Possible Models for the Micellar Aggregates of Glycocholate and

Monterotondo Stazione, Roma, Italy, and Centro di Studio per la Termodinamica Chimica alle. Alte Temperature c/o Dipartimento di Chimica, Universita` ...
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Langmuir 1996, 12, 4660-4667

Possible Models for the Micellar Aggregates of Glycocholate and Taurocholate Salts from Crystal Structures, QELS, and CD Measurements Angelo Antonio D’Archivio,† Luciano Galantini,† Enrico Gavuzzo,‡ Edoardo Giglio,*,† and Lucio Scaramuzza§ Dipartimento di Chimica, Universita` di Roma “La Sapienza”, P.le A. Moro 5, 00185 Roma, Italy, Istituto di Strutturistica Chimica “Giordano Giacomello” C. N. R., C. P. no. 10, 00016 Monterotondo Stazione, Roma, Italy, and Centro di Studio per la Termodinamica Chimica alle Alte Temperature c/o Dipartimento di Chimica, Universita` di Roma “La Sapienza”, P.le. A. Moro 5, 00185 Roma, Italy Received January 19, 1996. In Final Form: June 21, 1996X The structures of crystals of rubidium glycocholate (monoclinic) and rubidium taurocholate (monoclinic and tetragonal) were solved by X-ray diffraction analysis. These structures, together with those of the sodium salts formerly determined, were used to infer models for the corresponding micellar aggregates in aqueous solutions. The crystal packings of the sodium and rubidium salts were characterized by similar structural units. These were bilayers, 21 helices, distorted 21 helices, and units with a two-fold rotation axis. On the other hand, circular dichroism spectra of the sodium and rubidium salts with bilirubin-IXR in aqueous solution indicated the formation of very similar interaction complexes and micellar aggregates. This hypothesis was supported by quasi-elastic light scattering measurements carried out on aqueous micellar solutions as a function of the ionic strength, concentration, and temperature. Low values of the apparent hydrodynamic radius, corresponding to oligomers of small size, were obtained for all the salts. The micellar growth was practically independent of the concentration and temperature, within the range 20-60 °C, at low ionic strength, and slightly increased upon increasing the ionic strength. We studied the rubidium salts in place of the sodium ones by extended X-ray absorption fine structure spectroscopy when their structures are similar. The measurements at the Rb+ K-edge, previously accomplished on the crystal and the aqueous micellar solution of rubidium glycocholate, were in agreement with the model of the 21 helix and allowed us to discard the bilayer. Circular dichroism spectra, recorded as a function of the ionic strength, pointed out that bilirubin-IXR gives rise to a different enantioselective complexation with dihydroxy or trihydroxy salts. The different structure of the corresponding micellar aggregates was also supported by quasi-elastic light scattering measurements accomplished on dihydroxy and trihydroxy salts. The calculation of the hydrodynamic radius for the 21 helix, observed in the monoclinic crystal of sodium taurocholate, showed that there is a possible satisfactory agreement with the quasi-elastic light scattering data.

Introduction Some helices observed in crystals and fibers of sodium and rubidium deoxycholate (NaDC and RbDC),1-13 gly† ‡

Universita` di Roma “La Sapienza”. Istituto di Strutturistica Chimica “Giordano Giacomello” C. N.

R. § Centro di Studio per la Termodinamica Chimica alle Alte Temperature. X Abstract published in Advance ACS Abstracts, August 15, 1996.

(1) Conte, G.; Di Blasi, R.; Giglio, E.; Parretta, A.; Pavel, N. V. J. Phys. Chem. 1984, 88, 5720. (2) Campanelli, A. R.; Candeloro De Sanctis, S.; Giglio, E.; Petriconi, S. Acta Crystallogr., Sect. C 1984, C40, 631. (3) D’Alagni, M.; Forcellese, M. L.; Giglio, E. Colloid Polym. Sci. 1985, 263, 160. (4) Esposito, G.; Giglio, E.; Pavel, N. V.; Zanobi, A. J. Phys. Chem. 1987, 91, 356. (5) Esposito, G.; Zanobi, A.; Giglio, E.; Pavel, N. V.; Campbell, I. D. J. Phys. Chem. 1987, 91, 83. (6) Giglio, E.; Loreti, S.; Pavel, N. V. J. Phys. Chem. 1988, 92, 2858. (7) Campanelli, A. R.; Candeloro De Sanctis, S.; Giglio, E.; Pavel, N. V.; Quagliata, C. J. Inclusion Phenom. Mol. Recognit. Chem. 1989, 7, 391. (8) Chiessi, E.; D’Alagni, M.; Esposito, G.; Giglio, E. J. Inclusion Phenom. Mol. Recognit. Chem. 1991, 10, 453. (9) Burattini, E.; D’Angelo, P.; Giglio, E.; Pavel, N. V. J. Phys. Chem. 1991, 95, 7880. (10) D’Alagni, M.; Delfini, M.; Galantini, L.; Giglio, E. J. Phys. Chem. 1992, 96, 10520. (11) D’Alagni, M.; D’Archivio, A. A.; Giglio, E. Biopolymers 1993, 33, 1553. (12) Ascone, I.; D’Angelo, P.; Pavel, N. V. J. Phys. Chem. 1994, 98, 2982. (13) D’Angelo, P.; Di Nola, A.; Giglio, E.; Mangoni, M.; Pavel, N. V. J. Phys. Chem. 1995, 99, 5471.

