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Langmuir 1997, 13, 3090-3095
Calcium Ion Binding to Bile Salts Angelo Antonio D’Archivio,† Luciano Galantini,† Enrico Gavuzzo,‡ Edoardo Giglio,*,† and Fernando Mazza§ Dipartimento di Chimica, Universita` di Roma “La Sapienza”, P.le A. Moro 5, 00185 Roma, Italy, Istituto di Strutturistica Chimica “Giordano Giacomello” CNR, CP No. 10, 00016 Monterotondo Stazione, Roma, Italy, and Dipartimento di Chimica, Ingegneria Chimica e Materiali, Universita` di L’Aquila, 67010 L’Aquila, Italy Received July 10, 1996X The nature of the interactions between calcium ions and bile salt anions and the effect of the competition between calcium and sodium ions for possible formation of micellar aggregates are investigated. The crystal structures of calcium deoxycholate (CaDC) and glycodeoxycholate (CaGDC) and quasi-elastic lightscattering measurements, carried out on sodium deoxycholate (NaDC) and glycodeoxycholate (NaGDC) aqueous solutions containing CaCl2, are discussed. The calcium ions are coordinated to oxygen atoms of carboxylate groups and water molecules by means of ion-ion and ion-dipole interactions. The hydroxyl groups of the bile salt anions are not involved. The Ca2+-bile salt complexes fulfill both a 1:1 (CaDC) and a 1:2 (CaGDC) stoichiometry. An assembly of wedge-shaped bilayers, cemented with CaDC anions, or an assembly of irregular trimeric units, each one containing three anions, can be recognized in the crystal packing of CaDC. Ca2+ and Cl- ions and water molecules are located in liquid-like regions. The crystal packing of CaGDC is characterized by 21 helices and units with a 2-fold rotation axis. The quasi-elastic light-scattering measurements show that NaDC and NaGDC aqueous solutions with low concentrations of Ca2+ ions have micellar aggregates with apparent hydrodynamic radii remarkably greater than those of the solutions containing only Na+ ions. The Ca2+ ions seem to have a greater affinity for the deoxycholate and glycodeoxycholate anions than the Na+ ions, and the micellar aggregates with Na+ and Ca2+ ions seem to be more stable than those containing only Na+ ions. It is proposed that the Ca2+ ions act as aggregation centers of the sodium micellar aggregates.
Introduction Sodium ions, which are the predominant cations in bile, are assumed to be the cations primarily involved in the formation of bile salt micellar aggregates. Calcium ions in bile interact with various anions, including those of bile salts, reducing their activity. Physiological complexes of calcium ions with bile salts are observed in both hepatic and gallbladder bile. Generally, the calcium bile salts are stable and soluble under normal conditions.1 Experimental results have shown that a significant fraction of calcium ions in the bile is bound to bile salts, and it has been speculated that this binding may reduce the precipitation of insoluble calcium salts,2-6 which is a requisite event in the initiation and growth of pigment gallstones. Since the bile salts can act as buffers for calcium ions in bile, the complexation of calcium ions by bile salts in aqueous solution has been investigated by using Ca2+specific electrodes.2,4,5,7 It is well-known that the carboxylate group has a higher affinity for Ca2+ than other anionic groups. Surprisingly, a somewhat higher Ca2+ binding affinity for taurocholate than for glycocholate has been observed. This has been explained assuming that Ca2+ could be more easily interposed between a cholanic ring hydroxyl group and the sulfonate group of taurocholate rather than the carboxylate group of glycocholate, †
Universita` di Roma “La Sapienza”. Istituto di Strutturistica Chimica “Giordano Giacomello” CNR. § Universita ` di L’Aquila. X Abstract published in Advance ACS Abstracts, April 15, 1997. ‡
(1) Hofmann, A. F. Gastroenterology 1965, 48, 484. (2) Williamson, B. W. A.; Percy-Robb I. W. Biochem. J. 1979, 181, 61. (3) Williamson, B. W. A.; Percy-Robb I. W. Gastroenterology 1980, 78, 696. (4) Rajagopalan, N.; Lindenbaum, S. Biochim. Biophys. Acta 1982, 711, 66. (5) Moore, E.; Celic, L.; Ostrow, J. D. Gastroenterology 1982, 83, 1079. (6) Kahn, M. J.; Lakshminarayanajah, N.; Trotman, B. W.; Chun, P.; Kaplan, S. A.; Margulis, C. Hepathology 1982, 2, 732. (7) Moore, E. Hepathology 1984, 4, 228S.
