X-ray, Electrolytic Conductance, and Dielectric Studies of Bile Salt

Ingegneria Chimica e Materiali, Universita` di L'Aquila, 67010 Coppito Due, L'Aquila, Italy. Received June 16, 2000. In Final Form: September 15, 2000...
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Langmuir 2000, 16, 10436-10443

X-ray, Electrolytic Conductance, and Dielectric Studies of Bile Salt Micellar Aggregates Adalberto Bonincontro,† Angelo Antonio D’Archivio,§ Luciano Galantini,‡ Edoardo Giglio,*,‡ and Francesco Punzo‡ INFM, Dipartimento di Fisica, and 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 June 16, 2000. In Final Form: September 15, 2000 Previously, structural models, observed in fibers and crystals, were proposed for sodium deoxycholate (NaDC), glycodeoxycholate (NaGDC), taurodeoxycholate (NaTDC), and taurocholate (NaTC) micellar aggregates, and were verified in aqueous solutions by means of several techniques. Here we report the X-ray analysis of sodium glycocholate (NaGC) fibers, which indicates that NaGC micellar aggregates could be formed by dimers and octamers as in the case of NaTC. Moreover, we present electrolytic conductance and dielectric measurements on NaGDC, NaTC, and NaGC aqueous micellar solutions to verify our micellar aggregate models. Specific conductance values of 0.1 mol dm-3 NaDC, NaTDC, NaGDC, NaTC, and NaGC solutions containing NaCl at concentration ranging from 0 to 0.8 mol dm-3 practically do not depend on the particular bile salt. Comparison with NaCl values shows that bile salt contribution to conductance decreases by increasing NaCl concentration, is nearly zero around the concentration range 0.5-0.6 mol dm-3, and becomes negative at higher concentration. This behavior can be explained if Na+ ions strongly interact with bile salt anions and reinforce their interaction when micellar size increases. Even the inclusion of Na+ and Cl- ions, coming from NaCl, into micellar aggregates cannot be excluded, especially at high ionic strength. NaDC, NaTDC, NaGDC, NaTC, and NaGC present high values of the average electric dipole moment per monomer µ that can be justified by a remarkable hydration of their micellar aggregates. Reasonably, micellar aggregate composition and population change very slightly or do not change at all within the temperature range 15-45 °C, because µ is nearly constant in this interval. Results also suggest that Na+ ions are anchored to anions in dilute solution, thus forming ion pairs in the case of NaTC and NaGC, at least. Dihydroxy and trihydroxy bile salts are characterized by very similar cation-anion interaction strengths, even though their structures are different. The trend of µ, which moderately decreases by increasing bile salt concentration, agrees with our structural models and can be due to coexistence of two structures, at least.

Introduction Bile salts are natural steroids with surface-active and detergentlike properties capable of forming micellar aggregates and solubilizing in water many compounds,1-4 some of which are important from a biological and physiological point of view (for instance, bilirubin-IXR, cholesterol, fatty acids, phospholipids, and proteins). The structures of bile salt micellar aggregates are crucial for comprehending their physicochemical and biomedical properties. Bile salt anions have one relatively rigid (A, B, C, and D rings) and one flexible (the side chain that ends with a carboxylate or sulfonate group) moiety, possess an arched shape due to the cis fusion of A/B rings, and are two-faced, showing both polar (R face) and apolar (β face) sides (Figure 1). Bile salts relevant to this study have hydroxyl functions at positions 3R and 12R (dihydroxy salts) or 3R, 7R, and 12R (trihydroxy salts) on the R face, and methyl groups C18 and C19 on the β face. They are sodium salts of 3R,* Corresponding author. E-mail: [email protected]. † INFM. ‡ Dipartimento di Chimica, Universita ` di Roma “La Sapienza”. § Universita ` di L’Aquila. (1) Hofmann, A. F.; Small, D. M. Annu. Rev. Med. 1967, 18, 333. (2) Small, D. M. In The Bile Acids; Nair, P. P., Kritchevsky, D., Eds.; Plenum Press: New York, 1971; Vol. 1, pp 249-356. (3) Carey, M. C. In The Liver: Biology and Pathobiology; Arias, I. M., Popper, H., Schacter, D., Shafritz, D., Eds.; Raven Press: New York, 1982; pp 429-465. (4) Carey, M. C. In Sterols and Bile Acids; Danielsson, H., Sjøvall, J., Eds.; Elsevier/North-Holland Biomedical Press: Amsterdam, 1985; pp 345-403.