S0743-7463(96)00055-8 CCC: $12.00

codeoxycholate (NaGDC and RbGDC), and taurodeoxycholate (NaTDC and RbTDC)8,13-18 by means of X-ray diffraction analysis were checked as structural models of the micellar aggregates of these salts in aqueous solution. Some models were verified and sometimes supported by small-angle X-ray scattering, extended X-ray absorption fine structure (EXAFS), nuclear magnetic resonance, electron spin resonance, circular dichroism (CD), quasielastic light scattering (QELS), and electromotive force measurements, together with potential energy calculations. Although the rubidium salts are not important from a biological point of view, they were studied in place of the sodium salts by means of EXAFS spectroscopy, owing to the low K-edge absorption of the sodium ions. Of course, this technique was used to get a deeper insight into the structure of the micellar aggregates when similar models were inferred from the crystals or the fibers of the same sodium and rubidium salts.6,13 The trihydroxy salts sodium glycocholate and taurocholate (NaGC and NaTC) form much smaller micellar aggregates than the dihydroxy ones under the same conditions and, generally, show dissimilar physicochem(14) Campanelli, A. R.; Candeloro De Sanctis, S.; Giglio, E.; Scaramuzza, L. J. Lipid Res. 1987, 28, 483. (15) D’Alagni, M.; Giglio, E.; Petriconi, S. Colloid Polym. Sci. 1987, 265, 517. (16) Campanelli, A. R.; Candeloro De Sanctis, S.; Chiessi, E.; D’Alagni, M.; Giglio, E.; Scaramuzza, L. J. Phys. Chem. 1989, 93, 1536. (17) D’Alagni, M.; D’Archivio, A. A.; Giglio, E.; Scaramuzza, L. J. Phys. Chem. 1994, 98, 343. (18) Briganti, G.; D’Archivio, A. A.; Galantini, L.; Giglio, E. Langmuir 1996, 12, 1180.

© 1996 American Chemical Society

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Figure 1. Projection along (a) the 21 axis of the NaGC helix; (b) the 21 axis of the NaTC helix; (c) the twofold rotation axis of one NaTC structural unit; (d) a direction parallel to the plane in which the NaTC bilayer extends. A thicker line represents an anion nearer to the observer. Full or broken lines indicate ion-ion and ion-dipole or hydrogen-bonding interactions.

ical properties.19 Several experiments suggest that they self-associate in a noncooperative continuous manner in water and at low ionic strengths, whereas sometimes a cooperative association of a large number of molecules occurs, especially at high ionic strengths.19 NaTC, for example, shows a progressive aggregation on the basis of reliable surface tension and translational diffusion coefficient measurements.20 In this connection, it must be stressed that the helix satisfies both types of growth, since it is a one-dimensional open system containing monomers with unsaturated forces at the end points, if the solvent molecules are disregarded. In fact, the aggregate can grow by a stepwise addition of monomers or by welding of helices.7 In order to get models for the micellar aggregates of NaTC and NaGC, which are the most studied and most abundant conjugated bile salt in humans, respectively, we solved the crystal structure of a tetragonal phase of NaGC21 and that of a triclinic22 as well as a monoclinic23 phase of NaTC. Structural units characterized by a twofold screw or rotation axis or by bilayers were observed (Figure 1). The aim of this paper is to obtain possible structural models also for the micellar aggregates of the rubidium salts of glycocholic and taurocholic acids (RbGC or RbTC) by solving some crystal structures, to compare these models with those of NaGC and NaTC, and to check them indirectly by QELS and CD measurements. (19) For leading references, see: Carey, M. C. In Sterols and Bile Acids; Danielsson, H., Sjøvall, J., Eds.; Elsevier/North-Holland Biomedical Press: Amsterdam, 1985; Chapter 13, p 345. (20) Kratohvil, J. P.; Hsu, W. P.; Jacobs, M. A.; Aminabhavi, T. M.; Mukunoki, Y. Colloid Polym. Sci. 1983, 261, 781. (21) Campanelli, A. R.; Candeloro De Sanctis, S.; Galantini, L.; Giglio, E.; Scaramuzza, L. J. Inclusion Phenom. Mol. Recognit. Chem. 1991, 10, 367. (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.

Experimental Section Materials. RbGC was obtained by adding a little less than the equivalent amount of RbOH (Aldrich, about 99.9%) to glycocholic acid (Sigma) and filtering the resulting aqueous suspension. Subsequently, acetone was added until the solution became cloudy. RbTC was kindly supplied by Prodotti Chimici e Alimentari S. p. A., Basaluzzo (Al), Italy. RbGC was twice crystallized from a mixture of water and acetone by diffusion of acetone. RbTC was crystallized from a mixture of water, acetone, and diethyl ketone by diffusion of acetone. NaCl and RbCl (Merck, suprapur) were used. X-ray Analysis of Monoclinic RbGC, and Monoclinic and Tetragonal RbTC. Intensity X-ray data were collected at room temperature using the ω - 2θ mode in the range 5 e 2θ e 124° by means of a Rigaku four-circle diffractometer, equipped with a 12 kW rotating anode and a graphite monochromator, using Cu KR radiation (λ ) 1.5418 Å). The scan was carried out at variable and appropriate speeds as a function of the intensity of the reflections. Three standard reflections were monitored during the collection, but negligible decay was observed. The data were corrected for Lorentz and polarization effects. An empirical absorption correction, based on an azimuthal scan of several reflections, was applied to the intensities. Unit cell parameters were determined by least-squares refinement of the angular setting of 25 selected reflections. The density was measured by flotation in a chloroform/chlorobenzene mixture, the density of which was determined by means of a DMA 02C densimeter. The structures were refined by the full-matrix least-squares method with the program SIR-CAOS.24 The function minimized was ∑w(|Fo| - |Fc|)2 with w ) (sin θ/λ)2. Atomic scattering factors and anomalous dispersion coefficients were taken from ref 25. The hydrogen atoms were generated at the expected positions, except for the water and hydroxylic hydrogens, which were not taken into account in the calculations. Their atomic coordinates were not refined, and their thermal parameters were taken to be equal to the isotropic ones of the parent atoms. The structures were solved by direct methods by using the SIR92 (24) Camalli, M.; Capitani, D.; Cascarano, G.; Cerrini, S.; Giacovazzo, C.; Spagna, R. SIR-CAOS: User Guide; Istituto di Strutturistica Chimica CNR C. P. no. 10,00016 Monterotondo Stazione: Roma, 1986. (25) International Tables for X-ray Crystallography; Kinoch: Birmingham, England, 1974; Vol. IV.

