J. Phys. Chem. 1989, 93, 3321-3326
3321
Spin-Label Studies of Bile Salt Micelles Hideo Kawamura,t Yoshio Murata,*yt Takeo Yamaguchi,t Hirotsune Igimi,' Mitsuru Tanaka? Gohsuke Sugihara; and Josip P. Kratohvils Department of Chemistry, Faculty of Science, and First Department of Surgery, School of Medicine. Fukuoka University, Fukuoka City 814-01, Japan, and Institute of Colloid and Surface Science, Clarkson University, Potsdam, New York 13676 (Received: March 27, 1985)
The structure of micelles of trihydroxy bile salts (sodium cholate, sodium taurocholate) and dihydroxy bile salts (sodium deoxycholate,sodium taurodeoxycholate,sodium taurochenodeoxycholate,sodium tauroursodeoxycholate)has been investigated by the spin-label technique using stearic acid and its methyl ester nitroxide probes in borate buffer solutions of pH 7.8 and Na+ ion concentration of 60 mM at 25 OC. The immobilization degree of solubilized stearic acid spin probes was found to vary with the position of the nitroxide group in the sequence 12-doxylstearicacid >> 5-doxylstearicacid > 16-doxylstearic acid for all the bile salts investigated. This order suggests that the micelles have basically a similar shape irrespective of differences in the molecular structure of the respective bile salts. For dihydroxy bile salt micelles, ESR spectra of 12-doxylstearic acid methyl ester were characterized by the coexistence of a strongly immobilized component and a weakly immobilized one. This suggests that at least two kinds of micelles coexist in 100 mM dihydroxy bile salt solutions. On the other hand, trihydroxy bile salt micelles were found to show only the weakly immobilized component. This difference reflects differences in micellar structure between trihydroxy bile salts and dihydroxy bile salts despite their similar shape. Among the dihydroxy bile salts, the micelles of sodium tauroursodeoxycholate (NaUTDC) yield the largest value of the outer hyperfine splitting of the strongly immobilized component. This suggests that as a result of the hydrogen bonding between 7P-hydroxyl groups of adjacent monomers in a micelle, NaTUDC forms the most rigid micelles. These results may be correlated with the observation that NaTUDC micelles have less solubilizing power for cholesterol than the other dihydroxy bile salts.
Introduction
Bile salts are important biological surfactants acting as solubilizers for cholesterol and lipids. The physicochemical properties such as critical micelle concentration (cmc) and micellar aggregation numbers have been determined by a number of different methods including surface tension, light scattering, small-angle X-ray scattering, the solubilization of dyes, etc., for many bile salts.1-15 As for the micellar structure of bile salts, Small et al. have proposed a model in which the hydrophobic moieties of the monomers are oriented to the micellar interior and the hydrophilic groups are on the surface in contact with solvent.l~' Using quasi-elastic light-scattering spectroscopy, Mazer et al. determined the mean hydrodynamic radii and the apparent aggregation numbers as a function of the concentration of taurine-conjugated bile salts and concluded that the micelles polymerize in a linear fashion to form rodlike secondary micelles at high bile salt and NaCl concentration^.^.^ Rodlike micelles were also indicated in recent small-angle X-ray scattering studies.I5 Furthermore, studies of solubilization by bile salt micelles have also been extensively performed. Sugihara et al. have determined the cmc of sodium cholate (NaC) and sodium deoxycholate (NaDC) by measuring the solubilization of cholesterol and suggested that the micelles of NaDC have a larger size or a larger hydrophobic core and a more rigid structure than those of N a C 8 The first study of NaDC using the spin probe technique was done by Rabold2 by studying the solubilization of two kinds of fluorescence probes and an electron spin resonance probe. Recently, from measurements of the fluorescence spectra and the fluorescence decay, Zana and Guveli found the very interesting result that the environment of a pyrene molecule solubilized by bile salt micelles is much more apolar than that of a pyrene molecule solubilized by classical surfactants such as sodium dodecyl sulfate (NaDS).I3 For sodium chenodeoxycholate (NaCDC) and its 7P-hydroxyl epimer sodium ursodeoxycholate (NaUDC), used as an oral gallstone solubilizer, and for their taurine and glycine conjugates, Carey et al. have determined various physicochemical properties by quasi-elastic light scattering and high-performance reversedDepartment of Chemistry, Fukuoka University. 'School of Medicine, Fukuoka University. Clarkson University.
