Van de Vorst, A. J. Photochem. Photobiol. B: Biol. 1993, 17, 47. (22) Ju, C.; Bohne, C. J. Phys. Chem. 1996, 100, 3847. (23) Jover, A.; Meijide, F.; Rodrı´guez Nu´n˜ez, E.; Va´zquez Tato, J. Langmuir 1997, 13, 161. (24) Djavanbakht, A.; Kale, K. M.; Zana, R. J. Colloid Interface Sci. 1977, 59, 139.
10.1021/la9903705 CCC: $19.00 © 2000 American Chemical Society Published on Web 12/31/1999
Notes
Langmuir, Vol. 16, No. 4, 2000 2039
Transmission and absorption measurements were performed on a Shimadzu UV-2100 absorption spectrometer. Steady-state light scattering and fluorescence were measured with a RF-5000 Shimadzu fluorimeter at constant temperature (Haake FC-3 bath and Haake PG20 controller). Light scattering was measured at 360 nm with a 90° detection angle. Samples containing DPH were excited at 360 nm, and the emission was collected between 370 and 520 nm. The stopped-flow measurements were performed with an Applied Photophysics SX.18MV system. A 1:1 mixing ratio was employed, and the stock solutions in the syringes (5 mL) and the mixing chamber were kept at a constant temperature (Haake C25 bath). The light-scattering intensity was detected at a 90° angle at 360 nm using the fluorescence setup. Six to ten measurements were averaged for each experiment. Initiation of mixing, acquisition of kinetic traces, and analysis of data were performed with the Applied Photophysics SX.18MV software. The iodine-laser temperature-jump (ILTJ) setup has been described in detail.25,26 Iodine-laser photons (1315 nm) excite the rotational-vibrational states of water, leading to heating of the solution by approximately 1 °C in 2.4 µs. The long-time limit for measuring relaxation times is 1.5 s, and it is determined by the back-cooling rate of the solution. The relaxation kinetics was followed by light scattering at 360-380 nm (360 nm cutoff and Schott UG-11 filters) or fluorescence measurements (λexcitation ) 360-380 nm; 360 nm cutoff and Schott UG-11 filters and λemission > 400 nm; 400 nm cutoff and Andover Corporation SWP 550 filters) using a Xe/Hg 200 W arc lamp. The excitation and detection geometries are in a perpendicular arrangement. A shutter was employed between the lamp and the sample holder which was opened shortly before the laser pulse because DPH is reversibly bleached when irradiated by the arc lamp. Four to six traces were averaged for each relaxation experiment. Aqueous solutions in the presence or absence of NaCl were prepared for NaTDC (pH 4.5-5.0) and NaDC (pH 7.5-8.0). These samples formed gels when left standing, a feature that has been reported before.27-29 For NaTDC gel formation was observed after 16 h ([NaCl] e 0.8 M), whereas for NaDC gel formation was observed after 36 h ([NaCl] e 0.6 M). Gel formation was followed by measuring the transmission at 245 nm, where a marked decrease in the transmittance values was observed when the gels were formed. Reversible resolubilization of the bile salts was achieved by heating the solutions above 30 °C. All samples were heated for 1 h at 75 °C to ensure that no gel formation had been initiated. Samples containing DPH or TMA-DPH were prepared by injecting small amounts of a methanolic solution of the probes (0.5 mM) into 3 mL of the bile salt solutions. The samples containing the probes were again heated above 75 °C for at least 30 min.