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Figure 2. Atomic numbering of glycocholate and taurocholate anions. program.26 The common atom labeling of the bile salts was used (Figure 2). When a second molecule is present in the asymmetric unit, the atomic numbering is obtained by adding 35 to the atoms of the first molecule. Listings of observed and calculated structure factors, final fractional atomic coordinates and equivalent isotropic thermal parameters, and final bond lengths and bond angles have been deposited as Supporting Information (see the paragraph at the end of the paper). The average estimated standard deviations of the atomic parameters were rather high. However, the main aim of the present work is to identify the structural units which form the crystals in order to check them as models of micellar aggregates in aqueous solution. For this purpose the knowledge of a very precise molecular geometry is not needed. QELS Measurements. A Brookhaven instrument consisting of a BI-2030AT digital correlator with 136 channels and a BI200SM goniometer was used. The light source was an argon ion laser model 85 from Lexel Corporation operating at 514.5 nm. Dust was eliminated by means of a Brookhaven ultrafiltration unit (BIUU1) for flow-through cells, the volume of the flow cell being about 1.0 cm3. Nuclepore filters with a pore size of 0.1 µm were used. The samples were placed in the cell for at least 15 min prior to measurement to allow for thermal equilibration. Their temperature was kept constant within 0.5 °C by a circulating water bath. The time-dependent light scattering correlation function was analyzed only at the 90° scattering angle, since the observed apparent diffusion coefficients did not depend on the exchanged wave vector in the range 30-150° under our experimental conditions. The scattering decays were analyzed by means of cumulant expansion up to second order, because higher order contributions did not improve the statistics. The results are reported in terms of the apparent hydrodynamic radius obtained by the Stokes-Einstein relationship. CD Measurements. CD spectra were recorded on a JASCO J-500A spectropolarimeter at 25 °C by using a quartz cell with a path length of 1.0 cm and by flushing with ultrapurified nitrogen before and during the experiments. A slit program that gives a wavelength accuracy better than 0.5 nm was used. The instrument was calibrated with androsterone (1.69 × 10-3 M in dioxane) on the basis of a molar ellipticity [θ]304 ) 11 180 deg cm2 dmol-1. All the CD spectra were recorded within 30 min of solution preparation.

Results Crystal Structure of RbGC. Crystal data: RbC26H42NO6 + 2H2O, fw ) 586.1, monoclinic, C2, a ) 24.670(2) Å, (26) Altomare, A.; Cascarano, G.; Giacovazzo, C.; Guagliardi, A.; Burla, M. C.; Polidori, G.; Camalli, M. J. Appl. Crystallogr. 1994, 27, 435.

D’Archivio et al.

