Behaviors of Sodium Taurocholate and Sodium Taurodeoxycholate in

Mean aggregation numbers of mixed micelles for both systems abruptly .... NMR study of bile salts' molecules in binary mixed micelles of bile salt and...
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Langmuir 1996, 12, 5536-5540

Behaviors of Sodium Taurocholate and Sodium Taurodeoxycholate in Binary Mixed Micelles of Bile Salt and Nonionic Surfactant K. Suzuki,† T. Hasegawa,† Y. Takamura,† K. Takahashi,† H. Asano,† and M. Ueno*,†,‡ Department of Applied Chemistry, Faculty of Science, Science University of Tokyo, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162, Japan, and Institute of Science and Colloid Chemistry, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162, Japan Received February 2, 1996. In Final Form: June 3, 1996X Behavior of sodium taurocholate (NaTC) and sodium deoxycholate (NaTDC) in binary mixed micelles consisting of bile salt and octaethylene glycol mono-n-decyl ether (C10E8) has been studied on the basis of micellar compositions, polarities of interior of intramicelles, mean aggregation numbers, and 1H-NMR measurements, respectively. Micellar compositions for both the NaTC-C10E8 system and NaTDC-C10E8 system tended to change from C10E8-rich micelles to bile-salt-rich micelles with the mole fraction of bile salts from the results of both theoretical calculations using critical micellar concentration (cmc) and the micellar polarity, and also a microenvironment of intramicelles for the NaTDC-C10E8 system was more hydrophobic than that for the NaTC-C10E8 system. Mean aggregation numbers of mixed micelles for both systems abruptly decreased with the increase of mole fraction of bile salts in the range of the low mole fraction, but mean aggregation numbers for the NaTDC-C10E8 system were larger than those for NaTCC10E8 system. Furthermore, from the results of 1H-NMR measurements, the methyl group protons at the 18, 19, and 21 positions in NaTC molecules were found to have a slight freedom with an increase of mole fraction of NaTC, but those of NaTDC were found to be restricted with an increase of mole fraction of NaTDC.

Introduction Bile salt molecules form rigid back-to-back micelles in an aqueous solution owing to bulky hydrophobic parts with strong affinity among steriod rings in the molecules,1-7 while a nonionic surfactant has typical colloidal properties and forms loosely packed micelles; therefore in our laboratory, behaviors of various bile salts in solution which is mixed together with this nonionic surfactant have been investigated by using a surface tension, a theoretical analysis of micellar compositions, a steady state fluorescence of pyrene, and a quenching method.8-11 From the results of these measurements, a miscibility between the bile salt and the nonionic surfactant in the mixed micelles is found to be effected by the number of hydroxyl groups in the bile salt molecules and by the difference in terminal group. And also we have reported that the micellar shape or structure abruptly may change at some * To whom correspondence should be addressed at Department of Applied Chemistry, Faculty of Science, Science University of Tokyo. † Science University of Tokyo. ‡ Institute of Science and Colloid Chemistry. X Abstract published in Advance ACS Abstracts, October 15, 1996. (1) Small, D. M. In The Bile Acids; Nair, P. P., Kritchevsky, D., Eds.; Plenum Press: New York, 1971; Chapter 8, p 249. (2) Mazer, N. A.; Benedek, G. B.; Carey, M. C. Biochemistry 1980, 19, 601. (3) Carey, M. C.; Montet, J. C.; Phillips, M. C.; Armstrong, M. J.; Mazer, N. A. Biochemistry 1981, 20, 3637. (4) Kratohvil, J. P.; Hsu, W. P.; Jacobs, M. A.; Aminabhavi, T. M.; Mukunoki, Y. Colloid Polym. Sci. 1983, 261, 781. (5) Roda, A.; Hofmann, A. F.; Mysels, K. J. J. Biol. Chem. 1983, 258, 6362. (6) Roe, J. M.; Barry, B. W. J. Colloid Interface Sci. 1985, 107 (2), 398. (7) Hofmann, A. F.; Mysels, K. J. Colloid Surf. 1988, 30, 145. (8) Asano, H.; Aki, A.; Ueno, M. Colloid Polym. Sci. 1989, 267, 935. (9) Asano, H.; Murohashi, A.; Ueno, M. J. Am. Oil Chem. Soc. 1990, 67, 1002. (10) Asano, H.; Yamazaki, M.; Fujima, A.; Ueno, M. J. Jpn. Oil Chem. Soc. 1991, 40 (4), 293. (11) Asano, H.; Izumi, C.; Sano, Y.; Tabata, Y.; Ueno, M. J. Am. Oil Chem. Soc. 1993, 70 (7), 693.