12R-dihydroxy-5β-cholanic acid (sodium deoxycholate, NaDC), 3R,12R-dihydroxy-5β-cholanoyltaurine (sodium taurodeoxycholate, NaTDC), 3R,12R-dihydroxy-5β-cholanoylglycine (sodium glycodeoxycholate, NaGDC), 3R,7R,12R-trihydroxy-5β-cholanoyltaurine (sodium taurocholate, NaTC), and 3R,7R,12R-trihydroxy-5β-cholanoylglycine (sodium glycocholate, NaGC) (Figure 1). Corresponding rubidium and calcium salts will be hereafter indicated replacing Na with Rb and Ca, respectively. Aggregation models without a well-defined packing structure and valid for all bile salts (dihydroxy and trihydroxy salts) were proposed. Among the others, that of Small et al.5 and, to a less extent, those of Oakenfull and Fisher6 and Kawamura et al.7 have been frequently used to interpret experimental data. However, there are several discrepancies that cast doubt on their reliability. In particular, because dihydroxy and trihydroxy bile salts show very dissimilar physicochemical properties,4 it is highly improbable that their micellar aggregates possess a very similar structure. It was observed that NaDC8-10 and some other bile salts11-16 give rise to transitions [aqueous micellar solution]f[gel]f[fiber] and, sometimes, [fiber]f[crystal]. (5) Small, D. M.; Penkett, S. A.; Chapman, D. Biochim. Biophys. Acta 1969, 176, 178. (6) Oakenfull, D. G.; Fisher, L. R. J. Phys. Chem. 1977, 81, 1838. (7) Kawamura, H.; Murata, Y.; Yamaguchi, T.; Igimi, H.; Tanaka, M.; Sugihara, G.; Kratohvil, J. P. J. Phys. Chem. 1989, 93, 3321. (8) Rich, A.; Blow, D. M. Nature (London) 1958, 182, 423. (9) Blow, D. M.; Rich, A. J. Am. Chem. Soc. 1960, 82, 3566. (10) D’Archivio, A. A.; Galantini, L.; Giglio, E.; Jover, A. Langmuir 1998, 14, 4776.

10.1021/la000844w CCC: $19.00 © 2000 American Chemical Society Published on Web 11/21/2000

Bile Salt Micellar Aggregates

Figure 1. Carbon atom numbering of bile salt anions relevant to this work. Hydrogen atoms are omitted for the sake of clarity. From top to bottom: X ) H deoxycholate, glycodeoxycholate, taurodeoxycholate; X ) OH cholate, glycocholate, taurocholate.

The [aqueous micellar solution]f[gel] transition occurs by varying, for instance, ionic strength, pH, and concentration. Glassy and birefringent fibers can be drawn from the gel or the aqueous micellar solution near the gelation point,8-16 and crystals can be obtained from fibers by aging.17 Of course, the same structural unit, or a very similar one, could be present both in solid (fiber and crystal) and liquid state (aqueous micellar solution), as frequently occurs for macromolecular compounds. The solid state was used as a source of models that were verified and sometimes confirmed in the study of the aqueous micellar solutions. Models with definite packing structures, which can suitably represent those of micellar aggregates, were obtained by solving several crystal11,18-25 and fiber10-16 structures. Reasonably, fiber models are more reliable than crystal ones, because the fiber is a system nearer to the aqueous micellar solution than is the crystal. On the other hand, it is probable that (11) D’Alagni, M.; D’Archivio, A. A.; Giglio, E.; Scaramuzza, L. J. Phys. Chem. 1994, 98, 343. (12) Briganti, G.; D’Archivio, A. A.; Galantini, L.; Giglio, E. Langmuir 1996, 12, 1180. (13) D’Alagni, M.; D’Archivio, A. A.; Galantini, L.; Giglio, E. Langmuir 1997, 13, 5811. (14) D’Archivio, A. A.; Galantini, L.; Giglio, E. Langmuir 1997, 13, 4197. (15) Bottari, E.; D’Archivio, A. A.; Festa, M. R.; Galantini, L.; Giglio, E. Langmuir 1999, 15, 2996. (16) Bonincontro, A.; D’Archivio, A. A.; Galantini, L.; Giglio, E.; Punzo, F. J. Phys. Chem. B 1999, 103, 4986. (17) Conte, G.; Di Blasi, R.; Giglio, E.; Parretta, A.; Pavel, N. V. J. Phys. Chem. 1984, 88, 5720. (18) Campanelli, A. R.; Candeloro De Sanctis, S.; Giglio, E.; Petriconi, S. Acta Crystallogr., Sect. C 1984, C40, 631. (19) Campanelli, A. R.; Candeloro De Sanctis, S.; Giglio, E.; Scaramuzza, L. J. Lipid Res. 1987, 28, 483. (20) Campanelli, A. R.; Candeloro De Sanctis, S.; Chiessi, E.; D’Alagni, M.; Giglio, E.; Scaramuzza, L. J. Phys. Chem. 1989, 93, 1536. (21) Campanelli, A. R.; Candeloro De Sanctis, S.; Galantini, L.; Giglio, E.; Scaramuzza, L. J. Inclusion Phenom. Mol. Recognit. Chem. 1991, 10, 367.

