J. Phys. Chem. B 2005, 109, 9851-9852
Reply to the Comment on “Two-Dimensional NMR Study on the Structures of Micelles of Sodium Taurocholate” Noriaki Funasaki,* Makoto Fukuba, Tomohiro Kitagawa, Masao Nomura, Seiji Ishikawa, Shun Hirota, and Saburo Neya† Kyoto Pharmaceutical UniVersity, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan ReceiVed: September 30, 2004; In Final Form: February 28, 2005 Synthetic surfactants, sodium alkanoates, short-chain phospholipids, and other amphiphiles form normal micelles in aqueous media, whereas they can form reverse micelles in organic solvents. The normal micelle consists of the hydrophobic interior and the hydrophilic exterior to interact favorably with water. The reverse micelles are polar inside and apolar outside. The micellization of bile salts was explained by this traditional viewpoint: Small and others proposed the structures (back-toback) of the normal micelles of bile salts.1-6 However, Giglio and co-workers recently challenged it. They argued that bile salts form so-called reverse micelles (face-to-face) in aqueous media, similar to the structures found in their crystals.6-9 The interior region (backbone) of the helical structure contains the sodium (or alkali meatal) counterions. A close network of hydrogen bonds, together with Coulombic interactions between alkali metal cations and the carboxylate (or sulfonate) groups of the bile salt and ion-dipole interactions between the alkali metal cations and water molecules have been suggested to be factors stabilizing the helix. Surprisingly, the nonpolar faces (convex sides) of the bile salt molecules are oriented out toward the bulk aqueous medium.6 Furthermore, there are similar debates whether the dimer of bile salt has the back-to-back or face-to-face structure.3-10 The aggregation number of bile salt depends on the kind and concentration of bile salt, temperature, and the kind and concentration of inorganic salt. This topic is also controversial among many researchers.5,6 In a very recent paper, it has been revealed by two-dimensional NMR spectroscopy that the dimer and micelle of sodium taurocholate (TC) have the back-to-back structures.10 The comments by Galantini et al. on this paper may be summarized in four points.11 In the following, we state each of them and then answer it. Comment 1: Structure of the Dimer. Answer: The X-ray crystal structure of the face-to-face dimer (Supporting Information) given in Figure 5b of ref 10 was taken from ref 8 through the Cambridge Crystallographic Data Center. This is the same or close to one of the structures given in refs 8 and 9. Apart from the detailed structures, the structures of the dimer given in refs 8 and 9 look similar to that given in ref 10. These structures consist of the face-to-face association mainly by ion-ion interactions and intermolecular hydrogen bonds. Therefore, no structure of the face-to-face dimers can give so small distances (below ca. 0.6 nm) between the intermolecular * To whom correspondence should be addressed. Fax: +81-75-5954762. E-mail:
[email protected]. † Present address: Graduate School of Pharmaceutical Sciences, Chiba University, Inage-Yayoi, Chiba 263-8522, Japan.
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protons as to exhibit the NOE and ROE cross-peaks (Figure 6 in ref 10). On the other hand, the X-ray crystal structure of the backto-back dimer (Supporting Information) was taken from ref 7 through the Cambridge Crystallographic Data Center and then it was energy-optimized to give the back-to-back shown in Figure 5a of ref 10. This structure gives so small interproton distances (below ca. 0.6 nm) as to be able to exhibit the NOE and ROE cross-peaks (Figure 6 in ref 10). This structure is stabilized by hydrophobic interactions and is much distant from the face-to-face structure. Unfortunately, we do not know the detailed atomic coordinates of the TC dimers proposed in ref 9. If we had these atomic data on the TC dimer in crystals, we could calculate the interproton distances and could correlate them with our NOE data (Table 3 in ref 10). Our question is why Galantini et al. do not make these calculations to provide evidence for their comment. Comment 2: Structure of the Micelle. Answer: We could not determine the whole structures of micelles of TC by NMR and assigned typical six partial structures. Two of them (ABB and AFF) are accurately defined, but the other four structures are rather arbitrary. These are time average structures of micelles, because they change rapidly in aqueous media. Therefore, we could not determine the exact atomic coordinates of these partial and complete structures of the micelles and do not need detailed structures to estimate the presence or absence of NOE or ROE cross-peaks. The atomic coordinates of the six structures of TC dimers in ref 10 are given in the Supporting Information. In the ROESY spectrum the effect of TOCSY transfers or relays between J-coupling spins sometimes diminishes the intensity of ROE cross-peaks. For instance, the ROE/N value for the protons 1R and 1β of TC is null, because these protons are geminal-coupling with each other (Table 2 in ref 10).12,13 Comment 3: Degree of Sodium Ion Binding. Answer: The degree of counterion binding generally increases with increasing aggregation numbers, because the micellar surface charge density increases with increasing aggregation numbers. Bile salts form very small micelles, so that the degree of counterion