0022-3654/89/2093-3321!$0l.50/0
phase liquid chromatography, etce6 Those bile salts possessing an epimeric relation to each other differ strikingly in their capability of solubilizing cholesterol in spite of the similarity of aggregation numbers in the absence of cholesterol. The authors concluded that the micellar surface plays an important role in this capability to solubilize cholesterol. Judging from the similar therapeutic efficiency of NaCDC and NaUDC, Igimi indicated that bile salt micelles play the major role in NaCDC-rich human bile and that phosphatidylcholine bilayer vesicles are instrumental in the action of NaUDC-rich human bile in dissolving cholesterol.I2 The information concerning micellar interior construction as well as micellar size is necessary to interpret the behavior of cholesterol solubilization by bile salt micelles. At present, however, the information concerning such construction at the molecular level is lacking. We have tried to obtain some information on the micellar interior structure of bile salts by using the spin-label technique, which has already been applied to examine the interior states of biological membranes, liposomes, bile salt-lecithin mixed micelles, and synthetic surfactants such as NaDS and hexadecyltrimethylammonium bromide.16-21 (1) Small, D. M.; Penkett, S. A,; Chapman, D. Biochim. Biophys. Acta 1969, 176, 178. (2) Rabold, G. P. J . Polym. Sci., Part A-1 1969, 7, 1187. (3) Carey, M. C.; Small, D. M. A m . J . Med. 1970, 49, 590.
(4) Small, D. M. In The Bile Acids; Nair, P. P., Kritchevski, D., Eds.; Plenum Press: New York, 1971; Vol. 1, pp 249-356. (5) Mazer, N. A.; Carey, M. C.; Kwasnick, R. F.; Benedek, G. B. Biochemistry 1979, 18, 3064. (6) Carey, M. C.; Montet, J. C.; Phillips, M. C.; Armstrong, M. J.; Mazer, N. A. Biochemistry 1981, 20, 337. (7) Mazer, N. A,; Carey, M. C.; Benedek, G. B. In Solution Behauior of Surfactants-Theoretical and Applied Aspects; Mittal, K. L., Fendler, E. J., Eds., Plenum Press: New York, 1982; pp 595-610. (8) Sugihara, G.; Yamakawa, K.; Murata, Y.; Tanaka, M. J. Phys. Chem. 1982,86, 2784. (9) Roda, A,; Hofmann, A. F.; Mysels, K. J. J . Biot'. Chem. 1983, 258, 6362. (10) Kratohvil, J. P.; Hsu, W. P.; Jacobs, M. A.; Aminabhavi, T. M.; Mukunoki, Y. Colloid Polym. Sci. 1983, 261, 781. (1 1) Kratohvil, J. P. Hepatology (N.Y.) 1984, 4, 85s. (12) Igimi, H. Ph.D. Thesis, Fukuoka University, 1984. (13) Zana, R.; Guveli, G. J. Phys. Chem. 1985,89, 1687. (14) Kratohvil, J. P.; Hsu, W. P.; Kwok, D. I. Langmuir 1986, 2, 256. (1 5) Matsuoka, H.; Kratohvil, J. P.; Ise, N. J. Colloid Interface Sci. 1987, 118, 387. (16) Schreier, S.; Ernandes, J. R.; Cuccovia, I.; Chaimovich, H. J. Magn. Reson. 1978, 30, 283.
0 1989 American Chemical Society
Kawamura et al.