Results Determination of Experimental Conditions for Time-Resolved Studies. The size of bile salt aggregates increases when the bile salt concentration or the ionic strength of the medium is increased.4,30-32 The steadystate light-scattering intensity of NaTDC and NaDC increased when the bile salt concentration was raised from 10 to 100 mM or NaCl (e0.6 M) was added. The temperature dependence (15-75 °C) of the light-scattering intensity (90° detection angle) or the turbidity intensity (25) Holzwarth, J. F.; Schmidt, A.; Wolff, H.; Volk, R. J. Phys. Chem. 1977, 81, 2300. (26) Holzwarth, J. F.; Eck, V.; Genz, A. In Spectroscopy and the Dynamics of Molecular Biological Systems; Bayley, P. M., Dale, R. E., Eds.; Academic Press: London, 1985; p 351. (27) Sugihara, G.; Ueda, T.; Kaneshina, S.; Tanaka, M. Bull. Chem. Soc. Jpn. 1977, 50, 604. (28) Jover, A.; Meijide, F.; Rodrı´guez Nu´n˜ez, E.; Va´zquez Tato, J.; Mosquera, M.; Prieto, F. R. Langmuir 1996, 12, 1789. (29) Jover, A.; Meijide, F.; Rodrı´guez Nu´n˜ez, E.; Va´zquez Tato, J. Langmuir 1998, 14, 4359. (30) Mazer, N. A.; Carey, M. C.; Kwasnick, R. F.; Benedek, G. B. Biochemistry 1979, 18, 3064. (31) Shurtenberger, P.; Mazer, N.; Ka¨nzig, W. J. Phys. Chem. 1983, 87, 308. (32) Kratohvil, J. P.; Hsu, W. P.; Kwok, D. I. Langmuir 1986, 2, 256.
(0° detection angle) was studied with the objective of determining the temperature at which the rate of intensity change was maximized. This information is important to establish the optimum temperature for the ILTJ measurements. The light-scattering intensity of bile salt solutions (20-100 mM; 0 e [NaCl] e 0.6 M) decreased continuously when the temperature was raised (rate 1 °C/min). These intensities at constant bile salt and NaCl concentrations were always smaller for NaDC than NaTDC, and at a constant bile salt concentration the intensity increased when the NaCl concentration was raised. In all cases the intensity change over 1 °C in the whole temperature range studied was 0.5-0.8%. DPH and TMA-DPH were employed as fluorescent probes in the ILTJ experiments. DPH is frequently employed to investigate the fluidity of membranes,33 since it does not fluoresce in water due to the lower emission quantum yield in polar solvents34,35 and its low solubility in water. DPH and TMA-DPH solubilized in membranes were previously employed in ILTJ experiments to study membrane relaxation processes. In these experiments the fluorescence of the probes was continuously monitored to detect the relaxation processes induced by raising the temperature.36 DPH is a nonpolar molecule, whereas TMADPH is positively charged at one extremity and for this reason is amphiphilic. The incorporation of DPH into bile salt aggregates was studied to establish the concentration range that could be employed without leading to saturation phenomena. For the same amount of DPH (5 µM) the absorption maximum at 360 nm increased from 0.1 in the presence of 5 mM NaTDC to 0.67 and 0.70 at 20 mM and 100 mM, respectively. These results indicate that at 5 mM bile salt only a limited amount of DPH was incorporated, but above 20 mM full incorporation occurred. A linear relationship between the fluorescent intensity (433 nm, 20 mM NaTDC and 0.2 M NaCl) and the DPH concentration was observed at low probe concentrations, but a downward curvature appeared above 10 µM. For this reason all time-resolved experiments were done at a probe concentration of 5 µM. Stopped-Flow Experiments. The relaxation kinetics at 15 or 45 °C were followed by monitoring changes in the light-scattering intensities induced by altering the NaCl concentration during the mixing time. Solutions containing NaTDC or NaDC in the presence of 0.6 M NaCl where mixed with solutions containing only the bile salts, leading to a dilution of the NaCl but keeping the bile salt concentration constant. Alternatively, solutions containing only bile salts were mixed with solutions containing only NaCl and for the final mixture the concentrations of bile salt and NaCl were halved. In all cases we observed a change for the light-scattering intensity, indicating that the extent of aggregation was altered, but no relaxation kinetics could be resolved (g0.9 ms). This result suggests that the relaxation lifetimes are faster than 1 ms. Experiments were performed at progressively longer time ranges (e20 s) to ensure that long relaxation processes were not missed. The lack of a relaxation signal is not due to a small sensitivity for light-scattering measurements in the stopped-flow setup, since the amplitude defined by the intensity values before and after mixing of the solutions (Table 1) is large enough for a relaxation signal to be (33) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Plenum Press: New York, 1983. (34) Cehlnik, E. D.; Cundall, R. B.; Lockwood, J. R.; Palmer, T. F. J. Phys. Chem. 1975, 79, 1369. (35) Bondarev, S. L.; Bachilo, S. M. J. Photochem. Photobiol. A: Chem. 1991, 59, 273. (36) Genz, A.; Holzwarth, J. F. Colloid Polym. Sci. 1985, 263, 484.