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mixed ratios of bile salts, and the break points appear on the measured points. This suggests that conformations of bile salt molecules in the mixed micelles at the break point are abruptly changed by the differences in the orientation of hydroxyl groups and in the conjugation of the terminal group. On the other hand, nuclear magnetic resonance (NMR) spectroscopy has become the most important technique for the study inorganic and organic molecules in solutions, and also it can provide a wealth of information on molecular structure and physical properties of a compound.12-14 Accordingly the positions of bile salt molecules located among the nonionic surfactant molecules in the mixed micelle can be conformed by measuring the change of the relaxation time for bile salts in the mixed micelles with the increase of mole fraction of bile salts. In this paper, the micellar properties for two combination systems, where one consists of sodium taurocholate (NaTC) and octaethylene glycol mono-n-decyl ether (C10E8) and another system is sodium taurodeoxycholate (NaTDC) and C10E8, have been studied on the basis of critical micellar concentration (cmc), a polarity of interior of intramicelles, a mean aggregation number of micelles, and 1H-NMR measurements, respectively. And also, differences in the mixed micellar properties and in behaviors of bile salt molecules in the mixed micelles by the number of hydroxyl groups will be estimated. Experimental Section Materials. NaTC and NaTDC were supplied from Sigma Chemical. NaTC was purified by recrystallization with n-hexane as precipitant several times from a mixture of ethanol and (12) Barnes, S.; Kirk, D. N. In The Bile Acids; Setchell, K. D. R., Kritchevsky, D., Nair, P. P., Eds.; Plenum Press: New York, 1988; Volume 4, Chapter 3, p 65. (13) Kemp, W. In NMR in ChemistrysA Multinuclear Introduction; Macmillan Publishers, Ltd.: London, 1986. (14) Esposito, G.; Zanobi, A.; Giglio, E.; Pavel, N. V.; Champbell, I. D. J. Phys. Chem. 1987, 91, 83.

© 1996 American Chemical Society

Behavior of NaTC and NaTDC in Mixed Micelles

Langmuir, Vol. 12, No. 23, 1996 5537

methanol as dissolving solvent, followed by Soxhlet extraction with acetone for 72 h. For NaTDC, ethanol alone was used as the dissolving solvent for the recrystallization, and n-hexane was used as the solvent for the extraction. C10E8 (Nikko Chemicals) was purified by gel chromatography (Wakogel C-200, Wako Chemicals) with equivalent amounts of acetone and n-hexane as solvents. The single spot obtained from thin-layer chromatography indicates that the product is pure. Pyrene (Tokyo Kasei Kogyo) used as a probe and dodecylpyridinium chloride (Tokyo Kasei Kogyo) used as a quencher were purified by methods described in our previous papers.8-11 Aqueous solutions of surfactants were prepared with Tris/ HCl buffer solution adjusted at pH 9.00 ( 0.05 with ionic strength of 0.026 except for NMR measurements. Methods. Aqueous solutions for NaTC, NaTDC, and C10E8 alone were prepared and also for aqueous binary mixtures of bile salts and C10E8 with each mole fraction of 0.25, 0.50, 0.75, and 0.90, respectively. Surface tensions for obtaining cmc values of the mixed solutions, fluorescence emission spectra of pyrene in the mixed micelles, and mean aggregation numbers of the micelles were measured according to our previous works at 25.00 ( 0.05 °C. 1H-NMR measurements were carried out in deuterium oxide (D2O, 99.9 atom % D, Aldrich) solvent at about 25 °C and 4 µL of sodium deuterium (NaOD) solution (with about 40% NaOD in D2O, Merck) was always added to 6 mL of the D2O solvent in order to prevent the hydrolysis of bile salts. The mixed solutions of bile salts and C10E8 with constant total concentrations of 100 mM were prepared for bile salt mole fractions of 0.00, 0.25, 0.50, 0.75, 0.90, and 1.00, respectively, in an atmosphere of nitrogen. 1H-NMR spectra were determined at 400 MHz with JEOL JNMEX400, and also some spectra were recorded in the chemical shift range from 0 to 10 ppm for 1H-NMR. 1H chemical shifts were referred to internal TSP (sodium 2,2,3,3-tetradeuterio-3(trimethylsilyl)propionate, Aldrich) assigned a zero value.14 The 1H longitudinal relaxation times (T ) were measured by using 1 the inversion recovery method. The 1H transverse relaxation times (T2) were measured by using the Carr-Purcell-MeiboomBill sequence. In the series of 1H-NMR measurements, the signal of 1H of HOD was reduced by using a homogate decoupling method. If the relaxation of 1H nuclei arises predominantly from dipoledipole interaction, T1 and T2 values are represented by