S0743-7463(96)00676-2 CCC: $14.00
since the taurocholate side chain is longer and more flexible than that of glycocholate.8 In contrast, the analysis of lanthanide-induced paramagnetic nuclear magnetic resonance effects indicates that Ca2+ has a greater affinity for glycocholate than for taurocholate and suggests that Ca2+ is bound to the anionic group and not to any hydroxyl group of the bile salt anion.9 Moreover, the data obtained from measurements using Ca2+-specific electrodes5,7 and from the X-ray analysis of crystals, grown from mixtures of saturated CaCl2 and sodium cholate solutions,10 are consistent with a 1:1 stoichiometry of the Ca2+-bile salt complex. Valuable information about the solubility and the phase behavior of the calcium salts of some glycine conjugated bile acids is also available.11 Their precipitation is slow and often occurs after the formation of metastable viscoelastic gels, with a long time required to reach equilibrium. Furthermore, precipitation is prevented or reduced by calcium ion binding to monomers or micellar aggregates, whose formation is favored with increased sodium ion concentration.11 This work represents a preliminary study on the role of calcium in the biliary milieu.1-11 Some interesting facets are the nature of the interactions between calcium ions and bile salt anions, the size of their micellar aggregates compared with that of the sodium salts, and the effect of a possible competition between calcium and sodium ions for the formation of the micellar aggregates. Here we present the determination of the crystal structures of calcium deoxycholate (CaDC) and glycodeoxycholate (CaGDC) together with quasi-elastic light-scattering (8) Moore, E.; Ostrow, J. D. Gastroenterology 1985, 88, 1679. (9) Mukidjam, E.; Barnes, S.; Elgavish G. A. J. Am. Chem. Soc. 1986, 108, 7082. (10) Hogan, A.; Ealick, S. E.; Bugg, C. E.; Barnes, S. J. Lipid Res. 1984, 25, 791. (11) Jones, C. A.; Hofmann, A. F.; Mysels, K. J.; Roda, A. J. Colloid Interface Sci. 1986, 114, 452.
© 1997 American Chemical Society
Calcium Ions Binding to Bile Salts
(QELS) measurements carried out on sodium deoxycholate (NaDC) and glycodeoxycholate (NaGDC) aqueous micellar solutions with added CaCl2. Experimental Section Materials. NaDC and NaGDC (Sigma) were crystallized from a mixture of water and acetone. NaCl (Merck, Suprapur) and CaCl2‚2H2O (Sigma, purity about 99%) were used. CaDC and CaGDC were obtained by adding CaCl2 to aqueous solutions of NaDC and NaGDC. Crystals of CaDC were grown by slow evaporation of a mixture of water and acetone. Those of CaGDC were grown by slow evaporation of the same mixture (CaGDCA) or of a mixture of water, acetonitrile, and isopropyl alcohol (CaGDCB). Fibers of CaGDC were drawn from an aqueous suspension of CaGDC crystalline powder that swelled upon wetting. X-ray Measurements. X-ray data of CaDC and CaGDC crystals were collected at room temperature, using Cu KR radiation (λ ) 1.5418 Å), by means of a Rigaku AFC5R diffractometer, equipped with a 12 kW rotating anode and a graphite monochromator. Cell constants and an orientation matrix were obtained from a least-squares fit of the angular settings of 25 carefully centered reflections in the range 35.0 e 2θ e 47.5°. Intensity data were collected by the ω-2θ mode at a variable and appropriate speed to a maximum 2θ of 124°. Stationary background counts were recorded on each side of the reflection. The peak counting was twice that of the background. Reflections with I < 20σ(I) (CaGDCA), 10σ(I) (CaGDCB), and 25σ(I) (CaDC) were rescanned with an accumulation of counts to improve counting statistics. The refinement consisted of 3243 reflections with I > 2.0σ(I) (CaGDCA), 3361 with I > 1.5σ(I) (CaGDCB), and 5123 reflections with I > 4.5σ(I) (CaDC). The intensities of three standard reflections, measured after every 147 reflections, were found to be constant throughout data collection for CaGDCA, and therefore, no decay correction was applied. Since those of CaGDCB and CaDC decreased by about 4% and 23%, respectively, a linear correction factor was used. An empirical absorption correction, based on azimuthal scans of several reflections, was applied to CaGDCA and CaGDCB intensities. Owing to the low absorption, no correction was applied to CaDC intensities. The data were corrected for Lorentz and polarization effects. The structures were solved by direct methods using the program SIR9212 and refined anisotropically by the full-matrix least-squares method with the program SIR-CAOS13 (CaGDCA and CaGDCB) and TEXSAN14 (CaDC). Acetonitrile and isopropyl alcohol in the CaGDCB crystal as well as acetone and 11 oxygen atoms of water molecules in the CaDC crystal were refined isotropically. Since the atoms of acetone were not located in the CaGDCA crystal, an atomic scattering factor g was used in accordance with the Debye formula16 N