3322 The Journal of Physical Chemistry, Vol. 93, No. 8, 1989 COON0
NaC
R.=OH
2,o
HW' 1
co-'R3
"
R,-OH,
R,-OH
NaTt
R,-OH,
R3=H
NoTDC
R 2 = H,
R,-OH
NaTCDC
( b )
I O )
Figure 1. (a) Stuart-Briegleb molecular model of cholic acid with schematic longitudinal and cross-sectionalviews. (b) Chemical structure of bile salts employed in this work.
Experimental Section Materials. Sodium cholate (NaC), sodium deoxycholate (NaDC), sodium taurocholate (NaTC), sodium taurodeoxycholate (NaTDC) and sodium dodecyl sulfate (NaDS) were purchased from Nakarai Pure Chemicals Co. Ltd., Kyoto, Japan. Sodium taurochenodeoxycholate (NaTCDC) was obtained from Sigma Chemical Co., St. Louis, MO. Sodium Tauroursodeoxycholate (NaTUDC) was purchased from Calbiochem, San Diego, CA. After these bile salts and NaDS were recrystallized from ethanol, their purity was checked by measurements of surface tension and thin-layer chromatography. Figure 1 shows the molecular structure of bile salts employed in this work. Spin probes, 5-, 12-, and 16-doxylstearic acid (doxy1 = 4,4-dimethyl-3-oxazolidinyloxy; abbreviated as 5-NS, 12-NS, and 16-NS, respectively) and 12doxylstearic acid methyl ester (1 2-MeSL) were purchased from Sigma Chemical Co., St. Louis, MO, and used without further purifications. Other chemicals were of analytical grade. Sample Preparation. Solutions were prepared by dissolving the bile salts in a borate buffer (pH 7.8, concentration of Na' 0.06 M). The solutions were incubated there for 2 days at 37 "C. Spin probes dissolved in ethanol or in chloroform were evaporated in a sample tube to yield a thin film, onto which the bile salt solution was added. This concentration of the spin probe was kept constant at 0.1 mM ( lo4 mol dm") for all measurements except for the results shown in Figure 9 and 10. The number of solubilized spin-probe molecules per bile salt micelle was restricted to less than one for all sample solutions. Electron Spin Resonance ( E S R ) Measurement. The sample solutions were injected into capillary tubes after the incubation at 37 "C. Measurements were carried out by the use of a JEOL PE-1X or a JEOL FE-1X at 25 "C. The usual spectrometer settings were as follows: 100-kHz modulation amplitude, 1.O G; microwave power, 8 mW; scan range, 100 G; scan speed, 8 min. Results and Discussion To examine if the spin probes had been solubilized by bile salt micelles, we determined the rotational correlation time of the spin probe as a function of bile salt concentration above and below the critical micelle concentration (cmc). The expression presented by Cannon et al. was applied for the calculation of the correlation time when an isotropic ESR signal was obtained.22 T~
I
+
= 6.5 X 10-'oWo[(ho/h-l)1/2 ( h o / h + 1 ) 1 / 221
(17) Ernandes, J. R.; 1916, 16, 19.
(1)
Schreier, S . ; Chaimovich, H . Chem. Phys. Lipids
(18) Ernandes, J . R.; Chaimovich, H.; Schreier, S . Chem. Phys. Lipids
1977, 18, 304. (19) Spin Labeling, Theory and Applications; Berliner, L. J . , Ed., Aca-
demic Press: New York, 1976; Vol. 1. (20) Schwartz, H.M.; Bolton, J. R.; Borg, D. C. Biological Applications of Electron Spin Resonance; Wiley-Intersicence: New York, 1972. (21) Gajwiller, C.; von Planta, C.; Schmidt, D.; Steffon, H. Z . Naturforsch. 1977, 32c, 748.
0
20
10 Cb
(
30
nPl 1
Figure 2. Rotational correlation time (71) for 5-NS and 16-NS as a function of bile salt concentration (C,) at spin probe concentration 0.1 mM. 5-NS: (A) NaC; (0)NaDC. 16-NS: (A) NaC; ( 0 )NaDC.