b ) 7.639(1) Å, c ) 16.877(1) Å, β ) 111.73(1)°, V ) 2954.5(4) Å3, Z ) 4, Dc ) 1.32 g cm-3, Do ) 1.36 g cm-3, µ ) 29.3 cm-1, mp 510 K. A total of 1998 independent reflections with I > 2σ(I) were collected. The Fourier syntheses showed that the two highest peaks with similar electron density correspond to the Rb+ ion located in two positions with an occupancy factor of about 0.5. Three peaks, indicated as O(1), O(2), and O(3), were oxygen atoms of water molecules. An occupancy factor of 0.5 was assigned to O(2) and O(3), since they showed nearly half the electron density of O(1). The occupancy factors of 0.5 were justified, since Rb(1) at (x, y, z) and that at (1 - x, y, -z) as well as O(2) at (x, y, z) and O(3) at (1 - x, y, -z) give rise to unrealistic distances of 2.8 and 2.2 Å, respectively. The final agreement factors R and Rw converged to 0.076 and 0.080, respectively. The intramolecular bond lengths and bond angles were reliable and comparable to those observed in crystal structures of other bile salts. The average estimated standard deviations were 0.01 Å and 1° with maximum values of 0.02 Å and 1°. The side chain and ring D torsion angles are reported in Table 1 in agreement with the convention of Klyne and Prelog.27 The phase angle of pseudorotation ∆ and the maximum angle of torsion φm are calculated according to Altona, Geise, and Romers.28 The RbGC anion is characterized by a ring D very close to the half-chair symmetry28 and by a gauche conformation of C(17)-C(20)C(22)-C(23), at variance with the trans conformation of the NaGC anion.21 The first four torsion angles of Table 1 fit a minimum region of the potential energy map of the deoxycholic acid side chain.29 The atoms C(20), C(22), C(23), C(24), N(29), and C(30) are in a nearly extended conformation. Assuming that the three bonds to N(29) and the C(24)-O(28) bond lie in a plane, the deviation from planarity of the amide group is very small, since the O(28)-C(24)-N(29)-C(30) torsion angle is about 2°. The crystal packing (Figure 3) is almost similar to that found in the triclinic phase of NaTC22 (Figure 1d) and is characterized by bilayers developed in planes parallel to ab and held together along c by van der Waals forces involving mainly methyl groups. The bilayer is composed of rows of RbGC molecules which extend along a. The molecules in each row are linked chiefly by head-to-tail polar interactions. Translation of a row along b gives rise to a monolayer. Two antiparallel monolayers form one bilayer, stabilized mainly by ion-ion, ion-dipole, and hydrogen-bonding forces. Rb+ ions and water molecules fill hydrophilic channels with a small cross section centered on the twofold rotation axes. The inner surface of a channel is covered by hydroxyl, carboxyl, and carbonyl groups. These groups form hydrogen bonds involving O(25) with O(28) and O(32) with O(26) and O(27). The water molecules give rise to the following hydrogen bonds: O(1) with O(25), N(29) and O(33), O(2) with O(25), and O(3) with O(25) and O(28). The Rb(1) ion is coordinated within 4.0 Å with O(26), O(28), two O(32), two O(33), O(2), and two O(3), while Rb(2) is coordinated with O(26), O(27), O(32), O(33), O(1), O(2), and O(3). Crystal Structure of Monoclinic RbTC. Crystal data: 2RbC26H44NO7S + 4.5H2O, fw ) 1281.4, monoclinic, C2, a ) 25.530(2) Å, b ) 7.538(1) Å, c ) 35.203(4) Å, β ) 112.37(1)°, V ) 6265(1) Å3, Z ) 4, Dc ) 1.36 g cm-3, Do ) 1.40 g cm-3, µ ) 33.3 cm-1, mp 447 K. A total of 3667 independent reflections with I > 2.5σ(I) were collected. (27) Klyne, W.; Prelog, V. Experientia 1960, 16, 521. (28) Altona, C.; Geise, H. J.; Romers, C. Tetrahedron 1968, 24, 13. (29) Giglio, E.; Quagliata, C. Acta Crystallogr., Sect. B 1975, B31, 743.

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Table 1. Torsion Angles of RbGC and RbTC Side Chains and Rings D Together with ∆ and Om (deg)a RbTC monoclinic C(13)-C(17)-C(20)-C(21) C(13)-C(17)-C(20)-C(22) C(17)-C(20)-C(22)-C(23) C(20)-C(22)-C(23)-C(24) C(22)-C(23)-C(24)-O(28) C(22)-C(23)-C(24)-N(29) C(23)-C(24)-N(29)-C(30) C(24)-N(29)-C(30)-C(31) O(28)-C(24)-N(29)-C(30) N(29)-C(30)-C(31)-O(32) N(29)-C(30)-C(31)-O(33) N(29)-C(30)-C(31)-S(32) C(30)-C(31)-S(32)-O(33) C(30)-C(31)-S(32)-O(34) C(30)-C(31)-S(32)-O(35) C(17)-C(13)-C(14)-C(15) C(13)-C(14)-C(15)-C(16) C(14)-C(15)-C(16)-C(17) C(15)-C(16)-C(17)-C(13) C(16)-C(17)-C(13)-C(14) ∆ φm

RbTC tetragonal

RbGC

first mol.

second mol.

first mol.

second mol.

-52.8 -178.8 63.5 178.5 -17.6 165.6 179.2 -79.0 2.3 177.2 -0.4

-62.3 168.2 66.4 81.4 46.1 -134.3 -175.8 -146.2 3.7

-62.1 175.6 59.3 177.4 57.3 -112.7 170.5 116.1 0.5

-59.8 177.2 -173.5 162.2 31.3 -167.9 -175.4 -128.5 -12.6

-60.4 173.2 -167.3 -77.1 -59.6 120.1 -170.2 -89.8 9.5

-175.5 61.4 -55.6 -176.6 46.5 -33.4 7.5 20.7 -40.4 16.1 47.0

-177.0 90.5 -44.3 -161.5 47.2 -34.9 9.0 20.5 -41.0 13.8 47.5

-70.4 59.4 177.9 -64.5 45.6 -35.0 10.1 17.8 -37.2 8.1 45.7

-73.8 -45.2 74.3 -163.4 45.0 -32.9 8.6 19.1 -38.2 13.0 45.3

48.3 -37.9 11.7 17.3 -39.1 5.2 48.4

a The average estimated standard deviations for RbGC, monoclinic RbTC, and tetragonal RbTC are 1.1, 1.7, and 1.9°, with maximum values of 2.1, 4.7, and 4.1°, respectively.

Figure 3. Crystal packing of the RbGC crystal viewed along b.