g2 )
N
∑ ∑ f f sin(4πr i j
ij
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. g is calculated 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 ) (sinθ/λ)2. The function minimized was Σw(|Fo| - |Fc|)2, with w ) (sin θ/λ)2 (CaGDCA and CaGDCB) or w ) 1 (CaDC). Some other weighting schemes gave slightly worse results. (12) Altomare, A.; Cascarano, G.; Giacovazzo, C.; Guagliardi, A.; Burla, M. C.; Polidori, G.; Camalli, M. J. Appl. Crystallogr. 1994, 27, 435. (13) 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. (14) TEXSAN: TEXRAY Structure Analysis Package, Molecular Structure Corporation, 1985. (15) International Tables for X-ray Crystallography; Kynoch Press: Birmingham, England, 1974; Vol. IV. (16) Debye, P. Ann. Phys. (Leipzig) 1915, 46, 809.
Langmuir, Vol. 13, No. 12, 1997 3091 Atomic scattering factors and anomalous dispersion coefficients were taken from ref 15. The correction for the real and imaginary parts of the anomalous dispersion was applied. The hydrogen atoms were generated at the expected positions, except the water and hydroxyl hydrogens which were not taken into account in the calculations. Their atomic coordinates were not refined and their thermal parameters were set equal to the isotropic ones of the carrier atoms. The final agreement factors R and Rw were 0.068 and 0.078 for CaDC, 0.083 and 0.093 for CaGDCA, and 0.081 and 0.093 for CaGDCB, respectively. The bond distances and bond angles were in satisfactory agreement with those usually reported in the literature. Listings of the observed and calculated structure factors, atomic fractional coordinates together with equivalent isotropic thermal parameters, and bond lengths and bond angles are available as Supporting Information for CaDC and CaGDCB (see the paragraph at the end of the paper). Since there are up to three anions in the asymmetric units, the atomic numbers of the first, second, and third anion are obtained by adding 100, 200, and 300 to the common atom labeling of the bile salts, respectively. The CaGDC fiber is a preferentially oriented microcrystalline specimen. X-ray diffraction photographs were taken on air-dried fibers and recorded on flat films by means of a Buerger precession camera, using Ni-filtered Cu KR radiation. 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 the Lexel Corp. 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 at 25 ( 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° in our experimental conditions. The scattering decays were analyzed by means of cumulant expansion up to the second order, because higher order contributions did not improve the statistics. The results were reported in terms of the apparent hydrodynamic radius obtained by the Stokes-Einstein relationship.
Results Crystal Structure of CaDC. Crystal data: 3Ca2++ 3Cl- + 3(C24H39O4)- + 16H2O + C3H6O, triclinic, P1, a ) 18.089(4) Å, b ) 18.888(5) Å, c ) 7.411(3) Å, R ) 95.89(3)°, β ) 97.30(2)°, γ ) 68.48(2)°, V ) 2332(1) Å3, Z ) 1, Dc ) 1.24 g cm-3, µ ) 2.9 mm-1, mp 537 K. Fourier syntheses showed all the non-H atoms. Two Cl- were easily located. The coordinates of two peaks with nearly half the electron density of the two Cl- were attributed to the third Cl- (hereafter indicated as Cl-1/2). An occupancy factor of 0.5 was assigned to Cl-1/2 in the two positions, which were surrounded by oxygen atoms of water molecules. The Cl-‚‚‚O distances were typical of hydrogen bonding, being 3.0-3.3 Å long. It was observed that some oxygen atoms of water molecules interact with both Cl-1/2 and have high thermal parameters, since each of these oxygen atoms occupies two close sites. In Table 1 are listed the side chain and ring D torsion angles of the three CaDC anions in agreement with the convention of Klyne and Prelog,17 together with the phase angle of pseudorotation ∆ and the maximum angle of torsion φm according to Altona, Geise, and Romers.18 The conformation of ring D is intermediate between the halfchair and the β envelope symmetry for the first and the third CaDC anion and is very close to the half-chair symmetry for the second CaDC anion.18 The first four torsion angles of Table 1 fit two minimum regions of the (17) Klyne, W.; Prelog, V. Experientia 1960, 16, 521. (18) Altona, C.; Geise, H. J.; Romers, C. Tetrahedron 1968, 24,13.