2.0
1 C
0
20
10
Cb
(
30
niM 1
Figure 3. Rotational correlation time
( T , ) for 5-NS and 16-NS as a function of bile salt concentration ( c b ) at spin probe concentration 0.1 mM. 5-NS: (A)NaTC; (0)NaTDC. 16-NS: (A)NaTC; ( 0 )NaTDC.
where 71is the rotational correlation time of the probe molecule, Wois the peak-to-peak line width of the ESR midfield line (gauss), and ho, hWl,and h+l are peak-to-peak heights of the mid-, high-, and low-field lines, respectively. The parameter r1 increases abruptly at the cmc for a surfactant such as a N a D S 2 The results obtained for sodium cholate (NaC), sodium deoxycholate (NaDC), sodium taurocholate (NaTC), and sodium taurochenodeoxycholate (NaTCDC) solutions are shown in Figures 2 and 3 (spin-probe concentration at 0.1 mM). In agreement with the results for other surfactants studied by ESR,2 sharp rises in T~ were found around the cmc values already determined by a number of physicochemical means. From a detailed examination of Figures 2 and 3, it is found that dTl/dCb for dihydroxy bile salts is larger than that of trihydroxy bile compounds in the cmc region (as shown in Figure 2), although this trend is not as clear for the taurine conjugated bile salt (as shown in Figure 3). This suggests that the cooperativity of micelle formation for dihydroxy bile salts is greater than that for trihydroxy compounds. Figures 2 and 3 indicate that spin probes are well solubilized by bile salt micelles and are available for sensing the interior of micelles as well as the cmc determination. For the same probe used, the r l value above cmc for NaTDC is higher than that for NaTC, while little difference in T~ is observed below cmc. It may thus be stated that the difference in T~ reflects (22) Cannon, B.; Polnaszek, C. F.; Butler, K. W.; Erikson, L. E. G.; Smith, I . C. P. Arch. Biochem. Biophys. 1975, 167, 505.
The Journal of Physical Chemistry, Vol. 93, No. 8, 1989 3323
Spin Label Studies of Bile Salt Micelles TABLE I: Comparison of cmc Values Obtained in the Present Work and in Previous Studies at 25 OC cmc values, mM bile salts 5-NS" 16-NS' lit.b sodium cholate (NaC) 5.0 8.0 lld sodium deoxycholate (NaDC) 2.0 3.0 3d
sodium taurocholate (NaTC) sodium taurodeoxycholate (NaTDC)
5.0 3.2
7.0 3.2
sodium taurochencdeoxycholate(NaTCDC) sodium tauroursodeoxycholate(NaTUDC)
2.0
3.0
5.0
5.0
2.4' 6d 2.4d 1.6e 3d 2.2d
'Na+ concentration = 60 mM. *Na+ concentration = 150 mM. CNa+concentration = 150 mM and pH > 10. dFrom reference 9. From reference 14.
3,O -
"
v)
m 0
"2,O
-
i
P 1.0 -
O1
0
I
5 12 labelled carbon atom
I
1
16
Figure 4. Plot of 7 ,against position of a labeled carbon atom of the stearic acid spin probes in 100 mM bile salt solution: (0)NaC; ( 0 ) NaDC; (A) NaTC; (A) NaTDC; (V)NaTCDC; (V) NaTUDC; (0) NaDS.