Five peaks which appeared in Fourier syntheses were attributed to oxygen atoms of water molecules. One of these, O(2), lay in a special position with an occupancy factor of 0.5. The final agreement factors R and Rw converged to 0.072 and 0.077, respectively. Usual values were observed for intramolecular bond lengths and bond angles, except for those of the atoms belonging to the side chain of the second molecule starting from C(58). The average estimated standard deviations were 0.02 Å and 1° with maximum values of 0.06 Å and 3°. The torsion angles of ring D, reported in Table 1, show that the anions adopt a conformation intermediate between the half-chair and the β envelope symmetry, as in the case of the monoclinic NaTC,23 NaGC,21 tetragonal RbTC (see later), and NaGDC.16 The C(17)-C(20)C(22)-C(23) torsion angle is gauche. The first four torsion angles of the second molecule, listed in Table 1, fit the same minimum region of the potential energy map of the deoxycholic acid side chain29 as those of RbGC. The first molecule populates a region at higher energy, probably in order to satisfy some requirement for a better packing energy. As in the case of RbGC, the deviation from planarity of the amide group seems to be very small for

Figure 4. Crystal packing of the monoclinic RbTC crystal viewed along b.

both the anions, since the O(28)-C(24)-N(29)-C(30) torsion angles are about 4° and 1°. The crystal packing (Figure 4) is characterized by two types of bilayers developed in planes parallel to ab and held together along c by van der Waals forces involving mainly methyl groups. The bilayers closely resemble those of RbGC and the triclinic NaTC.22 Each bilayer is composed of rows of RbTC molecules which extend along a. Translation of a row along b gives rise to a monolayer. The bilayer is formed by two antiparallel monolayers and is stabilized mainly by ion-ion, ion-dipole, and hydrogenbonding forces. Rb+ ions and water molecules fill hydrophilic channels with a small cross section. The inner surface of a channel is covered by hydroxyl and sulfonate groups and in one of the two bilayers also by carbonyl groups. These groups form hydrogen bonds involving O(25) with O(28), O(33) with O(26) and O(27), O(60) with O(63), and O(62) with O(70). The water molecules are engaged in the following hydrogen bonds: O(1) with O(25) and O(3), O(2) with two N(29) and two O(35), O(3) with O(27) and O(34), O(4) with O(61), O(62), and O(5), and

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O(5) with O(60). The Rb(1) ion is coordinated within 4.0 Å with O(26), O(28), O(33), O(34), two O(1), and O(3), while Rb(2) is coordinated with O(61), O(68), two O(69), O(70), O(4), and O(5). The number and the distances of the interactions point out that the bilayer formed by the “first” molecules is more stable than that formed by the “second” molecules. This finding could explain the greater temperature factors and estimated standard deviations of the atomic parameters observed for the “second” molecule as compared with those of the “first” one. Crystal Structure of Tetragonal RbTC. Crystal data: 2RbC26H44NO7S + 2H2O + 0.5C2H6O, fw ) 1265.4, tetragonal, P42, a ) 28.418(4) Å, b ) 28.418(4) Å, c ) 7.623(1) Å, V ) 6156(1) Å3, Z ) 4, Dc ) 1.36 g cm-3, Do ) 1.38 g cm-3, µ ) 34.3 cm-1, mp 485 K. A total of 3091 independent reflections with I > 2.5σ(I) were collected. Two sharp peaks in addition to those of the two RbTC molecules appeared in the Fourier syntheses and were assigned to oxygen atoms of water molecules. Two large and broad peaks elongated along c, centered on the 42 axes at (a ) 0, b ) 0) and (a ) 1/2, b ) 1/2), were attributed to acetone molecules with an occupancy factor of 0.25. They fill channels, covered by nonpolar groups, around the 42 axes. Since it was impossible to locate the atoms of acetone, an atomic scattering factor g was used in accordance with the Debye formula30

D’Archivio et al.

Figure 5. Crystal packing of the tetragonal RbTC crystal viewed along c.

N N

g2 )

∑ ∑fifj sin(4πrij sin θ/λ)/(4πrij sin θ/λ) i)1 j)1

where N is the number of atoms, fi and fj are the atomic scattering factors of the atoms i and j, and rij is the distance between atoms i and j. The calculation of g was accomplished by means of the function g ) 20.0 exp(-115.0s) + 6.5 exp(-10.0s) + 2.5 exp(-80.0s) + 3.0, where s ) (sin θ/λ)2. Isotropic temperature factors were given to the carbon atoms and acetone molecules during the refinement. The final agreement factors R and Rw converged to 0.075 and 0.091, respectively. The low number of reflections collected, owing to the small size of the crystal (we were not able to get a better crystal), gave rise to a few unrealistic intramolecular bond lengths. The average estimated standard deviations for intramolecular bond lengths and bond angles were 0.03 Å and 1° with maximum values of 0.04 Å and 2°, respectively. The torsion angles reported in Table 1 show that the anions adopt a conformation of ring D intermediate between the half-chair and the β envelope symmetry and a trans conformation of C(17)-C(20)-C(22)-C(23), as in the case of monoclinic RbTC. The first four torsion angles of Table 1 fit two minimum regions of the potential energy map of the deoxycholic acid side chain.29 The deviation from planarity of the amide group seems to be a little higher for both the anions than in the case of RbGC and monoclinic RbTC. The atoms C(20), C(22), C(23), C(24), N(29), and C(30) are in a nearly extended conformation in the “first” molecule, which is more elongated than the “second” one. The torsion angles can assume values lying in wide ranges,2,14,16,17,21-23 except the first three of Table 129 and the O(28)-C(24)-N(29)-C(30) torsion angle. Thus, the side chain has a high conformational flexibility. The ability of the side chain to change its elongation can favor the formation of different structural units and crystal packings suitable from an energetic point of view. The crystal packing (Figure 5) is very similar to that of (30) Debye, P. Ann. Phys. (Leipzig) 1915, 46, 809.