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transitions [aqueous micellar solution]f[gel]f[fiber] occur without drastic structural changes, since the gel is obtained concentrating the aqueous micellar solution and the fiber is drawn from the gel by means of the weak force of gravity. Experimental data indicate that 8/1 or 7/1 helices observed in fibers satisfactorily represent micellar aggregates of lithium, sodium, potassium, and rubidium deoxycholate10 or those of NaTDC, RbTDC, NaGDC, RbGDC,11,12 and CaTDC14 (dihydroxy salts), whereas dimers and octamers of the type found in a NaTC crystal and fiber seem to be the building blocks of NaTC micellar aggregates (trihydroxy salt).15,23 These structures were verified in aqueous solutions containing micellar aggregates or their interaction complexes with some probe molecules by means of techniques commonly used in the study of micelles such as smallangle X-ray scattering,26 electron spin resonance,26 nuclear magnetic resonance,17,27-31 quasi-elastic lightscattering,10,11-14,16,23-25,32and electrolytic conductance.16 Moreover, we tested less conventional techniques to gain a better insight into micellar aggregate structures. Extended X-ray absorption fine structure33-36 (EXAFS), circular dichroism11,13,14,20,23,24,27,29,31,32,37 (CD), electromotive force12,15,38-41 (EMF), and dielectric16,32 measurements were accomplished. Unfortunately, cation location and, as a consequence, interactions between cations and anions in 8/1 and 7/1 helices are unknown. Here we describe electrolytic conductance and dielectric measurements that permit discrimination between structural models and establishment of whether cations weakly or strongly interact with anion structures. Because we have models for the NaDC, NaTDC, NaGDC, and NaTC micellar aggregate structures but that of NaGC is unknown, first we attempted to solve a NaGC fiber structure. Experimental Section Materials. NaDC, NaTDC, NaGDC, NaTC, and NaGC (Sigma) were twice crystallized from a mixture of water and acetone. Water and acetone were released from crystals by heating at 50 (22) Campanelli, A. R.; Candeloro De Sanctis, S.; D’Archivio, A. A.; Giglio, E.; Scaramuzza, L. J. Inclusion Phenom. Mol. Recognit. Chem. 1991, 11, 247. (23) D’Alagni, M.; Galantini, L.; Giglio, E.; Gavuzzo, E.; Scaramuzza, L. Trans. Faraday Soc. 1994, 90, 1523. (24) D’Archivio, A. A.; Galantini, L.; Gavuzzo, E.; Giglio, E.; Scaramuzza, L. Langmuir 1996, 12, 4660. (25) D’Archivio, A. A.; Galantini, L.; Gavuzzo, E.; Giglio, E.; Mazza, F. Langmuir 1997, 13, 3090. (26) Esposito, G.; Giglio, E.; Pavel, N. V.; Zanobi, A. J. Phys. Chem. 1987, 91, 356. (27) D’Alagni, M.; Forcellese, M. L.; Giglio, E. Colloid Polym. Sci. 1985, 263, 160. (28) Esposito, G.; Zanobi, A.; Giglio, E.; Pavel, N. V.; Campbell, I. D. J. Phys. Chem. 1987, 91, 83. (29) D’Alagni, M.; Giglio, E.; Petriconi, S. Colloid Polym. Sci. 1987, 265, 517. (30) Chiessi, E.; D’Alagni, M.; Esposito, G.; Giglio, E. J. Inclusion Phenom. Mol. Recognit. Chem. 1991, 10, 453. (31) D’Alagni, M.; Delfini, M.; Galantini, L.; Giglio, E. J. Phys. Chem. 1992, 96,10520. (32) Bonincontro, A.; Briganti, G.; D’Archivio, A. A.; Galantini, L.; Giglio, E. J. Phys. Chem. B 1997, 101, 10303. (33) Giglio, E.; Loreti, S.; Pavel, N. V. J. Phys. Chem. 1988, 92, 2858. (34) Burattini, E.; D’Angelo, P.; Giglio, E.; Pavel, N. V. J. Phys. Chem. 1991, 95,7880. (35) Ascone, I.; D’Angelo, P.; Pavel, N. V. J. Phys. Chem. 1994, 98, 2982. (36) D’Angelo, P.; Di Nola, A.; Giglio, E.; Mangoni, M.; Pavel, N. V. J. Phys. Chem. 1995, 99, 5471. (37) D’Alagni, M.; D’Archivio, A. A.; Giglio, E. Biopolymers 1993, 33, 1553. (38) Bottari, E.; Festa, M. R.; Jasionowska, R. J. Inclusion Phenom. Mol. Recognit. Chem. 1989, 7, 443. (39) Bottari, E.; Festa, M. R. Mh. Chem. 1993, 124, 1119. (40) Bottari, E.; Festa, M. R. Langmuir 1996, 12, 1777. (41) Bottari, E.; Festa, M. R.; Franco, M. Analyst 1999, 124, 887.

