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Langmuir 1999, 15, 2996-2998
Structure and Composition of Sodium Taurocholate Micellar Aggregates E. Bottari,† A. A. D’Archivio,‡ M. R. Festa,† L. Galantini,‡ and E. Giglio*,† Dipartimento di Chimica, Universita` di Roma “La Sapienza”, P.le A. Moro 5, 00185 Roma, Italy, and Dipartimento di Chimica, Ingegneria Chimica e Materiali, Universita` di L’Aquila, 67010 Coppito Due (L’Aquila), Italy Received August 3, 1998. In Final Form: February 6, 1999
Introduction Bile salts are very important natural detergents and give rise to interesting processes of aggregation and solubilization.1,2 They form micellar aggregates capable of solubilizing important biological compounds as, for example, cholesterol, bilirubin-IXR, phospholipids, and fatty acids. The knowledge of a bile salt micellar structure is crucial for understanding its physicochemical and biological properties. Common bile salts in man possess hydroxyl functions at positions 3R and 7R or 3R and 12R (dihydroxy salts) or 3R, 7R, and 12R (trihydroxy salts). Although some aggregation models valid for all the bile salts were proposed, the structure and the composition of their micellar aggregates remain an open question. Small assumed that few bile salt molecules aggregate by means of hydrophobic interactions (primary micelles). The primary micelles lead to the formation of larger secondary micelles via polymerization in a linear fashion by means of hydrogen bonds at higher concentrations.3 Oakenfull and Fisher proposed the initial formation of dimers through hydrogen bonding, even though the hydrogenbonded dimer was criticized by Zana.4 Kawamura et al. presented a disklike model with bile salt anions having their molecular long axes parallel or approximately parallel (probably 50% “up-pointing” and 50% “downpointing” anions).5 Despite these and other models, however, an aggregation model has yet to be agreed upon. We tried to solve this problem using the solid state as the source of the models, since the same structural unit, or a very similar one, could be present both in the solid and liquid state, as frequently occurs for macromolecular compounds. The models observed in the solid state (crystals and fibers) were verified and sometimes confirmed in the study of the micellar solutions. This strategy was successfully applied to some dihydroxy salts. The micellar aggregates of sodium and rubidium deoxycholate6 or taurodeoxycholate and glycodeoxycholate7,8 were sat* Corresponding author. Telephone: 6-4452993. Fax: 6-490631. E-mail:
[email protected]. † Universita ` di Roma “La Sapienza”. ‡ Universita ` di L’Aquila. (1) Small, D. M. In The Bile Acids; Nair, P. P., Kritchevsky, D., Eds.; Plenum Press: New York, 1971; Vol. 1, Chapter 8. (2) Carey, M. C. In Sterols and Bile Acids; Danielsson, H., Siøvall, J., Eds.; Elsevier/North-Holland Biomedical Press: Amsterdam, 1985; p 345. (3) Small, D. M.; Penkett, S. A.; Chapman, D. Biochim. Biophys. Acta 1969, 176, 178. (4) Oakenfull, D. G.; Fisher, L. R. J. Phys. Chem. 1977, 81, 1838. Zana, R. J. Phys. Chem. 1978, 82, 2440. Oakenfull, D. G.; Fisher, L. R. J. Phys. Chem. 1978, 82, 2443. Fisher, L. R.; Oakenfull, D. G. J. Phys. Chem. 1980, 84, 937. (5) Kawamura, H.; Murata, Y.; Yamaguchi, T.; Igimi, H.; Tanaka, M.; Sugihara, G.; Kratohvil, J. P. J. Phys. Chem. 1989, 93, 3321.