Figure 6. Projection of the RbTC structural unit along the a axis (top) and the c axis (bottom). A thicker line represents an anion nearer to the observer. Full or broken lines indicate ion-ion and ion-dipole or hydrogen-bonding interactions.

NaGC and is characterized by an aggregation of structural units resembling the 21 helices observed in the NaGC crystal.21 No symmetry axis is present in the structural unit (Figure 6), which can be considered as a distorted 21 helix. From now on this structural unit, the 21 helix, and the unit with a twofold rotation axis will be indicated as binary units. Aggregates of four binary units (tetramers) are formed around the twofold rotation axis by means of strong polar interactions involving the Rb+ ions. Three kind of channels are present in the ab plane (Figure 5),

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Langmuir, Vol. 12, No. 20, 1996 4665

Figure 7. Rh as a function of the concentration for aqueous solutions of NaGC, RbGC, NaTC, and RbTC (bottom) and NaGDC, RbGDC, NaTDC, and RbTDC (top). The average standard deviation is (0.3 and (0.2 Å for the trihydroxy and dihydroxy salts, respectively.

Figure 9. Rh as a function of the NaCl or RbCl molar concentration for 0.1 M aqueous solutions of NaGC, RbGC, NaTC, and RbTC (bottom) and NaGDC, RbGDC, NaTDC, and RbTDC (top). The average standard deviation is (0.3 Å for both the trihydroxy and dihydroxy salts.

Figure 8. Rh as a function of the temperature for 0.5 M NaGC, RbGC, NaTC, RbTC, NaGDC, RbGDC, NaTDC, and RbTDC aqueous solutions. The average standard deviation is (0.3 Å for both the trihydroxy and dihydroxy salts.

two nonpolar at (0, 0) and (1/2, 1/2) centered on the 42 axes and one polar at (0, 1/2) centered on a twofold rotation axis. The nonpolar ones, covered by methyl groups and filled by acetone molecules, are generated by the packing of tetramers. The polar one has a small cross section and is filled by hydroxyl and sulfonate groups together with Rb+ ions. The hydrogen bonds in the crystal structure involve O(25) with O(70), O(26) with O(63), O(27) with O(63), O(28) with O(61) and O(62), O(33) with N(29), O(35) with O(60), O(60) with N(64), and, in the case of the water molecules, O(1) with O(25), N(29), and O(62), and O(2) with O(26) and O(33). The Rb(1) ion is coordinated within 4.0 Å with O(25), O(33), O(34), O(35), O(60), O(68), two O(69), O(70), and O(1), while Rb(2) is coordinated with O(33), two O(34), O(35), O(68), O(69), O(70), and O(2). QELS Study of Dihydroxy and Trihydroxy Salts. The apparent hydrodynamic radius Rh of NaGC, RbGC, NaTC, RbTC, NaGDC, RbGDC, NaTDC, and RbTDC micellar aggregates in aqueous solutions is reported as a function of the concentration in Figure 7. The Rh values are low and slightly increase passing from a concentration of 0.1 to 0.5 M for both the dihydroxy and trihydroxy salts. The effect of the temperature is shown in Figure 8. The Rh values of a 0.5 M aqueous solution of the same salt remain constant or sligthly decrease by increasing the temperature within the range 20-60 °C. The trend of Rh as a function of the ionic strength is shown in Figure 9. The Rh values of 0.1 M NaGC, RbGC, NaTC, and RbTC aqueous solutions, containing NaCl or RbCl, are compared with those of NaGDC, RbGDC,

NaTDC, and RbTDC already published in another paper.18 Our data agree with the previous ones obtained for NaTDC with 0.15, 0.6,31 and 0.8 M NaCl32 and for NaTC with 0.15 and 0.6 M NaCl.31 CD Study of NaTDC- and NaTC-BR Aqueous Solutions. Previously, CD measurements were carried out on the interaction complexes of bilirubin-IXR (BR) with NaTDC, RbTDC, NaGC, and NaTC micellar aggregates as a function of pH and BR concentration.17,23 In the present paper we report some data on the enantioselective complexation of BR to NaTDC and NaTC as a function of added NaCl, namely under conditions corresponding to a bigger micellar size, especially for NaTDC. The BR and bile salt concentrations are 4.6 × 10-6 and 0.1 M, respectively. The pH of the aqueous solutions is within the narrow range 6.4-7.0, using a 0.05 M phosphate buffer. Within this range BR is present mainly as a biacid species33 with a ridge tile conformation.34-40 Two CD spectra of NaTDC and NaTC are shown as an example in Figure 10. Two dichroic bands of opposite sign centered at about 410-415 and 460-465 nm and a cross-over point at about 435-440 nm characterize the spectra. They, very likely, do not sensibly suffer the effects of the BR self-association, owing to its very low concentration (4.6 (31) Mazer, N. A.; Carey, M. C.; Kwasnick, R. F.; Benedek, G. B. Biochemistry 1979, 18, 3064. (32) Schurtenberger, P.; Mazer, N.; Ka˚nzig, W. J. Phys. Chem. 1983, 87, 308. (33) Hansen, P. E.; Thiessen, H.; Brodersen, R. Acta Chem. Scand. 1979, B33, 281. (34) Bonnett, R.; Davies, J. E.; Hursthouse, M. B.; Sheldrick, G. M. Proc. R. Soc. London, B 1978, 202, 249. (35) Le Bas, G.; Allegret, A.; Mauguen, Y.; De Rango, C.; Bailly, M. Acta Crystallogr., Sect. B 1980, B36, 3007. (36) Mugnoli, A.; Manitto, P.; Monti, D. Acta Crystallogr., Sect. C 1983, C39, 1287. (37) Kaplan, D.; Navon, G. Biochem. J. 1982, 201, 605. (38) Trull, F. R.; Ma, J.-S.; Landen, G. L.; Lightner, D. A. Isr. J. Chem. 1983, 23, 211. (39) Navon, G.; Frank, S.; Kaplan, D. J. Chem. Soc., Perkin Trans. 2 1984, 1145. (40) Hsieh, Y.-Z.; Morris, M. D. J. Am. Chem. Soc. 1988, 110, 1949.