a difference in properties of these micelles. The cmc values obtained by ESR were determined from the starting point of deviation of T~ from the linear line in the dilute solution region (see Figures 2 and 3). The cmc data determined by the spin-label technique are compared with those by other t e c h n i q ~ e s ~in* Table '~ I . Considering the variation in cmc values due to the particular techniques used, especially for unconjugated bile salts such as NaC and NaDC,4,9v'1 it follows that the values from the spin-label technique are in near agreement with those obtained by using other techniques. For minimization of possible modification of micellar structure by the addition of a spin probe, subsequent measurements were made at bile salts concentrations of 100 mM (IO-I mol d d ) , which is much higher than the cmc values. The spin-probe concentration was fixed at 0.1 mM. Since micelles increases in size with increasing bile salt concentration above the cmc, it is assumed that at concentrations 13-60 times the cmc, depending on the type of bile salt, the effect of the solubilized spin probe on the size of micelles is minimal. As is well-known, T ] can be a measure of the immobilization degree of a spin probe. Figure 4 shows the dependence of calculated from the eq 1 on the position of a labeled carbon atom of a stearic spin probe. For comparison, T~ for a NaDS micelle is also shown. Looking at the data with 5-NS, T~ for NaDS is in good agreement with those for NaDC and NaTC. This suggests that the polar portion of NaDC and NaTC micelles is similar to that of a NaDS micelle, although their structures are quite dif-
( a )
( b )
Figure 5. (a) Schematic disklike micellar model. (b) Schematic model for 12-NS solubilized by a bile salt micelle. This figure was depicted as a longitudinal cross section to emphasize the solubilized spin probe.
ferent from each other. Comparing 5-NS with 12-NS, T~ of 12-NS for a NaDS micelle is smaller than that of 5-NS, and the T, values of 12-NS for bile salts micelles are much larger than those of 5-NS. This is a significant contrast. The t , value for a NaDS solution decreases in order of 5-NS > 12-NS > 16-NS, showing that the closer to the micelle core the location of the nitroxide ring is, the larger is the degree of its mobility. On the other hand, T, of 16-NS for bile salt solutions is rather close to T, of 5-NS. T~ values of 12-NS and 16-NS for bile salt solutions are significantly larger compared with those for NaDS. Therefore, it can be inferred that the micellar structure of bile salts is markedly different from that of NaDS. This different behavior of 12-NS for bile salt solutions is observed even for biomembranes and model membranes. 12-NS incorporated by erythrocyte membrane reflects the lipid-protein interaction more strongly than the other probes. Furthermore, the nitroxide ring in the membrane is close to the rigid steroid framework of cholesterol which is incorporated into the lipid bilayer so that this probe is easily subject to the influence of c h ~ l e s t e r o l . ~Thus ~ * ~ we ~ may consider that 12-NS is most suitable for searching the interior of bile salt micelles. However, in the case of taurine conjugates of dihydroxy bile salts, it was not possible to estimate the mobility of this probe because its immobilization was too strong to apply eq 1 (see Figure 6). Figure 4 shows that the observed behavior of T~ is qualitatively similar for all the bile salts. According to Small: and Carey,3,s-6 and Kratohvill' the aggregation numbers for the bile salts used in this work range from as low as four for NaTC and NaC to several tens and more for NaTDC, NaTCDC, and NaDC. Precise values of aggregation numbers depend on the concentrations of the bile salt and the counterions and temperature. The results of this work, however, suggest that the structure probed by the nitroxide spin compounds under the conditions of the experiment was rather insensitive to the size of the aggregates and the molecular structure of bile salt monomers. A model of a spherical micelle, such as that for a NaDS micelle, is not applicable to bile salts. If such a spherical micelle were formed, the hydrophilic moiety of bile salt monomers would be incorporated into the micellar interior. The immobilization degree for the spin probes in Figure 4 cannot be explained in terms of the model of spherical micelles. To explain the experimental results, we propose a disklike micellar model. The micellar particles are assumed to consist of bile salt monomers oriented with their hydrophobic surfaces toward the micelle interior and hydrophilic surfaces toward the solvent as shown in Figure 5a. (1) It can be applied to micelles of low aggregation numbers (trihydroxy bile salts) as well as to larger micelles such as dihydroxy compounds, and (2) the disklike micelle can provide a similar environment for the solubilized spin probes irrespective of the differences of aggregation number. A stearic acid spin probe can be considered to penetrate through the micelle as shown in Figure 5b, by taking account of the fact that the molecular length of the spin probe is longer than that of bile salts. (23) Taylor, M. G.; Smith, L. C . P . Biochim. Biophys. Acfo 1980, 599, 140. (24) Yamaguchi, T.; Kuroki, S.; Tanaka, M.; Kimoto, E. J . Biochem. 1982, 92, 673.