isfactorily represented by 8/1 or 7/1 helices observed in their fibers, which were drawn from gels obtained in aqueous solutions. The basic building block of the helices was a trimer with a 3-fold rotation axis. Accordingly, the micellar aggregation numbers of sodium taurodeoxycholate, determined by electromotive force (emf) measurements as a function of pH, ionic strength, and bile salt concentration, were mostly multiples of three.8 The trihydroxy salts form much smaller micellar aggregates than the dihydroxy ones under the same conditions and show dissimilar physicochemical properties.2 Little is known about the structure of their micellar aggregates and conflicting results were reported about their size distribution. This paper deals with the structure and composition of sodium taurocholate (NaTC), a very important and carefully studied trihydroxy bile salt in humans. Previously, dynamic fluorescence anisotropy measurements suggested the presence of a NaTC dimer “sandwich” with perylene in aqueous solution. Moreover, a primary-secondary aggregation model was proposed in which the dimer is the building block.9 Experimental Section X-ray Measurements. NaTC (Sigma) was twice crystallized from a mixture of water and acetone. These molecules were released from the crystals by heating. Aqueous solutions of the resulting material gradually increased their viscosity until gels were obtained. These gels were drawn out into long glassy and brittle fibers, which are preferentially oriented microcrystalline specimens. Their X-ray diffraction photographs were recorded on flat films by means of the Buerger precession camera, using Ni-filtered Cu KR radiation (λ ) 1.5418 Å). The most intense NaCl interplanar spacings, which are known with great precision, were used to determine those of the NaTC fiber. emf Measurements. N(CH3)4Cl (Fluka) was crystallized from a mixture of water and ethanol. Tetramethylammonium taurocholate, necessary to vary the ratio between sodium and taurocholate ions, was prepared from N(CH3)4OH and taurocholic acid. This compound was obtained by diffusion of HCl vapor into a propanolic solution of NaTC. The H+- and Na+-free concentrations were measured by means of galvanic cells containing glass electrodes reversible to H+ and Na+. The galvanic cells were calibrated by using H+ and Na+ standard solutions in the selected ionic medium. Radiometer pH M64 and Metrohm 654 potentiometers equipped with radiometer and metrohm glass electrodes were used.
Results and Discussion Some crystal structures of trihydroxy salts were solved.10-12 Their structural units have an approximate shape of an elliptic cylinder and a roughly elliptical cross (6) Conte, G.; Di Blasi, R.; Giglio, E.; Parretta, A.; Pavel, N. V. J. Phys. Chem. 1984, 88, 5720. Esposito, G.; Giglio, E.; Pavel, N. V.; Zanobi, A. J. Phys. Chem. 1987, 91, 356. Giglio, E.; Loreti, S.; Pavel, N. V. J. Phys. Chem. 1988, 92, 2858. D’Alagni, M.; D’Archivio, A. A.; Galantini, L.; Giglio, E. Langmuir 1997, 13, 5811. D’Archivio, A. A.; Galantini, L.; Giglio, E.; Jover, A. Langmuir 1998 14, 4776. (7) Briganti, G.; D’Archivio, A. A.; Galantini, L.; Giglio, E. Langmuir 1996, 12, 1180. (8) Bottari, E.; Festa, M. R. Langmuir 1996, 12, 1777. (9) Li, G.; McGown, L. B. J. Phys. Chem. 1993, 97, 6745. (10) Campanelli, A. R.; Candeloro De Sanctis, S.; Galantini, L.; Giglio, E.; Scaramuzza, L. J. Inclusion Phenom. Mol. Recognit. Chem. 1991, 10, 367. Campanelli, A. R.; Candeloro De Sanctis, S.; D’Archivio, A. A.; Giglio, E.; Scaramuzza, L. J. Inclusion Phenom. Mol. Recognit. Chem. 1991, 11, 247. (11) D’Alagni, M.; Galantini, L.; Giglio, E.; Gavuzzo, E.; Scaramuzza, L.Trans. Faraday Soc. 1994, 90, 1523. (12) D’Archivio, A. A.; Galantini, L.; Gavuzzo, E.; Giglio, E.; Scaramuzza, L. Langmuir 1996, 12, 4660.
10.1021/la9809630 CCC: $18.00 © 1999 American Chemical Society Published on Web 03/26/1999
Notes
Figure 1. NaTC crystal packing viewed along b. A thicker line represents an anion nearer to the observer. Broken or thin full lines indicate hydrogen bonds or ion-ion and ion-dipole interactions.