4666 Langmuir, Vol. 12, No. 20, 1996

Figure 10. CD spectra of BR in NaTDC and NaTC aqueous solution without added NaCl.

Figure 11. θn as a function of added NaCl.

× 10-6 M). A preferential complexation of the left- or right-handed BR enantiomer is suggested by a bisignate circular dichroism Cotton effect (CE). The spectra of Figure 10 indicate, on the basis of the exciton coupling theory,41 that a slight enantioselective complexation of the left-handed BR enantiomer occurs, since there is a (-) longer wavelength CE followed by a (+) shorter wavelength CE. CD spectra have been recorded as a function of the ionic strength by adding NaCl within the concentration range 0.1-0.8 M. The ratio θn between the ellipticity of a sample and that of the sample without added NaCl (see Figure 10) at the point of the minimum is shown in Figure 11. Discussion All the several crystal structures of the trihydroxy salts studied so far are formed by assemblies of binary units or bilayers (see Figures 1 and 3-5). Alternatively, the bilayers (see Figures 1d, 3, and 4) are composed of an assembly of binary units, such as those of Figure 1a-c and 5, with a roughly ellipsoidal cross section. The growth of these units along a direction perpendicular to the cross section is controlled by a twofold screw axis (RbGC), a twofold rotation axis (monoclinic RbTC), and no symmetry axis (triclinic NaTC). These units are held together in each assembly mainly by strong polar interactions involving the Na+ and Rb+ ions, and are related in each assembly by twofold screw axes (the two types of bilayers of monoclinic RbTC), twofold rotation axes (RbGC), and no symmetry axes (triclinic NaTC). Therefore, the binary units can be recognized as the fundamental units in the crystal packing of the trihydroxy salts. They have an approximate shape of an elliptic cylinder and can be permeable to the water molecules in aqueous solution, (41) Blauer, G.; Wagnie´re, G. J. Am. Chem. Soc. 1975, 97, 1949.

D’Archivio et al.

since there is enough room between two adjacent anions related by a translation along the axis perpendicular to the roughly ellipsoidal cross section. The binary units are stabilized mainly by ion-ion, ion-dipole, and hydrogen-bonding forces. They have an outer surface which is polar near the end regions of the semimajor axis of the rough ellipsoidal cross section and nonpolar elsewhere. The cations are located near the polar outer surface, on the outside of the binary units, in contact with the aqueous environment. Several experimental results agree with this model. The EXAFS spectra of the monoclinic RbGC crystals are in accordance with the Rb+ coordination observed in the crystal structure presented in this paper.13 Those of the aqueous solutions, containing micellar aggregates of small size, show that the Rb+ ions are exposed to or dipped into the solvent. Their local environment and coordination are like those of RbOH in water, as is expected for aggregates with the structure of binary units.13 The structure of the bilayer can be discarded, since the Rb+ ions are inside the aggregate (see Figures 1d, 3, and 4). Moreover, crystals and micellar solution spectra satisfactorily fit a theoretical signal calculated for a coordination model of a Rb+ ion surrounded by water molecules by using a Rb-O radial distribution function obtained from molecular dynamics simulations.13 The QELS data show that the dihydroxy and trihydroxy salts without NaCl and RbCl have rather equal Rh values (Figure 7). Their estimated standard deviations (about 0.2-0.3 Å) do not allow us to substantiate sensible differences among them. However, the Rh values of the dihydroxy salts seem to be constantly a little greater than those of the trihydroxy ones (about 0.5 Å). Although the low Rh values observed for all the salts could be affected by polar and/or apolar interactions among aggregates, the experimental data point out that micellar aggregates of small size are present. Very probably, polar interactions mainly stabilize the small aggregates (Figure 8). In fact, Rh should increase as a function of the temperature within the range 20-60 °C if the growth of the micellar aggregates were controlled by hydrophobic interactions. The X-ray patterns of the dihydroxy salt fibers were interpreted by means of 7/1 helices formed by trimers. It was proposed that these helices adequately represent the corresponding micellar structures.18 Electromotive force measurements carried out on NaTDC solutions at constant ionic medium provided the distribution of micellar sizes. Most of the aggregates had aggregation numbers which were multiples of 3. The calculations of mean hydrodynamic radii using the helical model were in satisfactory agreement with QELS data at low bile salt concentration. It was also found that the Na+ ions had a greater affinity for the micellar aggregates than the Rb+ and N(CH3)4+ ions at high ionic strength. Furthermore, the X-ray and QELS data suggested a similar helical structure of the anions for the NaTDC, RbTDC, NaGDC, RbGDC, and tetramethylammonium taurodeoxycholate micellar aggregates.18 The Rh increase of the trihydroxy salts is much more moderate than that of the dihydroxy ones (Figure 9). Since NaTDC and RbTDC do not differ more than 5% up to a 0.7 M NaCl or RbCl concentration, the aggregation does not depend much on the interactions of the Na+ and Rb+ ions up to this concentration. A similar behavior is displayed by NaTC and RbTC. The Rh values of NaGDC and RbGDC are practically equal up to a 0.2 M NaCl or RbCl concentration, while NaGDC shows a faster increase than RbGDC at higher NaCl or RbCl concentration. NaGC and RbGC follow the same trend even though less markedly. Generally, the Rh values of the sodium salts