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Table 1. Torsion Angles (deg) of CaDC and CaGDCB Side Chains and Rings D Together with ∆ and Om CaDC 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(27) C(22)-C(23)-C(24)-O(28) C(22)-C(23)-C(24)-N(28) C(23)-C(24)-N(28)-C(29) C(24)-N(28)-C(29)-C(30) O(27)-C(24)-N(28)-C(29) N(28)-C(29)-C(30)-O(31) N(28)-C(29)-C(30)-O(32) 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
CaGDCB
first anion
second anion
third anion
first anion
second anion
-55(2) -179(1) -173(1) 179(1) -42(2) 143(1)
-61(2) 176(1) -162(1) 171(1) 167(1) -17(2)
-61(2) 174(1) 177(1) 62(2) -116(2) 63(2)
-58(1) 175(1) -161(1) 177(1) -9(2)
-63(2) 174(1) -167(1) -174(2) 48(3)
174(1) 177(1) -118(1) 0(2) -31(2) 153(1) 45(1) -32(1) 7(1) 20(1) -39(1) 16 45
-132(2) -179(2) 86(2) 2(3) 13(2) -169(1) 46(1) -34(2) 7(2) 20(1) -40(1) 15 46
47(1) -35(1) 8(1) 20(2) -40(1) 13 47
46(1) -38(1) 14(1) 13(2) -35(1) -3 46
Figure 1. Crystal packing of the CaDC crystal viewed along c.
potential energy map of the deoxycholic acid side chain.19 The side chain of two anions is in a nearly extended conformation, whereas that of the third one assumes a gauche conformation. The crystal packing (Figure 1) is characterized by wedgeshaped bilayers, which extend into planes parallel to bc. The bilayers contain Ca2+ and Cl- ions, acetone, and water molecules in their interior, and give rise to empty channels, running along c, which are filled by deoxycholate anions. Two Ca2+ ions strongly interact with carboxylate groups and water molecules and are more stable than the third Ca2+ ion, which is coordinated only to water molecules (Figure 2). Probably, the positions of this Ca2+ ion and its coordinated water molecules can slightly change, depending on which Cl-1/2 is present. Alternatively, the
44(1) -29(1) 4(2) 23(2) -40(1) 25 45
crystal packing is characterized by an assembly of trimers. Two types of trimers (T1 and T2) are the most stable. T1 is composed by the molecules of the asymmetric unit (indicated as A, B, and C in Figure 1), and T2 by B, C and the molecule A at x + 1. T1 and T2 are stabilized mainly by polar interactions. Ca2+ and Cl- ions, and water molecules form liquid-like regions, incorporated within the wedge-shaped bilayers, and give rise to ion-ion and ion-dipole interactions together with an efficient network of hydrogen bonds. Acetone is firmly anchored by hydrogen bonds to two water molecules. Crystal Structures of CaGDC. Crystal data for CaGDCB: Ca2+ + 2(C26H42NO5)- + 3H2O + C3H8O + 0.5C2H3N, monoclinic, C2, a ) 42.130(6) Å, b ) 7.741(1) Å, c ) 20.214(3) Å, β ) 116.42(1)°, V ) 5904(1) Å3, Z ) 4, Dc ) 1.21 g cm-3, µ ) 1.4 mm-1, mp 479 K. Crystal data for CaGDCA: Ca2+ + 2(C26H42NO5)- + 3H2O + C3H6O, monoclinic, C2, a ) 42.260(6) Å, b ) 7.724(1) Å, c ) 20.147(4) Å, β ) 116.54(2)°, V ) 5884(2) Å3, Z ) 4, Dc ) 1.19 g cm-3, µ ) 1.3 mm-1, mp 479 K. Acetonitrile is absent in CaGDCA. Acetone in CaGDCA replaces isopropyl alcohol of CaGDCB. The atomic coordinates of CaGDC and of the oxygen atoms of water molecules in the two crystal structures are practically equal. Therefore, only the CaGDCB crystal structure is reported. All the atoms were located by Fourier syntheses. An occupancy factor of 0.5 was assigned to the atoms of acetonitrile found at a ) c ) 0. The atoms of acetonitrile and isopropyl alcohol did not form close contacts with the surrounding atoms, and had high thermal parameters. The two CaGDC