Figure 2. NaTC structural unit (R) with a 2-fold screw axis projected (A) along a direction perpendicular to the helical axis and (B) along the helical axis. The meaning of the lines is as in Figure 1.
section, are formed by dimers, and were proposed as models of the corresponding micellar aggregates.11,12 The crystal structure of a NaTC monoclinic crystal was reported in a previous paper.11 The relevant crystal data were a ) 40.20, b ) 7.66, c ) 23.04 Å, β ) 92.8°, space group C2, 8NaTC + 46H2O + 4CH3-CO-CH3. Its packing is shown in Figure 1. Two structural units are recognizable. One (R) has a 2-fold screw axis (Figure 2) and the other (β) has a 2-fold rotation axis (Figure 3). Four units (two R and two β) are located around hydrophilic channels, filled with Na+ ions and water molecules, and held together by means of strong polar interactions (Figure 1).11
Langmuir, Vol. 15, No. 8, 1999 2997
Figure 3. NaTC structural unit (β) with a 2-fold rotation axis projected (A) along a direction perpendicular to the 2-fold rotation axis and (B) along the 2-fold rotation axis. The meaning of the lines is as in Figure 1.
Recently, a fiber has been drawn from a gel obtained by concentrating a NaTC aqueous solution. The X-ray diffraction pattern of an air-dried fiber can be interpreted by means of the unit cell constants a ) 19.65, b ) 7.65, c ) 25.70 Å, R ) 90°, β ) 97°, and γ ) 90°, strictly related to that of the monoclinic crystal11 (a is halved). The periodicity along the b fiber axis (7.65 Å), measured by averaging the values of the first- and second-layer line, agrees with that of the crystal dimers (7.66 Å).11 The relevant data of the equatorial reflections are reported in Table 1. Very probably the space group is monoclinic (P2 or P21). Thermogravimetric and density measurements give a formula unit NaTC + 5.3H2O (NaTC + 5.75H2O in the crystal). These results indicate that the fiber structure is very similar to that of the crystal, and therefore, Figures 1-3 represent with good approximation also the fiber structure. The dimers shown in Figures 2B and 3B are the basic building blocks and form R and β by translation of about 7.7 Å along an axis. Their outer surfaces are polar near the end regions of the semimajor axis and apolar around the angular methyl groups, which are the most protruding outside as in the case of the 7/1 and 8/1 helices.6,7 The dimers as well as the R and β structural units are strongly stabilized mainly by ion-ion and iondipole interactions, and by hydrogen bonds (Figures 2 and 3). Two facing antiparallel anions of a dimer are linked by head-to-tail hydrogen bonds, together with Na+‚‚‚SO3Coulombic interactions. The cations show a distorted octahedral coordination (one or two SO3- groups and five or four water molecules). Both R and β are permeable to the aqueous solvent since the separation of about 7.7 Å between two adjacent anions is sufficiently large (Figures 2A and 3A). Different side chain conformations of the anions and different locations of the cations, which are more embedded in β, characterize R and β. It is important to stress that the R and β dimers are very different from those previously proposed.4,9 The hydrogen-bonded dimer of ref 4, formed by two cholate anions, presents three hydrogen bonds between hydroxyl groups. The β dimer, for instance, forms five and seven hydrogen bonds by means of the polar head and the hydroxyl groups, respectively. Moreover, water molecules
2998 Langmuir, Vol. 15, No. 8, 1999
Notes
Table 1. Observed (do) and Calculated (dc) Spacings (Å) and Miller Indices (h l) of NaTC Equatorial Reflections hl
do
dc
hl
do
dc
hl
do
dc
hl
do
dc
10 2 -1 3 -3
19.5 9.5 5.4
19.5 9.5 5.5
1 -1 04 05
16.5 6.4 5.1
16.5 6.4 5.1
02 23 15
12.8 6.0 4.8
12.8 6.0 4.8
1 -2 32 06
11.3 5.5 4.3
11.3 5.5 4.3