Aggregates of Glycocholate and Taurocholate Salts

Figure 12. Experimental Rh of 0.2 M NaTC as a function of NaCl molar concentration (open circles: their size corresponds to an average standard deviation of (0.3 Å). Calculated Rh for the model of the NaTC helix (Figure 1b) as a function of the aggregation number (black dots).

are larger than those of the corresponding rubidium salts. Again the Na+ ions seem to have a greater affinity than the Rb+ ions for the micellar aggregates at high ionic strength. The CD spectra of the dihydroxy salts differ remarkably from those of the trihydroxy ones,17,18 even though the spectra are recorded under conditions corresponding to small micellar sizes. By increasing the ionic strength, NaTC decreases its enantioselective ability, whereas NaTDC shows a maximum at 0.3 M NaCl (Figure 11). The presence of a maximum can be related to a structural change of the aggregates, which can assume disklike, globular, and rodlike shapes.18 These results confirm the formation of dissimilar interaction complexes between BR and a dihydroxy or trihydroxy salt. Therefore, the trihydroxy salts have a structure of the micellar aggregates different from that of the 7/1 helix proposed for NaTDC and RbTDC.17,23 On the other hand, potential energy calculations, accomplished with the 21 helix of the NaGC crystal and the left- or right-handed BR enantiomer, provided models of the NaGC-BR interaction complexes in agreement with the enantioselective complexation of BR from NaGC and NaTC observed in the CD spectra.23 The calculation of Rh for the 21 helix observed in the monoclinic crystal of NaTC23 has been accomplished in order to verify whether or not there is a possible agreement with the experimental data. The Rh values of a 0.2 M NaTC aqueous solution as a function of added NaCl are shown in Figure 12. The data are reported starting from a 0.2 M NaCl concentration in order to decrease the interparticle correlation effects due to charge interactions. The rather high NaTC concentration ensures that the size of the micellar aggregates is not too small. Rh has been computed for monodisperse systems as a function of the aggregation number assuming that the aggregates

Langmuir, Vol. 12, No. 20, 1996 4667

are prolate ellipsoids.42 The semiminor axis is 7.3 Å, the length of the radius of the circle with an area equivalent to that of the roughly ellipsoidal cross section shown in Figure 1b. The semimajor axis coincides with the 21 helical axis. The contribution of a dimer such as that of Figure 1b to the length of the prolate ellipsoid along the 21 axis is 7.7 Å, the value of the b axis (helical axis) in the crystal structure of the monoclinic NaTC.23 For all the aggregates a water shell with a 2 Å thickness has been added. The linear dependence of Rh on the aggregation number and a possible satisfactory best fit between calculated and experimental data are shown in Figure 12. A last point deserves some attention. The study of a NaTDC aqueous solution pointed out that its micellar aggregates can be represented by two types of structures with approximate shapes of oblate and prolate ellipsoids.18 The model of the binary unit proposed for the trihydroxy salts represents one type of structure different from those of NaTDC. These findings can agree with the results of a spin-label study of dihydroxy and trihydroxy bile salts.43 It was inferred that two kinds and one kind of micelle are present in the dihydroxy and trihydroxy bile salts, respectively. In fact, a strongly and a weakly immobilized probe molecule coexist in dihydroxy bile salt solutions, whereas only the weakly immobilized component was found in trihydroxy bile salt solutions. A possible explanation is that the strongly immobilized component occurs in the presence of NaTDC micellar aggregates with a helical structure,18 since it is considered to result from solubilization of the spin probe by the larger micelles,43 and that the weakly immobilized component arises in the presence of NaTDC monomers and small oligomers (such as trimers and hexamers) or trihydroxy bile salt monomers and binary units. Acknowledgment. This work was sponsored by the Italian Consiglio Nazionale delle Ricerche, Progetto Finalizzato Chimica Fine e Secondaria, and by the Italian Ministero per l’Universita` e per la Ricerca Scientifica e Tecnologica. Supporting Information Available: Listings of the final fractional atomic coordinates, the equivalent isotropic thermal parameters, and the final bond lengths and bond angles for RbGC, monoclinc RbTC, and tetragonal RbTC (16 pages); listings of observed and calculated structure factors for RbGC, monoclinic RbTC, and tetragonal RbTC (75 pages). Ordering information is given on any current masthead page. LA9600558 (42) Chu, B. Laser Light Scattering; Academic Press: New York, 1974; p 212. (43) Kawamura, H.; Murata, Y.; Yamaguchi, T.; Igimi, H.; Tanaka, M.; Sugihara, G.; Kratohvil, J. P. J. Phys. Chem. 1989, 93, 3321.