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°C and under a pressure of 10-2 mm Hg. RbTDC was prepared as previously reported.11 NaCl and RbCl were from Merck (suprapur). Glassy and brittle fibers were drawn from a gel obtained by concentrating a NaGC aqueous solution by means of the weak force of gravity. X-ray Fiber Diffraction Measurements. X-ray diffraction patterns of NaGC fibers were recorded on flat films by means of a 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 NaGC fibers. Electrolytic Conductance Measurements. Impedance analyzer HP 4194 A, which covers the frequency range 10 kHz-100 MHz, was used. Impedance of the cell filled with the sample was measured in the narrow range of frequencies 10100 kHz. Within this interval the phase angle is nearly zero, because impedance is the sample electric resistance and it is inversely proportional to its conductance. Frequencies in this interval are so much lower than those of solute and, even more so, of solvent dielectric relaxation, as to exclude any contribution of dielectric loss to measured resistance. This ensures that ionic or static conductance of the sample is measured. Cell constant was determined measuring standard NaCl aqueous solutions. Conductivity measurements of these solutions show a relative accuracy and reproducibility less than ( 2%. Dielectric Measurements. Impedance analyzer HP 4291 A was used in the frequency range 1.0 MHz-1.8 GHz. Measured reflection coefficient and phase angle at the interface with the sample were converted to real and imaginary parts of dielectric constant by an interpolation method based on dielectric measurements of electrolyte solutions that have conductance near those of investigated samples.42 Dielectric loss values were obtained by subtraction of ionic conductance contribution according to the known formula:

′′ ) ′′T - σo/(oω) where ′′, ′′T, σo, o, and ω are dielectric loss, observed imaginary part of complex dielectric constant, ionic conductance of solution, dielectric constant in a vacuum, and angular frequency, respectively. Conductance σo was measured as described in the previous paragraph. The sample holder was thermostated within 0.1 °C. Measurements of permittivity and dielectric loss show a relative accuracy and reproducibility of about (1% and (2%, respectively.

Results and Discussion X-ray Analysis of NaGC Fiber. The structure of a NaGC tetragonal crystal, which was grown by diffusion of acetone vapor into a bile salt aqueous solution, was previously solved.21 Relevant crystal data are: a ) b ) 27.793(4), c ) 7.937(1) Å, Z ) 8, space group I4, asymmetric unit NaGC + 3.375 H2O + 0.25 acetone. Crystal structure is characterized by a packing of 21 helices (Figure 2), which can be generated by translation along c of a dimer (Figure 3). Recently, two types of glassy and brittle fibers (fibers A and B) have been drawn from aqueous solutions without acetone. X-ray diffraction patterns of an air-dried fiber A resemble those of a powder formed by microcrystals oriented at random and can be interpreted by means of unit cell constants a ) b ) 27.8, c ) 7.9 Å (Table 1), practically equal to those of the NaGC tetragonal crystal. However, the condition for possible reflection h + k + l ) 2n of I4 is not satisfied (Table 1) and, therefore, crystal and fiber space groups are different. Because values of unit cell constants do not change, it is highly probable that crystal and fiber structures vary very little, that of the fiber being of lower symmetry. The most reasonable candidates for fiber A are space groups P4 and P42, which can be obtained from I4 removing two-fold screw axes. (42) Athey, T. W.; Stuchly, M. A.; Stuchly, S. S. IEEE 1982, MTT 30, 82.

Bonincontro et al.

Figure 2. NaGC crystal packing viewed along c. Thicker lines represent an anion nearer to the observer. Ow is water molecule oxygen atom. Broken lines indicate ion-ion and ion-dipole interactions or hydrogen bonds.

Therefore, 21 helices disappear in the fiber and the two monomers of a dimer are no longer related by a two-fold screw axis. This hypothesis is supported by a RbTC crystal structure.24 It crystallizes in space group P42 and shows packing and unit cell constants [a ) b ) 28.418(4), c ) 7.623(1) Å] very similar to those of the NaGC crystal. X-ray diffraction patterns of an air-dried fiber B are characteristic of a poorly oriented fiber, resemble those of a previously studied NaTC fiber,15 and can be interpreted by unit cell constants a ) 19.8, b ) 7.66, c ) 24.8 Å, R ) γ ) 90°, β ) 94° (those of NaTC fiber are a ) 19.65, b ) 7.65, c ) 25.70 Å, R ) γ ) 90°, β ) 97°) (Table 2). Very probably, the space group is P2 or P21 (Z ) 4). NaTC fiber structure and packing15 are strictly related to those of a NaTC monoclinic crystal.23 On the other hand, NaGC and NaTC crystal packings are very similar. Thermogravimetric and density measurements for fiber A or B give a formula unit NaGC + 3.8 H2O or 3.3 H2O, respectively, in good agreement with that of the NaGC crystal. The dimer structural motif often recurs in bile salt crystal structures. It was observed for NaTC,23 RbTC,24 NaGC,21 and RbGC24 (trihydroxy salts) as well as for RbTDC11 and CaGDC25 (dihydroxy salts). The two monomers of the dimer are always related by a two-fold screw axis (Figure 3) or a two-fold rotation axis except for RbTC where no symmetry axis is present. Three types of structural unit, obtained by translation of these three dimers along the two-fold screw or rotation axis or along the growing direction of the structural unit, are recognizable. These structural units have an approximate shape of elliptic cylinder with a roughly elliptical cross-section, and are mainly stabilized by ion-ion and ion-dipole interactions and by hydrogen bonds. Their outer surface is polar near the end regions of the elliptical cross-section semimajor axis and apolar around C18 and C19, which are the most protruding groups outside. They are permeable to water molecules in aqueous solution, because separation between two adjacent anions along the growing direction is sufficiently large (about 7.5-8.0 Å). Each dimer is