Table 2. NaTC (CTC) and N(CH3)4Cl (CS) Concentrations (mol dm-3) in Aqueous Solutions at pH 8.0 [Aggregation Number of Na+ Ions (N+), Taurocholate Anions (N-, Bold-Faced Numerals), and P (in Parentheses)] CTC
CS
N+, N- (P)
0.02 0.02 0.05 0.02 0.05 0.08
0.08 0.48 0.45 0.78 0.75 0.72
0,1 (79.6); 1,2 (11.5); 2,2 (6.0); 1,4 (2.8) 0,1 (42.5); 2,2 (20.8); 3,4 (26.9); 4,6 (7.3); 5,8 (2.5) 0,1 (16.7); 2,2 (19.7); 3,4 (38.9); 4,6 (16.2); 5,8 (8.5) 0,1 (37.8); 2,2 (32.4); 3,4 (7.9); 5,8 (16.7); 8,12 (2.2); 10,16 (2.8); 15,24 (0.1) 0,1 (14.6); 2,2 (23.5); 3,4 (7.4); 5,8 (26.5); 8,12 (8.3); 10,16 (17.5); 15,24 (2.2) 0,1 (9.1); 2,2 (17.8); 3,4 (6.1); 5,8 (25.6); 8,12 (10.4); 10,16 (25.9); 15,24 (5.0)
and sodium ions are essential in stabilizing the β dimer by means of ion-ion, ion-dipole, and hydrogen-bonding interactions.11 On the other hand, the dimer of ref 9 is hydrophobic and is stabilized by interactions between the NaTC apolar faces and the apolar perylene molecule: the dynamic fluorescence anisotropy measurements do not give information on the geometry of the dimer structure without perylene. emf measurements at 25 °C of galvanic cells, carried out on aqueous solutions of NaTC as a function of pH, ionic strength, and bile salt concentration, provide the reagent concentrations.8,13 The constant ionic medium method14 has been used, adding N(CH3)4Cl, to minimize the variation of the activity coefficients despite the change of the reagent concentrations, so that concentrations can substitute activities in the calculation of the aggregation constants βqpr ) [NaqHpTCr]/[Na+]q[H+]p[TC-]r. The distribution of the micellar aggregation numbers is independent of pH within the range 6-11. The micellar species necessary to satisfactorily fit the emf data, together with their percentages (P) expressed as a function of the taurocholate anion concentration, are shown in Table 2 and Figure 4 for some of the many solutions investigated. Since all the aggregates have aggregation numbers of the anions (N- ) that are multiples of two (in agreement with the R and β structures), a model formed by the dimers is validated. However, the fiber, which is drawn from the gel, is submitted to a very moderate elongational stress, and the gel is obtained by concentrating the aqueous micellar solution. Therefore, it is probable that the transitions [aqueous micellar solution] f [gel] f [fiber] occur without drastic structural changes and that a model, similar to that of the fiber, is present in solution. If this is the case, the β dimer seems the best candidate to represent the dimer in solution because it is more stable than the R one. Another point in favor of the β dimer is that each Na+ links two monomers belonging to the same dimer in β and to different dimers in R. Furthermore, assemblies of four dimers (Figure 1) could exist under conditions which favor aggregates of large size as in prefiber states. At the higher ionic strength (see Table 2 and Figure 4) aggregates with N- ) 8, 16, and 24 are present, whereas those with N- ) 6, 10, 14, 18, 20, and (13) Bottari, E.; Festa, M. R.; Jasionowska, R. J. Inclusion Phenom. Mol. Recognit. Chem. 1989, 7, 443. Bottari, E.; Festa, M. R. Monatsh. Chem. 1993, 124, 1119. (14) Biedermann, G.; Sillen, L. G. Ark. Kemi 1953, 5, 425.
Figure 4. Percentages (P) of the NaTC micellar aggregates, expressed as taurocholate anion concentration, as a function of the aggregation number of the anions (N-) at pH ) 8.0. CTC and CS are the NaTC and N(CH3)4Cl concentrations (mol dm-3), respectively.
22 are unobserved (N- ) 12 is a minor component in comparison to N- ) 8 and 16). Thus, an octamer (four dimers, probably all β) similar to that of Figure 1 and the β dimer could be the building blocks of the biggest (N- ) 16 and 24) and smallest (N- ) 4, 6, and 12) aggregates, respectively. Acknowledgment. This work was sponsored by the Italian Ministero per l’Universita` e per la Ricerca Scientifica e Tecnologica (Cofin. MURST 97 CFSIB). LA9809630