anions (Table 1) adopt a conformation of ring D intermediate between the half-chair and the β envelope symmetry.18 The first four torsion angles of Table 1 fit the lowest minimum of the potential energy map of the deoxycholic acid side chain.19 The side chain of the first and second anion is in a nearly extended conformation up to C29 and C24, respectively. The side chain conformation of the second anion is somewhat similar to that observed in the crystal structures of sodium glycodeoxycholate20 and glycocholate.21 The flexibility of the side (19) Giglio, E.; Quagliata, C. Acta Crystallogr., Sect. B 1975, B31, 743. (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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Figure 3. Crystal packing of the CaGDCB crystal viewed along b.
Figure 2. View of the coordination of the calcium ions with the nearest neighbor oxygen atoms in the CaDC crystal. The atomic numbering of the oxygen atoms is shown. The Ca2+‚‚‚O distances (Å) are reported.
chain, verified in many other crystal structures,10,20-29 plays an important role in the formation of various structural patterns which can accommodate different cations. A 21 helix and a unit with a 2-fold rotation axis (2-fold unit), previously observed in several crystal structures of (22) D’Alagni, M.; D’Archivio, A. A.; Giglio, E.; Scaramuzza, L. J. Phys. Chem. 1994, 98, 343. (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.; Giglio, E.; Gavuzzo, E.; Scaramuzza, L. Langmuir 1996, 12, 4660. (25) Cobbledick, R. E.; Einstein, F. W. B. Acta Crystallogr., Sect. B 1980, B36, 287. (26) Coiro, V. M.; Giglio, E.; Morosetti, S.; Palleschi, A. Acta Crystallogr., Sect. B 1980, B36, 1478. (27) Campanelli, A. R.; Candeloro De Sanctis, S.; Giglio, E.; Petriconi, S. Acta Crystallogr., Sect. C 1984, C40, 631. (28) Campanelli A. R.; Candeloro De Sanctis, S.; Giglio E.; Scaramuzza, L. J. Lipid Res. 1987, 28, 483. (29) Campanelli, A. R.; Candeloro De Sanctis, S.; D’Archivio, A. A.; Giglio, E.; Scaramuzza, L. J. Inclusion Phenom. Mol. Recognit. Chem. 1991, 11, 247.
Figure 4. Projection of the CaGDCB 21 helix along an axis perpendicular to the helical axis (top) and along the helical axis (bottom). A thicker line represents an anion nearer to the observer. Broken lines indicate hydrogen bonds.
dihydroxy and trihydroxy salts of bile acids,21-24 can be recognized in the crystal packing (Figure 3). The 21 helix, which is chiefly stabilized by hydrogen bonds (Figure 4), resembles those of sodium glycocholate21 and taurocholate,23 and to less extent those of rubidium taurodeoxycholate.22 It has an approximate ellipsoidal cross section perpendicular to the helical axis and an outer surface which is mainly polar around the end regions of the semimajor axis and nonpolar elsewhere. The helix can be permeable to the water molecules of the solvent because of the sufficient separation between two adjacent anions related by a translation of 7.74 Å along the helical axis (Figure 4, top) and can be easily filled with water molecules in an aqueous medium. The 2-fold unit (Figure 5) presents the same characteristics. However, it accommodates acetonitrile and isopropyl alcohol in its interior, giving rise to a semiminor axis longer than those of similar units
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Figure 7. Rh as a function of NaCl concentration for the 0.1 M NaDC and NaGDC aqueous solutions. The maximum standard deviation is (0.2 Å.