Bile Salt Micellar Aggregates

Langmuir, Vol. 16, No. 26, 2000 10439 Table 2. Observed (do) and Calculated (dc) Spacings (Å) and Miller Indices (h k l) of Reflections of a Typical NaGC Fiber B hkl

do

dc

100 1 0 -1 002 1 0 -2 2 0 -1 201 004 020

19.8 16.0 12.4 10.8 9.4 9.0 6.2 3.8

19.8 16.0 12.4 10.8 9.4 9.0 6.2 3.8

Table 3. Specific Conductance (S m-1) of Bile Salts (0.1 mol dm-3) with Different Concentrations of NaCl at 25 °C NaCl concentration (mol dm-3) NaCl NaDC NaTDC NaGDC NaTC NaGC

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.56 0.56 0.56 0.53 0.56

1.07 1.49 1.48 1.43 1.50 1.46

2.03 2.38 2.34 2.31 2.33 2.28

2.95 3.20 3.10 3.09 3.17 3.06

3.83 3.91 3.89 3.95 4.06 3.93

4.68 4.64 4.78 4.71 4.78 4.62

5.50 5.59 5.52 5.45 5.48 5.35

6.30 6.21 6.06 6.19 6.20 6.16

7.08 6.97 6.72 6.90 6.92 6.81

Specific conductance of NaCl is reported for comparison.

Figure 3. NaGC structural unit with two-fold screw axis projected along a direction (a) perpendicular and (b) parallel to the two-fold screw axis. Meaning of lines is as in Figure 2. Table 1. Observed (do) and Calculateda (dc) Spacings (Å) and Miller Indices (h k l) of NaGC Fiber A Reflections hkl

do

dc

1 2 0, 2 1 0 0 3 0, 3 0 0 2 3 0, 3 2 0 0 1 1, 1 0 1 1 4 0, 4 1 0, 1 2 1, 2 1 1 330 1 3 1, 3 1 1 0 5 0, 5 0 0, 3 4 0, 4 3 0 0 4 1, 4 0 1, 2 5 0, 5 2 0

12.4 9.3 7.7 7.6 6.7 6.6 5.9 5.6 5.2

12.4 9.3 7.7 7.6 6.7 6.6 5.9 5.6 5.2

a The d spacings are equal to those calculated by using unit cell c constants of NaGC tetragonal crystal.

formed by two antiparallel monomers facing their polar R faces. Different side-chain conformations and cation locations have been observed. Cations are strongly bound to two monomers near the end regions of the elliptical cross-section semimajor axis, are coordinated to water molecules, hydroxyl, and carboxylate or sulfonate groups, and generally give rise to five-fold or six-fold coordination polyhedra.

NaTC and NaGC show nearly equal physicochemical properties.15,23,24 Similarity between NaTC and NaGC fiber structures strongly suggests that structures and compositions of NaTC and NaGC micellar aggregates should be very similar. Dimers, together with aggregates having anion aggregation numbers that are always multiples of two, were observed in NaTC aqueous solutions by EMF measurements.15,41 The most stable dimer in solution, as far as internal energy is concerned, presents a two-fold rotation axis, since only in this case the two cations of the dimer are equivalent and link two monomers belonging to the same dimer. Moreover, a very stable octamer, formed by four dimers held together by polar interactions around hydrophilic channels containing Na+ ions and water molecules, is observed both in NaTC15 and NaGC fiber structure (Figure 2). Because at high ionic strength and NaTC concentration are present mainly octamers, hexadecamers, and, to a less extent, tetraeicosamers, the octamer could be the building block of the biggest aggregates also in the case of NaGC. In support of dimer and octamer structures it must be noticed that they provide suitable models for enantioselective complexation of bilirubin-IXR, in agreement with CD spectra and potential energy calculations.23 Electrolytic Conductance Study An important problem in the structure of bile salt micellar aggregates regards cation binding to anion aggregates. Previously, specific conductance measurements were accomplished on 0.1 mol dm-3 NaDC and NaTDC aqueous solutions at 25 °C as a function of NaCl up to 0.6 mol dm-3 concentration.16 NaDC and NaTDC were chosen as representative compounds of 8/1 and 7/1 helices, respectively. Experimental values were practically equal for NaDC and NaTDC (Table 3), thus supporting a close interaction energy between Na+ ions and anion aggregates in the very similar 8/1 and 7/1 helices.10,12 Here we have investigated NaGDC (7/1 helix), NaTC, and NaGC (dimer and octamer), and also NaDC and NaTDC up to a 0.8 mol dm-3 NaCl concentration. The aim is to ascertain if NaGDC behaves as NaTDC and NaDC, to determine trihydroxy bile salt behavior, and to compare it with that of dihydroxy ones. Results are collected in Table 3 where those of NaCl are reported for comparison. All bile salts show nearly equal values of specific conductance and, for this reason, only NaGC + NaCl data versus NaCl are shown as an example in Figure