3324 The Journal of Physical Chemistry, Vol. 93, No. 8, 1989
Kawamura et al.
10 G
Figure 6. ESR spectra of 12-NS in 100 mM bile salt solutions: (a) NaTC; (b) NaTDC; (c) NaTCDC; (d) NaTUDC. Solid and broken arrows indicate the strongly immobilized component and the component resulting from a bulk solution, respectively.
Figure 5b depicts the longitudinal cross section, emphasizing the location and orientation of the solubilized spin probe. Based on this model, 12-nitroxide stearic acid (12-NS) is the most strongly immobilized due to steric hindrance between the nitroxide ring of the probe and the hydrophobic moiety of bile salt molecules. On the other hand, the nitroxide ring of 5-doxylstearic acid (5-NS) is located closer to the polar head group and is only partly in contact with the hydrophobic moiety of bile salt. The ring of 16-doxylstearic acid (16-NS) protrudes to the outside of the micelle. 5-NS may be, therefore, somewhat more strongly immobilized than 16-NS. Thus, the disklike model assumed here for the common structure of the various bile salt micelles can give a reasonable explanation of the patterns in Figure 4. The fact that the counterion dissociation of bile salt micelles is much larger than that of NaDS m i ~ e l l ecan ' ~ ~also ~ ~be explained by the same model. The polar head-group charge density on a micellar surface is lower, and, consequently, the degree of counterion binding is smaller for the disklike micelles than in the case of spherical micelles. Similar molecular orientation as in our model was observed in the crystals of ursodeoxycholic acid by X-ray diffraction method.26 However, this model does not exclude the possibility of other models, such as rods, to give a reasonable explanation for the ESR results. Under the condition of our experiments (0.06 M Na+) the aggregation number of even dihydroxy bile salts are too small to form rods. As shown in Figure 6, there are differences in the ESR spectra of 12-NS solubilized in the micelles of dihydroxy and trihydroxy bile salts. A strongly immobilized component (represented by solid arrows in Figure 6) was observed for micelles of dihydroxy bile (25) Murata, Y . ;Okawauchi, M.; Kawamura, H.; Sugihara, G.; Tanaka,
M. Surfacrants in Solution;Mittal, K. L., Bothorel, P., Eds.; Plenum Press: New York, 1987; Vol. 5, pp 861-872. (26) Higuchi, T.; Kamitori, S.;Hirotsu, K.; Takeda, H. Yakugaku Zasshi ( J . Pharm. SOC.Jpn.) 1985, 105, 1 1 15.
Spin Label Studies of Bile Salt Micelles
The Journal of Physical Chemistry, Vol. 93, No. 8, 1989 3325
1.0 9 0
O
Q
n
0
0 3
? 4
0.5
0( -
1.5
1.0
0.5
2,0
(mM)
CD
Figure 9. Probe concentration dependence of As/Aw (0) and 2T- (A for a weakly immobilized component, A for a strongly immobilized component) of a NaTUDC micelle with C, = 100 mM at 20 O C .