( )[ ( )[

2γ4I(I + 1) h 1 ) T1 2π 5r6 γ4I(I + 1) h 1 ) T2 2π 5r6

2

2

3τc +

]

4τc

τc

1 + ω2τc2

+

5τc 2

(1)

1 + 4ω2τc2

]

2τc

1 + ω τc

2

+

1 + 4ω2τc2

Figure 1. Dotted curve of theoretical mole fraction of bile salts M in one mixed micelle of an ideal state, Xideal , vs apparent mole fraction of bile salts in the solution, Xbile. Plots of theoretical mole fraction of bile salts in one micelle calculated by excess M , vs Xbile: (a) NaTC-C10E8 thermodynamic quantities, Xexcess system; (b) NaTDC-C10E8 system. M Table 1. Values of Cmc, Xexcess , I1/I3, and Mean Aggregation Number for Each Bile Salt-C10E8 System

mole fraction of bile salt

(2)

where γ is the magnetogyric ratio, I is the nuclear spin quantum number, r is the internuclear distance, h is Planck’s constant, ω is the Larmor frequency, and τc is the molecular mobility.15 The T1/T2 ratio does not contain, γ, I, and r, only τc and ω. The τc value can be calculated from the ratio T1/T2 because the ω value is still known.

Results and Discussion Composition in the Mixed Micelles. Each cmc for NaTC, NaTDC, and C10E8 alone and also for these binary mixtures with various mole fractions was obtained from each curve of the surface tension vs logarithm of apparent concentration of surfactant, and then these cmc’s were used for the theoretical calculation of micellar compositions in an ideal state16 and in a nonideal state by the excess thermodynamic quantities of Motomura.17 Figure 1 shows each plot of the mole fraction of bile salts in the mixed micelles which is calculated by (15) Kazuyuki, A.; Toshiaki, I. Farra Becker: Pulse and Fourier Transform NMR; Yoshioka Press: Kyoto, 1976. (16) Rubingh, D. N. In Solution Chemistry of Surfactants; Mittal, K. L., Ed.; Plenum: New York, 1980; Vol. 1. (17) Motomura, K.; Yamanaka, M.; Aratono, M. Colloid Polm. Sci. 1984, 262, 948.

item

system

NaTC-C10E8 NaTDC-C10E8 NaTC-C10E8 NaTDC-C10E8 NaTC-C10E8 I1/I3 (100 mM) NaTDC-C10E8 mean aggregation NaTC-C10E8 no. (50 mM) NaTDC-C10E8 cmc (mM) M Xexcess