2040
Langmuir, Vol. 16, No. 4, 2000
Notes Table 2. Observed Relaxation Lifetimes Obtained from ILTJ Experiments for NaDC and NaTDC in the Presence of 5 µM TMA-DPHa bile salt
[bile salt]/mM
[NaCl]/M
τobs/µs
NaDC NaDC NaTDC NaTDC NaTDC
20 100 20 100 100
0.2 0.2 0.2 0.2 0.4
3.1 ( 0.3 (3) 2.9 ( 0.4 (5) 3.4 ( 0.3 (3) 4.1 ( 0.6 (3) 4.2 ( 1.8 (7)
a Measurements performed at 15 and 45 °C. The number in parentheses corresponds to the number of experiments averaged. The errors correspond to standard deviations.
higher purity bile salts were within 15% of those measured for the less pure bile salts. Within experimental errors the same τobs values were measured at 15 and 45 °C. In addition, the lifetimes were shorter for NaDC than NaTDC. No other relaxations were observed for longer collection times (e1 s). Discussion
Figure 1. ILTJ relaxation kinetics for NaDC (A, 15 °C) and NaTDC (B, 45 °C) in the presence of DPH and TMA-DPH: (A) 20 mM NaDC, 0.2 M NaCl, and 5 µM DPH (1, b) or 5 µM TMA-DPH (2, O); (B) 100 mM NaTDC, 0.2 M NaCl, and 5 µM DPH (1, O) or 5 µM TMA-DPH (3, b), and 100 mM NaTDC, 0.4 M NaCl, and 5 µM TMA-DPH (2, 4). The signals for the traces were normalized to the same pre-laser intensity level. The positive signals correspond to an artifact due to the emission from hot spots created by the laser hitting black bodies (dust particles) in solution. The flat portion of the positive signal is due to the limits of the observation window. Table 1. Light-Scattering Intensities Measured for NaTDC Solutions in a Shimadzu Fluorimeter and an Applied Photophysics Stopped-Flow System light scattering solution water 10 mM NaTDC + 0.3 M NaCl 10 mM NaTDC + 0.6 M NaCl 20 mM NaTDC 20 mM NaTDC + 0.3 M NaCl 20 mM NaTDC + 0.6 M NaCl
fluorimeter (au)
stopped-flow system (au)
25 45 100 160
0.71 0.77 0.75 0.90 0.98
observable. In addition, the intensity changes parallel qualitatively the changes observed for the steady-state measurements on the fluorimeter (Table 1). A quantitative comparison is not warranted, since the excitation and emission geometries were not the same. Iodine-Laser Temperature-Jump (ILTJ) Experiments. No changes in the light-scattering intensities were observed when bile salt solutions were subjected to ILTJ experiments. This result is due to the small intensity gradient with temperature observed in the steady-state experiments. To overcome this problem, probes had to be employed. DPH and TMA-DPH have fluorescence quantum yields that decrease in polar environments. For DPH, a very small decrease of the fluorescence intensity was observed after the laser pulse, but no relaxation process longer than the intrinsic relaxation of the system (2.4 µs) was apparent (Figure 1). In contrast, for TMA-DPH a negative amplitude was observed (Figure 1), and the lifetimes (τobs) were longer than the instrumental relaxation (Table 2). For experiments performed on the same day the relaxation lifetimes for samples containing the
The results for the steady-state light-scattering experiments are in qualitative agreement with the detailed experiments described in the literature4,30,31 with respect to the fact that secondary aggregates are continuously growing as the bile salt or NaCl concentrations are raised. The continuous decrease of the intensity with temperature shows that the aggregates get smaller, but no phase transition is observed. For salt jump experiments the changes in the light-scattering intensity were large enough to detect changes in the secondary aggregation of the bile salts. Unfortunately, the relaxation processes were faster than 1 ms and we cannot establish if this relaxation is mainly determined by the association or dissociation processes of the secondary aggregates. Fluorescent probe molecules were employed for the ILTJ experiments because light-scattering measurements were not possible due to the very small amplitudes inside the noise level. DPH is probably included in the primary aggregate, since it is very hydrophobic. This assignment is based on the fact that DPH is solubilized in membranes36 and in the hydrophobic portion of block-copolymer micelles.37 Since DPH and TMA-DPH lead to different relaxation processes in the ILTJ experiments, they are likely to be located in different microenvironments of the bile salt aggregates. TMA-DPH is probably located in the secondary aggregates due to its higher polarity when compared to that of DPH. The use of probes leads to an intrinsic problem, since