t f
I1 II
v
3
d
s 0.5
-
Figure 8. ESR spectra of 12-MeSL in 100 mM bile salt solutions: (a) NaTC; (b) NaTDC; (c) NaTCDC; (d) NaTUDC. Arrows indicate the strongly immobilized component. of ESR the continuous distribution of aggregates may be divided into two groups. Furthermore the exchange (or diffusion) rate of a spin probe from one kind of micelle to another kind should be slower than that occurring between different sites in the same micelle. If we adopt interpretation 2, the results of Figures 7 and 8 can be explained as follows. Trihydroxy bile salts such as N a C and NaTC, which form small micelle^>^^'^ yield only the weakly immobilized component (derived from isotropic motion of a spin probe). On the contrary, dihydroxy compounds (NaDC, NaTDC, NaTCDC, and NaTUDC), which form larger micelles than the trihydroxy bile salts, give not only the weakly immobilized component but also the strongly immobilized one. In this sense the strongly immobilized component is considered to result from solubilization of the spin probe by the larger micelles. To discuss the micellar state of each bile salt more quantitively, we use two parameters obtainable from the ESR spectra. The first parameter is the ratio of the amplitudes of the strongly immobilized components to the weakly immobilized ones, A,/ Aw.29-31This parameter represents the molar ratio of the spin probe in these two states. The degree of immobilization of the spin probe in the micelles was assessed by the values of the maximum hyperfine splitting, 2T,,,,,. Two experiments were undertaken with a NaTUDC micelle at 20 OC. The first experiment was designed to measure how ASIA, varies for a fixed bile salt concentration (100 mM) as the probe concentration is varied from 0.1 to 1.5 m M (this should detect if the probe is biasing the distribution of two sites). In this condition there are (29) Yamaguchi, T.; Koga, M.; Takehara, H.; Kimoto, E. FEBS Letr. 1982, 141, 53.
(30) Yamaguchi, T.; Koga, M.; Fujita, Y.; Kimoto, E. J . Biochem. 1982, 91, 1299. (31) Yamaguchi, T.; Takehara, H.: Shibata, E.; Kimoto, E. Biochim. Biophys. Acta 1983, 736, 150.
0,
0
100
50
Cb
(mM)
Figure 10. Bile salt concentration dependence of As/Aw (0)and 2T(A for a weakly immobilized component, A for a strongly immobilized component) of a NaTUDC micelle with C,/C, = 0.01 at 20 OC. many vacant (i.e., those lacking spin probes) micelles. If more spin probes were to be solubilized in one site than the other, ASIA, should change with the probe concentration. The results are shown in Figure 9; A,/Aw did not change with the probe concentration. This result indicates that the spin probe is equally distributed in strongly immobilized sites and weakly immobilized sites. ASIA, is therefore equivalent to the molar ratio of the strongly immobilized sites to weakly immobilized sites. The second experiment was carried out to see how ASIA, varies as a function of bile salt concentrations as the probe concentration is varied with a fixed ratio of spin probe/bile salt (0.01). A strongly immobilized component could not be observed up to a bile salt concentration around 30 mM. In other words, A,/Aw was zero up to this concentration as shown in Figure 10. Thus the strongly immobilized sites do not exist up to about 30 mM. Beyond this concentration their fraction increases and finally tends to saturate. Furthermore, 2Tm,, for the weakly immobilized component is constant irrespective of the concentration of NaTUDC. This means that the state of the wealky immobilized sites is not influenced by the existence of the strongly immobilized sites. These results exclude the possibility of the existence of two kinds of sites located in one micelle. Consequently, we consider that at least two kinds of micelles coexist in a 100 mM dihydroxy bile salt solution. The results shown in Figures 9 and 10 were obtained at 20 O C by using unpurified NaTUDC, although the results in general are similar
3326 The Journal of Physical Chemistry, Vol. 93, No. 8, 1989
A
A
A
0
0
A
A
0
A
A n
A
A
Figure 11. ASIA, ratio and 2Tmxvalue for various bile salt determined by using 12-MeSL: (0)ASIA,, (A)2Tmxof the strongly immobilized component; (A) 2T,,,,, of the weakly immobilized component.