0.00 0.25 0.50 0.75 0.90 1.00 1.00 1.00 0.00 0.00 1.05 1.05 69 69

1.17 1.09 0.06 0.10 0.99 0.98 33 40

1.53 1.28 0.17 0.21 0.94 0.90 27 30

2.04 1.57 0.37 0.43 0.89 0.79 22 23

2.72 2.00 0.66 0.72 0.83 0.69 18 19

4.33 2.98 1.00 1.00 0.78 0.65 15 16

M ) as a function of the apparent Motomura’s theory (Xexcess mole fraction of bile salts in the solution (Xbile) for the NaTC-C10E8 system in (a) and the NaTDC-C10E8 system in (b), respectively, and each dotted curve shows the calculated mole fraction of bile salt in the mixed micelles M ) for the each system. The cmc’s of the ideal state (Xideal M and these Xexcess values are listed in Table 1. M Each Xexcess value for both systems deviated negatively M from each dotted curve of Xideal . These negative deviations suggest that the nonionic rich micelles are formed in these binary mixed solutions and both systems are nonideal mixtures. However, the extents of deviations of M curves for both systems are smaller than those the Xexcess for sodium cholate (NaC)-C10E8 and NaGC-C10E8 systems in our previous works.9 These reasons are that the

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a

Figure 3. Plots of I1/I3 values vs apparent mole fraction of bile salts.

b

Figure 2. Curves of I1/I3 values vs total surfactant concentration with various mole fraction of bile salts: (a) NaTC-C10E8 system; (b) NaTDC-C10E8 system.

miscibilities of NaTC and NaTDC molecules in the mixed micelles are similar to those of C10E8 molecules due to the hydrated water around the taurine group or the polyethylene region. Polarities and Mean Aggregation Number. Polarities of an intramicelle can be estimated from a measurement of a ratio of the first and third vibronic peak, I1/I3, in a monomeric fluorescence emission spectrum of pyrene.18-20 This I1/I3 value is low for a nonpolar microenvironment around the solubilized pyrene and high for a polar microenvironment. On the other hand, a mean aggregation number of one micelle can be examined from a luminescence quenching method using the pyrene as a probe and dodecylpyridinium chloride as a quencher.8-11,21,22 Figure 2 shows dependencies of I1/I3 values on the total surfactant concentrations above cmc’s for the NaTC-C10E8 system in (a) and for the NaTDC-C10E8 system in (b), and Figure 3 shows plots of I1/I3 values for various mole fractions of bile salts at constant total surfactant concentration of 100 mM. These I1/I3 values at 100 mM are listed in Table 1. In Figure 2, the I1/I3 values of each mixed system show the tendency approaching to that of (18) Kalyanasundaram, K.; Thomas, J. K. J. Am. Chem. Soc. 1977, 99 (7), 2039. (19) Thomas, J. K. Chem. Rev. 1980, 80 (4), 283. (20) Zana, R.; Guveli, D. J. Phys. Chem. 1985, 89 (9), 1687. (21) Turro, N. J.; Yekta, A. J. Am. Chem. Soc. 1978 100 (18), 5951. (22) Turro, N. J.; Gratzel, M.; Braun, A. M. Angew. Chem., Int. Ed. Engl. 1980, 19, 675.

C10E8 near the cmc. This suggests that the microenvironments of the intramicelles for each mixed system are nearly the same as that for the C10E8 micelle near the cmc, in the agreement with the tendencies of the micellar composition in Figure 1. Furthermore, the I1/I3 curves with mole fractions of 0.75 and 0.90 of bile salt in a NaTDC-C10E8 system become obviously lower than those in a NaTC-C10E8 system, and also, this tendency is more clear as shown in Figure 3. In other words the I1/I3 values of mole fraction of 0.25, 0.50, 0.75, 0.90, and 1.00 of NaTDC are lower than those of NaTC. These facts indicate that NaTDC molecules tend to form the nonpolar micelles with C10E8 molecules in aqueous solutions and suggest that the intramicelles of NaTDC alone and the NaTDC-C10E8 system solubilizing the pyrene molecules are in more hydrophobic condition than those of NaTC alone and the NaTC-C10E8 system owing to the deoxidation at the 7-position of the NaTDC molecule. However, there is another interesting feature in Figure 3. It is the point that the I1/I3 values have showed break points at the mole fraction of 0.72 for the NaTC-C10E8 system and 0.54 for the NaTDC-C10E8 system in the case of the mixed NaTC-C10E8 or NaTDC-C10E8 system, respectively. These break points in each curve corresponding to a transition point suggest that these mixed micelles change from the C10E8 rich to the bile salt rich ones and micellar shapes may change from spherical to lamellar micelles below and above each break point. On the other hand, abrupt decreases of the mean aggregation number at 50 mM of total surfactant concentration for both mixed systems were observed in the range of the low mole fraction of bile salts in analogy with the previous results of the mean aggregation number, as shown in Figure 4. These values are listed in Table 1. These decreases may be attributed to the difference in molecular cohesion and in bulkiness between bile salt and C10E8. Furthermore, values of the mean aggregation number for the NaTDC-C10E8 system were larger than those of the NaTC-C10E8 system in all ranges of the mole fractions of bile salts. This may be due to the high hydrophobicity of NaTDC molecules as shown in the results of I1/I3 measurements. Behaviors of Me18, Me19, and Me21 of Bile Salt Molecules into the Mixed Micelles. When bile salt alone was mixed with C10E8 in D2O solutions, the resolved signals in the 1H-NMR spectrum of bile salt alone which were assigned to methyl group protons of 18, 19, and 21 position for the molecular structure of bile salt (hereafter referred to as Me18, Me19, and Me21, respectively) did