the probes can alter the structure of the aggregates. However, currently the use of probes is the only option, since other direct detection measurements, such as light-scattering failed. No relaxation signal was resolved for DPH, and only a very small decrease of the emission intensity was observed, which can be explained by the decrease of the DPH fluorescence quantum yields observed when the temperature is raised.34 DPH is located in the primary aggregates, and the lack of relaxation signals suggests that no relaxation process that exposes DPH to the aqueous phase was detected in the ILTJ experiments. However, the lack of a signal does not exclude a relaxation process involving the primary aggregates in the time range investigated (100 µs to 1 s); it only indicates that DPH is not sensitive to such a process. The TMA-DPH relaxation signal with a negative amplitude indicates that the probe is exposed to a more (37) Kositza, M. J.; Bohne, C.; Alexandridis, P.; Hatton, T. A.; Holzwarth, J. F. Macromolecules 1999, 32, 5539.
Notes
Langmuir, Vol. 16, No. 4, 2000 2041
polar environment when the temperature is raised. The observed relaxation times (τobs) are close to the instrumental relaxation (τinstrumental ) 2.4 µs), and in principle a biexponential decay is expected. However, such an analysis is not justified with the signal-to-noise ratios observed. A simplified equation, used previously for ILTJ experiments,38 can be employed to estimate the relaxation lifetimes for the bile salt aggregate system (τbile salt) when the data are fitted to a single-exponential function:
τbile salt ) [(τobs)2 - (τinstrument)2]0.5
(1)
The calculated relaxation times (τbile salt) for NaDC are around 2 µs, and those for NaTDC are approximately 3 µs. These values should be seen as estimates because of the errors in the determination of τobs and the approximations introduced by using eq 1. The TMA-DPH relaxation lifetimes are of the same magnitude as the reciprocals of the exit rate constants for guests bound to bile salt aggregates.22 Thus, the TMADPH relaxations could be due to the probe redistribution between the aggregate and water. Although we cannot completely discard this possibility, it is unlikely that TMADPH redistributes in microseconds because it has a substantial hydrophobic moiety and its positive charge will be strongly attracted to the negative moiety of the bile salts. The latter argument is strengthened by the fact that the dielectric constant in the environment where TMA-DPH is located is certainly below that of water, causing a very strong electrostatic attachment. Consequently, the redistribution of the probe should be slow. (38) Holzwarth, J. F.; Meyer, F.; Pickard, M.; Dunford, H. B. Biochemistry 1988, 27, 6628.
The relaxation lifetimes are shorter for NaDC than for NaTDC. However, any quantitative analysis is not warranted due to the experimental errors observed. The fact that a relaxation process was observed for TMA-DPH but not for DPH suggests that TMA-DPH is probing changes for the degree of aggregation of the secondary structures, showing that ILTJ is a powerful technique to study the aggregation dynamics for bile salts. Unfortunately, the current technology does not provide the possibility for timeresolved laser temperature-jump studies with fluorescence detection in the sub-microsecond time domain and detailed mechanistic studies will have to wait for further technological development. In conclusion, stopped flow results showed that the dynamics for secondary aggregation of NaTDC and NaDC occurs on time scales faster than milliseconds. ILTJ experiments established that the relaxation lifetimes probed with TMA-DPH occur between 2 and 4 µs. Our experiments provide the first estimate for the aggregation dynamics of bile salts and establish that the secondary aggregation process is very dynamic. The fast dynamics established for secondary aggregation contrasts the observation of guest binding to a rigid environment,12-14,19,20 which is probably related to the guest solubilization in the primary aggregates. These results further strengthen the evidence provided previously22 that bile salts have domains with different properties. Acknowledgment. C. Bohne thanks the Alexander von Humboldt Foundation for a scholarship during her study leave in Berlin and the Natural Sciences and Engineering Research Council of Canada (NSERC) for continued financial support of her research. LA9903705