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Figure 4. Specific conductance of NaGC + NaCl as a function of added NaCl. That of NaCl is shown for comparison (upper panel). ∆σ vs NaCl concentration (lower panel).

4 (upper panel). Systematically, specific conductance difference of NaCl aqueous solutions with and without 0.1 mol dm-3 bile salt decreases by increasing NaCl amount, is nearly zero around the concentration range 0.5-0.6 mol dm-3, and becomes negative at higher NaCl concentration. It seems that bile salts give no contribution around the NaCl concentration range 0.5-0.6 mol dm-3 and reduce NaCl contribution at higher concentration. In this connection, it must be stressed that standard deviations of conductance measurements do not allow us to be strictly sure of previous deductions concerning the high ionic strength range. However, the very similar trends observed for all bile salts encourage us to have trust in them. As suggested by a reviewer, it is interesting to notice that the difference between the average specific conductance of the bile salts and that of NaCl (∆σ) for each column of Table 3 versus NaCl concentration gives rise to a linear trend (Figure 4, lower panel). The equation of the straight line is: y ) -0.8543 x + 0.4649 (regression value 0.996). The NaDC + NaCl conductance nonadditivity could depend on three possible effects, arising from ionic strength change. The first regards the interaction strength between anion aggregates and Na+ ions, which give a contribution to conductance greater than that of anion aggregates. Cations can interact with big aggregates utilizing a number of strong binding sites (mainly anion polar heads) greater than that cations can use with monomers and

Bonincontro et al.

small oligomers. The number of big aggregates increases by increasing ionic strength.16,24,38-41 Reasonably, interaction strength is enhanced and, as a consequence, cation mobility and conductivity as well as aggregate charge is lowered. The second could be due to migrating ions that should experience obstruction depending on micellar aggregate sizes and shapes, and could take different detours and paths between electrodes. The more the aggregate charge tends to zero, the more the obstruction effect should affect conductance. However, dihydroxy and trihydroxy salts show nearly the same values of specific conductance, although they have very different micellar sizes and shapes, especially at high ionic strength.24 Hence, obstruction effect due to micellar size and shape seems to be negligible. The third effect depends on possible inclusion of Na+ and Cl- ions, coming from NaCl, into micellar aggregates or micellar aggregate associations at high ionic strength. Micellar aggregate structures are not in disagreement with this hypothesis. The 8/1 (NaDC) and 7/1 (NaGDC and NaTDC) helices of dihydroxy bile salts are constructed using as repetitive unit a trimer (three bile salt molecules related by a three-fold rotation axis) that is rotated 45° (8/1 helix) or 34.29° (7/1 helix) around and translated about 6.5 Å along the helical axis, and have a radius of about 10 Å.10,12 These helices are mainly polar inside and apolar outside and their inner hydrophilic region is filled by cations, anion polar heads, and water molecules. Na+ and Cl- ions can be easily accommodated along the helical axis either by replacing water molecules or by slightly lengthening the helical radius. The octamers of trihydroxy bile salts (NaTC and NaGC) are formed by association of four dimers around hydrophilic channels at a ) b ) 1/2 (Figure 2). Replacement of water molecules with Na+ and Cl- ions in these channels can be accomplished with a very slight change of structure. On the other hand, possible inclusion of inorganic salt ions is supported by crystals grown from aqueous micellar solutions containing NaDC and CaCl2 or RbTDC and RbCl. In the case of NaDC + CaCl2 the crystals are formed by deoxycholate, Ca2+, and Cl- ions,25 whereas in the case of RbTDC + RbCl by taurodeoxycholate, Rb+, and Cl- ions. In this latter case we have solved but not published the crystal structure because the agreement factor is high, owing to a partial change of structure during intensity collection. However, X-ray analysis and density measurements cast no doubt about the presence of Rb+ and Clions coming from RbCl. In conclusion, NaDC and NaCl conductance nonadditivity can be ascribed to the first effect and, possibly, to the third one. Therefore, a very large fraction of counterions is strongly bound to anion aggregates in accordance with our structural models. Dielectric Study. Dielectric measurements have been carried out at 15, 25, 35, and 45 °C on 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, and 0.10 mol dm-3 NaGDC, NaTC, and NaGC aqueous solutions. The complex dielectric constant * ) ′- j′′ has been measured as a function of frequency. As an example, permittivity ′ and dielectric loss ′′ of 0.06 mol dm-3 NaGDC solution are shown as a function of frequency at 15, 25, 35, and 45 °C in Figures 5 and 6, respectively. Experimental point trends clearly show the presence of dielectric relaxation, usually termed β dispersion, which is observed at these frequencies in macromolecular solutions.43-46 Lines superimposed to (43) Grant, E. H.; Keefe, S. E.; Takashima, S. J. Phys. Chem. 1968, 72, 4373. (44) Takashima, S.; Gabriel, C.; Sheppard, R. J.; Grant, E. H. Biophys. J. 1984, 46, 29. (45) Bonincontro, A.; Caneva, R.; Pedone, F. J. Non-Cryst. Solids 1991, 133, 1186.