Behavior of NaTC and NaTDC in Mixed Micelles

Langmuir, Vol. 12, No. 23, 1996 5539 Table 2. Values of 1H Longitudinal Relaxation Timesa for Each Bile Salt-C10E8 System position of bile salt Me18 Me19 Me21 a

system NaTC-C10E8 NaTDC-C10E8 NaTC-C10E8 NaTDC-C10E8 NaTC-C10E8 NaTDC-C10E8

mole fraction of bile salt in the solution 0.25 0.50 0.75 0.90 1.00 521 473 808 999 415 386

519 527 732 593 425 445

514 561 535 574 428 478

491 592 491 555 422 502

461 610 461 584 419 544

Values in milliseconds.

a

Figure 4. Plots of mean aggregation number vs apparent mole fraction of bile salts.

a

b

b

Figure 6. Plots of 1H transverse relaxation times vs apparent mole fraction of bile salts: (a) NaTC-C10E8 system; (b) NaTDCC10E8 system.

Figure 5. Plots of 1H longitudinal relaxation times vs apparent mole fraction of bile salts: (a) NaTC-C10E8 system; (b) NaTDCC10E8 system.

not overlap with the spectrum of C10E8. As values of correlation time (τc) of Me18, Me19, and Me21, which were calculated by using 1H longitudinal relaxation time (T1) and 1H transverse relaxation times (T2), were changed with the addition of C10E8, these τc values were used to derive information about interactions between bile salt and C10E8 molecules and conformations for bile salt molecules in the mixed micelles. Figure 5 shows plots of 1H longitudinal relaxation times (T1) of Me18, Me19, and Me21, respectively, for each mixed

system at constant total surfactant concentration of 100 mM as a function of the mole fraction of bile salts for the NaTC-C10E8 system in (a) and for the NaTDC-C10E8 system in (b) and these values are listed in Table 2. The T1 values of Me18 and Me21 for the NaTC-C10E8 system were almost unchanged, but those for the NaTDC-C10E8 system became slightly smaller with the increase of mole fraction of C10E8. However, those of Me19 for both NaTCC10E8 and NaTDC-C10E8 systems became larger, and especially, the NaTDC-C10E8 system at the low mole fraction of NaTDC changed abruptly. Figure 6 shows plots of 1H transverse relaxation times (T2) of Me18, Me19, and Me21, respectively, for each mixed system at constant total surfactant concentration of 100 mM as a function of the mole fraction of bile salts for the NaTC-C10E8 system in (a) and for the NaTDC-C10E8 system in (b), and these values are listed in Table 3. The

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Table 3. Values of 1H Transverse Relaxation Timesa for Each Bile Salt-C10E8 System position of bile salt Me18 Me19 Me21 a

system NaTC-C10E8 NaTDC-C10E8 NaTC-C10E8 NaTDC-C10E8 NaTC-C10E8 NaTDC-C10E8

mole fraction of bile salt in the solution 0.25 0.50 0.75 0.90 1.00 53.2 260 149 127 46.3 39.3

63.8 183 119 80.0 52.2 47.8

80.5 60.8 103 76.1 65.0 53.1

91.8 54.3 104 60.3 69.6 48.6

103 44.4 99.6 43.8 79.5 39.8

Values in milliseconds.