Figure 5. Projection of the CaGDCB 2-fold unit along an axis perpendicular to the 2-fold rotation axis (top) and along the 2-fold rotation axis (bottom). A thicker line represents an anion nearer to the observer. Broken lines indicate hydrogen bonds.
Figure 8. Rh as a function of CaCl2 concentration for the 0.1 M NaDC aqueous solution. The maximum standard deviation is (0.3 Å.
induces precipitation after a short time in 0.1 M NaDC and NaGDC aqueous solutions when the Ca2+ concentration is greater than 2.5 and 35 mM, respectively. Since it was reported that under different conditions the precipitation occurs slowly after the intermediate formation of metastable, nonmicellar viscoelastic gels,11 the QELS data were collected within 45 min of solution preparation. The Rh values of 0.1 M NaDC and NaGDC aqueous solutions vs CaCl2 concentration are shown in Figures 8 and 9, respectively. Figure 6. View of the coordination of the calcium ion with the nearest neighbor oxygen atoms in the CaGDCB crystal. The atomic numbering of the oxygen atoms is shown. The Ca2+‚‚‚O distances (Å) are reported.
observed in the crystal of sodium taurocholate.23 The guest molecules form some hydrogen bonds (Figure 5) but are weakly anchored to the CaGDC anions. The Ca2+ ions are piled into narrow hydrophilic channels, as that at a ) 1/4 and c ) 1/2 of Figure 3, and are coordinated to oxygen atoms of carboxylate groups and water molecules (Figure 6). Each Ca2+ ion acts as a center of strong attraction for several bile salt anions. QELS Study of NaDC and NaGDC Aqueous Solutions with CaCl2. The apparent hydrodynamic radius Rh of 0.1 M NaDC and NaGDC aqueous solutions vs NaCl concentration is shown in Figure 7. The trend of Rh is very similar for the two salts. The addition of CaCl2 gives information about the effect of the Ca2+ ions on the size of the NaDC and NaGDC micellar aggregates. CaCl2
Discussion The CaDC, CaGDCA, and CaGDCB crystal structures are examples of a 1:1 (CaDC) and a 1:2 (CaGDC) stoichiometric ratio of the Ca2+-bile salt complexes. However, the “local” stoichiometric ratio is lower. In fact, a “local” stoichiometric ratio 2:3 (CaDC) and 1:4 (CaGDC) can be recognized by inspection of the crystal packings (Figures 1 and 3). The Ca2+ ions of CaGDC, located within the hydrophilic channel at a ) 1/4 and c ) 1/2 (Figure 3), promote a strong aggregation of pairs of 21 helices and 2-fold units (two Ca2+ ions bind eight anions). The CaDC packing can be considered as an assembly of T1 or T2 trimers. T1 is mainly stabilized by strong ion-ion and ion-dipole interactions involving two Ca2+ ions. Each Ca2+ ion is bound to two or three carboxylate groups and to water molecules (Figure 2). T2 is stabilized by less polar and more apolar interactions than T1 (Figure 1). In conclusion, the CaDC, CaGDCA, and CaGDCB crystal structures are examples of the remarkable ability of the Ca2+ ions to interact with several carboxylate groups and
Calcium Ions Binding to Bile Salts
Figure 9. Rh as a function of CaCl2 concentration for the 0.1 M NaGDC aqueous solution. The maximum standard deviation is (0.3 Å.