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of bile salt micellar aggregates:49-52

µ2 ) 2kToM ∆/Noc

Figure 5. Permittivity of 0.06 mol dm-3 NaGDC solution vs frequency at 15 (circles), 25 (squares), 35 (triangles), and 45 (diamonds) °C.

Figure 6. Dielectric loss of 0.06 mol dm-3 NaGDC solution vs frequency at 15 (circles), 25 (squares), 35 (triangles), and 45 (diamonds) °C.

experimental points represent the best fit in terms of a Cole-Cole dispersion applying the known equation:

* ) ∞ + ∆/[1 + (jωτ)1-R] where ∞, ∆, τ, and R are high-frequency limit permittivity, dielectric increment, relaxation time, and an empirical parameter that indicates a spread of relaxation times, respectively. Plainly, disagreement in the frequency region near 1 GHz (Figure 6) originates from water contribution to dielectric loss at these higher frequencies, especially for lower temperatures. Apparent electric dipole moment of solute can be estimated from dielectric increment by means of the Oncley formula,47,48 verified in many solutions with solutes of molecular weights comparable with those (46) Pedone, F.; Bonincontro, A. Biochim. Biophys. Acta 1991, 1073, 580. (47) Oncley, J. L. In Proteins, Amino Acids and Peptides; Cohn, E. J., Edsall, J. T., Eds.; Reinhold: New York, 1943; p 543. (48) Pethig R.; Kell, D. B. Phys. Med. Biol. 1987, 32, 933.

where µ, k, T, M, No, and c are “effective” electric dipole moment, Boltzmann constant, absolute temperature, molecular weight of a bile salt monomer, Avogadro’s number, and solute concentration in g m-3, respectively. This relation assumes that dielectric relaxation is due to orientational polarizability of polar molecules or assemblies of molecules in solution. For instance, cations, aggregates, and their coordinated water molecules can move and can be oriented when an electric field is applied. The “effective” µ value is calculated in the limit of noninteracting micellar aggregates for bile salt monomer in its specific aggregation distribution function of the solution and not for micellar aggregates, because M is monomer molecular weight. It must be noticed that dielectric results refer to solutions without NaCl, containing aggregates of small size. The µ values, computed by the Oncley formula, are only depicted at 15 °C versus bile salt concentration in Figure 7, since they could be considered independent of temperature at a fixed concentration within the range 15-45 °C, owing to their standard deviations (for NaTDC see ref 32). As an example, µ values of NaTC, NaGC, and NaGDC solutions at concentrations 0.02, 0.06, and 0.10 mol dm-3 are reported as a function of temperature in Figure 8. Reasonably, each sample within the temperature range 15-45 °C has the same, or nearly the same, composition and population of micellar aggregates, which are stable and do not undergo remarkable structural change. High µ values could be explained by a contribution of hydration water. EXAFS measurements, carried out on RbTDC, RbGDC, RbGC,36 and RbDC33,36 aqueous solutions, support this hypothesis because cations result coordinated to several water molecules. By inspection of Figure 7, there is an evident similarity between µ trends of NaTDC and NaGDC on one hand and those of NaTC and NaGC on the other, in agreement with their close micellar aggregate structures. Those of NaTDC and NaGDC are characterized by aggregation numbers that are multiples of three and by 7/1 helices,12,39,40 whereas those of NaTC and NaGC by dimers and octamers.15,41 These findings are also supported by relaxation frequency values, which are very similar for dihydroxy salts (NaTDC, NaGDC, and NaDC) on one hand and for trihydroxy salts (NaTC and NaGC) on the other. Trihydroxy salts show, generally, higher values than dihydroxy ones under the same conditions, according to their smaller aggregate sizes. NaTC, NaGC, NaTDC, NaGDC, and NaDC16 µ values increase passing from bigger aggregates to monomeric species. Moreover, all bile salts show a moderate decrease of µ by increasing their concentration. These trends and the high µ values permit the discarding of models that correspond to low or zero µ values as, for example, a model that is disordered or has a center or a pseudocenter of symmetry. Both NaTDC and NaGDC experimental points can be fitted by curves with slope changes at a concentration of (49) Takashima, S. In Physical Principles and Techniques of Protein Chemistry, Part A; Leach, A. J., Ed.; Academic: New York, 1969; p 291. (50) Gerber, B. R.; Routledge, L. M.; Takashima, S. J. Mol. Biol. 1972, 71, 317. (51) Grant, E. H.; South, G. P.; Walker, I. O. Biochem. J. 1971, 122, 765. (52) Pethig, R. In Dielectric and Electronic Properties of Biological Materials; Wiley: Chichester, U.K., 1979; pp 70-99.