a

b

Table 4. Values of Correlation Timesa for Each Bile Salt-C10E8 System position of bile salt Me18 Me19 Me21 a

mole fraction of bile salt in the solution 0.25 0.50 0.75 0.90 1.00

system NaTC-C10E8 NaTDC-C10E8 NaTC-C10E8 NaTDC-C10E8 NaTC-C10E8 NaTDC-C10E8

1.30 0.30 0.88 1.13 1.23 1.30

1.16 0.52 0.96 1.09 1.16 1.30

0.99 1.25 0.86 1.10 1.00 1.23

0.87 1.39 0.79 1.25 0.95 1.34

0.76 1.59 0.78 1.56 0.86 1.58

Values in nanoseconds.

of Me18 and Me21 for the NaTC-C10E8 system became slightly smaller with the increase of mole fraction of NaTC, and those of Me19 were almost unchanged. The slight lost strength of the interaction between each of Me18 and Me21 of NaTC and the hydrophobic part of C10E8 can be estimated from the fact that the mobility of Me18 and Me21 becomes slightly free by location near hydrophilic groups of C10E8 in mixed micelles and also that Me19 is located near hydrophilic groups of C10E8. On the other hand, the τc values of Me18, Me19, and Me21 for the NaTDC-C10E8 system became larger from 0.75 of bile NaTDC’s mole fraction to 1.00 with the increase of mole fraction of NaTDC, and only those of Me18 became abruptly larger from 0.50 of NaTDC’s mole fraction to 0.75. The interaction between only Me18 of NaTDC and the hydrophobic part of C10E8 became abruptly stronger from 0.50 of the NaTDC’s mole fraction to 0.75. The eager strength of the interaction between each Me18, Me19, and Me21 of NaTDC and the hydrophobic part of C10E8 from 0.50 of NaTDC’s mole fraction to 0.75 can be estimated from the fact that the mobility of Me18, Me19, and Me21 is restricted by location in mixed micelles indicates that all Me18, Me19, and Me21 are located at hydrophobic groups of the C10E8 molecule. And also the abrupt change of Me18 τc values around 0.50 of NaTDC’s mole fraction corresponding to a transition point suggest that these mixed micelles change from C10E8 rich to bile salt rich. Conclusions

Figure 7. Plots of correlation times vs apparent mole fraction of bile salts: (a) NaTC-C10E8 system; (b) NaTDC-C10E8 system.

T2 values of Me18 and Me21 for the NaTC-C10E8 system became slightly smaller, but those of Me19 became larger with the increase of mole fraction of C10E8. Those of Me18 and Me19 for the NaTDC-C10E8 system became larger with the increase of mole fraction of C10E8, and especially, Me18 changed abruptly at low mole fraction of C10E8, and those of Me21 were almost unchanged. Figure 7 shows plots of correlation time (τc) of Me18, Me19, and Me21, respectively, for each mixed system at constant total surfactant concentration of 100 mM as a function of the mole fraction of bile salts for the NaTCC10E8 system in (a) and for the NaTDC-C10E8 system in (b), and these values are listed in Table 4. The τc values

From these results, properties of binary mixed micelles of the NaTC-C10E8 system and the NaTDC-C10E8 system have been concluded as follows: (1) Both NaTC-C10E8 and NaTDC-C10E8 systems tend to form the nonionic rich micelles in the binary mixed solutions. (2) The I1/I3 values for NaTDC alone and NaTDC-C10E8 mixtures is lower than those for NaTC alone and NaTC-C10E8. This may be due to the deoxidation at the 7 position of bile salt molecules and suggests that NaTDC molecules form more hydrophobic and rigid micelles than NaTC molecules. (3) Mean aggregation numbers of both NaTC-C10E8 and NaTDC-C10E8 systems tend to decrease abruptly in the range of low mole fraction of bile salts, and those of the NaTDC-C10E8 system are larger than those of the NaTCC10E8 system owing to high hydrophobicity of NaTDC molecules. (4) From results of 1H-NMR measurements, the behaviors of NaTC and NaTDC molecules in mixed micelles were found to be different in orientation due to the number of hydroxyl groups in one molecule of bile salt. LA960102W