to those for the purified sample. The results shown in Figure 7 and 8 for the two parameters are summarized for all the bile salts in Figure 1 1. In the case of micelles of trihydroxy bile salts, the strongly immobilized component was not observed, and thus the A,/Aw values for NaC and NaTC are zero. A,/Aw values (shown by circles in Figure 11) for dihydroxy bile salts are large, corresponding to a relatively high proportion of larger micelles. On the other hand, the values of the 2Tm (shown by open triangles) for the weakly immobilized component of trihydroxy bile salts are almost the same as those of dihydroxy bile salts (as shown in Figure 11). Considering the low average aggregation numbers of trihydroxy bile salt micelles, the weakly immobilized component observed for dihydroxy bile salts suggests the presence of the smaller micelles having the same order of aggregation number as the trihydroxy bile salt micelles in solution. According to Mazer and care^,^^^ the cmc's and the aggregation numbers at 100 mM of NaTDC, NaTCDC, and NaTUDC micelles are similar. However, we found higher A,/Aw and 2Tm,, values of the strongly immobilized component (shown by closed triangles) for NaTUDC micelles than for other bile salts. The largest value of 2Tm,, for NaTUDC suggests that the large micelles of this bile salt provide the most rigid environment for the 12-MeSL probe. The special nature of NaTUDC micelles was also observed by fluorescence polarization for the 12-(9anthroy1oxy)stearic acid and 2Tm, for the 3-doxylcholestane spin probe.32 It appears that the results obtained are independent of (32) Kawamura, H.; Manabe, M.; Narikiyo, T.; Igimi, H.; Murata, Y . ; Sugihara, G.; Tanaka, M. J . Solution Chem. 1987, Id, 433.
Kawamura et al. the structure of spin probes used in the ESR experiments. The rigidity of NaTUDC micelles may results from the hydrogen bonding between 7/3-hydroxyl groups of adjacent molecules in a micelle. The hydrogen bonding was observed in the crystal of ursodeoxycholic acid by X-ray diffraction.26 Igimi et al. have observed that the amount of cholesterol solubilized by NaTUDC is about 10 times smaller than that by NaTCDC, an epimer of NaTUDC.33-35 This result cannot be explained in terms of similarity of their aggregation number^.^^^ As discussed above, NaTUDC micellar solutions seem to contain the largest fraction of larger micelles of the most rigid structure. Since cholesterol molecules must penetrate into the micellar interior to be solubilized and will break the intermolecular bile salt hydrogen bonding, the NaTUDC micelles with cholesterol would be more unstable than the pure NaTUDC micelles. Thus the information on the interior structure of micelles as well as the micellar size must be considered to explain the differences in the amount of cholesterol solubilized by various bile salts. On the basis of the results obtained by using nitroxide stearic acid spin probes, we conclude that a common micellar structure, probably disklike, exists for all bile salt micelles under condition of our experiments. Furthermore, the trihydroxy bile salts form only one type of micelle of comparatively low aggregation numbers and loose structure. For dihydroxy bile salts two distinguishable types o f f micelles coexist in solutions at 100 mM, total concentration of Na+ = 60 mM and pH 7.8: micelles of larger aggregation number and more rigid structure than that of trihydroxy compounds and smaller micelles similar in properties to the trihydroxy bile salt micelles. The special properties of NaTUDC among the dihydroxy bile salts results from the hydrogen bonding between 7P-hydroxyl groups of adjacent molecules in a micelle. Acknowledgment. We thank Professor Keishiro Shirahama and Dr. Noboru Takisawa, Saga University, for the use of ESR spectroscopy. We are also grateful to Tadashi Narikiyo, who contributed his technical assistance to the accomplishment of the experimental program. The experiments of Figures 9 and 10 were suggested by a reviewer of this paper. We express our deepest thanks to the reviewers for the fruitful suggestions and the refinement of the English of this paper. We thank John W. Scott for checking the English of this paper. The present work was in part supported by the Central Research Institute of Fukuoka University and by Grant in Aid for Scientific Research No. 6047001 1 from the Ministry of Education, Science and Culture. Registry No. NaC, 361-09-1; NaTC, 145-42-6; NaDC, 302-95-4; NaTDC, 1180-95-6;NaTCDC, 6009-98-9; NaTUDC, 35807-85-3. (33) Igimi, H.; Carey, M. C. J . Lipid Res. 1980, 21, 72. (34) Igimi, H.; Carey, M. C. J . Lipid Res. 1981, 22, 254. (35) Salvioll, G.; Igimi, H.; Carey, M. C. J . Lipid Res. 1983, 24, 701.