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Bonincontro et al.

Figure 8. Average electric dipole moment per bile salt monomer as a function of temperature. Concentration (mol dm-3): 0.02 circles; 0.06 squares; 0.10 diamonds.

Figure 7. Average electric dipole moment per bile salt monomer as a function of concentration c (mM) at 15 °C. Standard deviations are shown.

about 0.05 mol dm-3, in accordance with the two-structure model and equilibrium proposed for NaTDC.32 The first structure, which prevails up to a concentration of about 0.05 mol dm-3, is mainly characterized by aggregation numbers less than or equal to 12 (from one to four trimers). The first-structure trimer has an approximate shape of an oblate ellipsoid, being formed by three bile salt molecules, approximately in fully extended conformation (semimajor axis of about 20-22 Å), related by a three-fold rotation axis. The second structure (7/1 helix), which should remarkably contribute to µ within a concentration

range from about 0.05 to 0.10 mol dm-3, is mainly characterized by aggregation numbers greater than or equal to 15 (from five trimers upward), since it was supposed that cooperative effects in 7/1 helix formation arise starting from pentadecamer. This hypothesis is reasonable because the 7/1 helix strongest interactions are those between trimer j and trimers j ( 1 and j ( 2. Hence, the smallest aggregate that gives rise to all four of these interactions is formed by five trimers.12,32 The second structure trimer, that is, the 7/1 helix repetitive unit, has a cylindrical shape with a radius of about 10 Å. Therefore, equilibrium is shifted from oblate to cylindrical aggregates by increasing concentration. Both NaTC and NaGC experimental points can be fitted by curves that present a maximum at concentration 0.06 mol dm-3. These curves resemble those of NaDC16 and can be similarly interpreted, assuming a two-structure model and equilibrium as in the case of NaTDC, NaGDC, and NaDC. The first structure, which prevails up to a concentration of about 0.05 mol dm-3, could be mainly constituted by monomers, dimers, and perhaps tetramers on the basis of NaTC EMF results.15,41 For instance, 0.02 mol dm-3 NaTC aqueous solution containing 0.08 mol dm-3

Bile Salt Micellar Aggregates

N(CH3)4Cl has monomer, dimer, and tetramer percentages, calculated as the ratio between oligomer concentration, expressed as a function of monomer, and NaTC total concentration, equal to 0.80, 0.17, and 0.03, respectively. The second structure, which should remarkably contribute to µ within the concentration range from about 0.05 to 0.10 mol dm-3, could be mainly constituted by tetramers and by oligomers bigger than a tetramer. The octamer, together with its multiples, is very stable (Figure 2) and predominant among oligomers when aggregate size increases, as can be inferred from NaTC EMF measurements.15 For this reason, the second-structure main component could be the octamer. The µ increase between 0.05 and 0.06 mol dm-3 can be explained by assuming that in this region octamers begin to form and their µ is greater than the average µ of smaller oligomers that are present in the same region. Average electric dipole moment per bile salt monomer is about 50 D for 0.02 mol dm-3 NaTC and NaGC solution (Figure 8). Monomer percentage at this concentration must be greater than 0.80, that is, the percentage obtained for a solution containing 0.08 mol dm-3 N(CH3)4Cl. Therefore, the very high intrinsic µ value of a single monomer (about 50 D) indicates that each Na+ ion is anchored to one anion in dilute solution, giving rise to ion pair formation. In conclusion, X-ray, electrolytic conductance, and dielectric results converge in outlining structural models

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having cations strongly interacting by means of polar forces with anion aggregates. These models are ordered and very stable, present high cation fractional bindings in agreement with EMF data,12,38-41 and are characterized by very similar cation-anion interaction strengths, even though dihydroxy and trihydroxy aggregate structures are different. Hence, Na+ ions of dihydroxy salts should be coordinated to water molecules and hydroxyl and carboxylate or sulfonate groups, forming with oxygen atoms five-fold or six-fold coordination polyhedra, as in the case of trihydroxy salts. This hypothesis is supported by NaTDC19 and NaGDC20 crystal structures, where sixfold coordination polyhedra, similar to those of trihydroxy salts, have been observed. Acknowledgment. This work was sponsored by Italian Ministero per l’Universita` e per la Ricerca Scientifica e Tecnologica. Supporting Information Available: Three tables detailing the dieletric data of NaGDC, NaGC, and NaTC aqueous solutions at different temperatures. The relaxation frequency fo, the dielectric increment ∆, and the average electric dipole moment µ are reported. This material is available free of charge via the Internet at http://pubs.acs.org. LA000844W