provide possible coordination models of the Ca2+ ions in aqueous micellar solutions. No Ca2+ binding to any of the hydroxyl groups of the anions is observed, in agreement with an NMR study of the binding of Na+ and Ca2+ to glycocholate and taurocholate anions,9 and with the crystal structure of calcium cholate chloride heptahydrate.10 On the other hand, neither the Na+ nor the Rb+ ion binds simultaneously to the carboxylate or sulfonate group and to a hydroxyl group of the same anion in the crystal structures of sodium and rubidium salts of cholic, deoxycholic, glycocholic, taurocholic, glycodeoxycholic, and taurodeoxycholic acids solved up to now.20-29 Therefore, the interposition of a Ca2+ ion between the carboxylate or sulfonate group and a cholanic ring hydroxyl group of the same anion7,8 seems to be improbable. The free glycocholate and taurocholate anions (and perhaps small oligomers as dimers and trimers) have much higher affinity for Ca2+ than their respective micellar aggregates.7 Of course, a Ca2+ ion can interact more easily with the carboxylate groups of free anions than with those of ordered micellar aggregates of the sodium salt, since in the latter case the carboxylate groups could be approached by the Ca2+ ions with difficulty. Thus, the addition of Ca2+ ions to NaDC and NaGDC micellar solutions could give rise initially to the preferential binding of the Ca2+ ions to free anions and small oligomers and, subsequently, to the binding to micellar aggregates. NaDC and NaGDC (0.1 M ) aqueous solutions show practically equal Rh values as a function of NaCl concentration (Figure 7). However, NaGDC displays a greater resistance to precipitation than NaDC when CaCl2 is added. The Rh values of NaDC slightly increase as a function of CaCl2 concentration (Figure 8). On the contrary, they remain practically constant up to 7.5 mM NaCl concentration, which approximately corresponds to 2.5 mM CaCl2 concentration as far as the ionic strength is concerned. A much larger effect is shown by NaGDC (Figure 9). The Rh value suddenly increases from 20 to 78 Å passing from 20 to 35 mM CaCl2 concentration. The value of 78 Å is greater than that corresponding to a 0.7 M NaCl concentration (Figure 7). This somewhat surprising result seems to indicate that NaGDC micellar aggregates are held together by means of Ca2+ ions. Two linear trends with different slopes can be recognized for the data of Figure 9. One, corresponding to lower CaCl2
Langmuir, Vol. 13, No. 12, 1997 3095
concentrations, could originate from the initial binding of the Ca2+ ions mainly to free anions and to small oligomers of NaGDC. The other could reflect the aggregation of the NaGDC micellar aggregates by means of the Ca2+ ions. In fact, a mere substitution of the Na+ ions with the Ca2+ ions cannot be invoked, because the ratio Ca2+/ Na+ is less than 0.5 (not more than 0.35). If this hypothesis is correct, NaDC shows only the preferential binding of the Ca2+ ions to its free anions and small oligomers, owing to the low concentrations of added CaCl2 (Figure 8). A last point deserves some attention. The structural units of a fiber are more similar to the micellar aggregates than those of a crystal. An identity period of 7.0 Å has been observed in the helical fiber of CaGDC. This value is different from those of the 21 helices and 2-fold units found in the crystals of CaGDC (7.74 Å) and in some sodium and rubidium salts of bile acids (7.54-7.94 Å)21-24 and is nearly equal to that observed in the helices of a fiber obtained when HCl is added to aqueous micellar solutions of NaTDC30 as well as in the 31 helices of a NaTDC crystal (7.1 Å).28 Thus, the structural units observed in the CaGDC crystals do not represent the structure of possible CaGDC micellar aggregates, and it is more probable that the CaGDC helix is formed by trimers with a 3, 31, or 32 symmetry axis. Conclusions The main results of this preliminary study can be summarized as follows: (i) The coordination of Ca2+ ions, found in the CaDC and CaGDC crystal structures, reveals that each Ca2+ ion is a center of strong attraction for the anions. The ability of the Ca2+ ions to interact with several anions is inferred from the “local” stoichiometric ratio Ca2+/anion, which is as low as 1/4 for CaGDC. The Ca2+ ions are not bound to hydroxyl groups of the anions. (ii) The structural units observed in the CaGDC crystals are not representative models of possible CaGDC micellar aggregates as shown by the X-ray pattern of the CaGDC fiber. (iii) Very probably, the addition of Ca2+ ions to NaDC and NaGDC aqueous micellar solutions causes the binding of the Ca2+ ions initially to free anions and small oligomers and, subsequently, to micellar aggregates. The QELS data can be reasonably explained if NaGDC micellar aggregates are held together by means of Ca2+ ions. 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 and equivalent isotropic thermal parameters for CaDC, and CaGDCB, and listings of the final bond lengths and bond angles for CaDC and CaGDCB (12 pages); listings of observed and calculated structure factors for CaDC and CaGDCB (60 pages). Ordering information is given on any current masthead page. LA9606761 (30) Briganti, G.; D’Archivio, A. A.; Galantini, L.; Giglio, E. Langmuir 1996, 12, 1180.