binding is small.14-17 The degrees of sodium binding are 0.003-0.16 (pNa)14 and 0.1-0.2 (self-diffusion).16 Using a taurocholate selective electrode, Ryu et al. showed that sodium taurocholate forms small micelles with little sodium ion binding,17 as has been mentioned.18 Therefore we neglected sodium ions in molecular mechanics calculations of the TC dimer. The TC micelles could bind some ions. Bottari et al. reported large sodium ion binding of TC micelles in the presence of tetramethylammonium chloride,9 in contradiction with the measurements of pNa, self-diffusion, and a taurocholate selective electrode.15-17 As has been mentioned,18 tetramethylammonium ions as well as sodium ions could be bound to the TC micelles in the presence of large amounts of tetramethylammonium chloride, for instance, to form N(CH3)4TC4. The degrees of counterion binding reported by Bottari et al. seem too large, even if this tetramethylammonium ion binding was not taken into consideration.9 Comment 4: Aggregation Models and Aggregation Numbers. Answer: As was described on p 442 of ref 10, NMR chemical shifts are worse for estimating the aggregation model than gel filtration chromatography (GFC).5,18 The GFC data had been analyzed on the basis of stepwise aggregation models,
10.1021/jp0455374 CCC: $30.25 © 2005 American Chemical Society Published on Web 04/19/2005
9852 J. Phys. Chem. B, Vol. 109, No. 19, 2005 whereas the NMR data were analyzed on the basis of dimer plus single aggregate models. This approximation causes rather rough aggregation numbers and aggregation constants. Furthermore, the GFC experiments had been carried out in 154 mM sodium chloride solutions, whereas the NMR experiments were performed in deuterium oxide without salt. The NMR chemical shift is not an appropriate quantity to prove the absence of aggregates different from dimers and pentamers, as had been reported in ref 10. Because one can never know the “true” value of a physical quantity such as a binding constant, one must attempt to eliminate all significant systematic errors in its determination and then express its reliability in terms of a measure of its reproducibility, usually standard deviation or a related quantity such as a confidence interval. When binding constants reported by different workers, or measured by different methods are compared, it is not uncommon to find that they are (statistically) significantly different.19 According to our experiences, the dimerization constant is much more independent of aggregation models employed than those of multimerization constants. We have shown that the dimerization constants of methylene blue and chlorpromazine hydrochloride determined by GFC are close to those obtained by absorbance measurements, respectively.20 The reported dimerization constants of TC are 0.0062 (NMR),10 0.0061 (GFC),5 0.035 (light scattering),21 and 0.1 (dialysis rate) mM-1.22 Thus, the former two values are more reliable than the latter, because of their proximity. Thanks very much to Galantini et al. for pointing out several mistakes and some unclear statements in ref 10 and for giving the opportunity to correct and clarify them. The circles and triangles in Figure 3 of ref 10 should be interchanged. Reference 7 does not deal with NMR data of TC and does not propose that the TC micelles are composed of multiples of trimers. The deff value for H5β-H21 should read 0.70 nm, instead of 0.469 nm reported in ref 10. In conclusion, the dimer and micelles of TC in aqueous media have the structures of back-to-back association (normal micelle) driven by hydrophobic interactions, instead of those of faceto-face association (reverse micelle) driven by ionic interactions and hydrogen bonds. The detailed face-to-face structure of dimer of TC in Figure 5b of ref 10 would be different from that reported in refs 8 and 9. This difference, however, does not reverse the above conclusion, because it is minor in comparison with a major difference between the back-to-back and face-toface structures.
Comments Supporting Information Available: The face-to-face and initial back-to-back structures of the TC dimer and the atomic coordinates of the six dimeric structures reported in ref 10 are available free of charge via the Internet at http://pubs.acs.org. The face-to-face and initial back-to-back structures of the TC dimer were drawn on the basis of atomic coordinates given in refs 7 and 8. References and Notes (1) Small, D. M. The Physical Chemistry of Cholanic Acids. In Chemistry; The Bile Acids; Nair, P. P., Kritchevsky, D., Eds.; Plenum Press: New York, 1971; Vol. 1, Chapter 8. (2) Carey, M. C. Physical-Chemical Properties of Bile Acids and Salts. In Sterols and Bile Acids; Danielsson, H., Sjovall, J., Eds.; Elsevier: Amsterdam, The Netherlands, 1985; Chapter 8. (3) Mazer, N. A.; Carey, M. C.; Kwasnick, R. F.; Benedek, G. B. Biochemistry 1979, 18, 3064. (4) Li, G.; McGown, L. B. J. Phys. Chem. 1994, 98, 13711. (5) Funasaki, N.; Ueshiba, R.; Hada, S.; Neya, S. J. Phys. Chem. 1994, 98, 11541. (6) Hinze, W. L.; Hu, W.; Quina, F. H.; Mohammadzai, I. U. Bile Acid/Salt Surfactant Systems. In Bile Acid/Salt Surfactant Systems; Hinze, W. L., Ed.; Organized Assemblies in Chemical Analysis; Jai Press: Stamford, CT, 2000; Vol. 2, Chapter 1. (7) Campanelli, A. R.; Candeloro De Sanctis, S.; Galantini, L.; Giglio, E.; Scaramuzza, L. J. Inclusion Phenom. Mol. Recognit. Chem. 1991, 11, 247. (8) D’Alagni, M.; Galantini, L.; Giglio, E.; Gavuzzo, E.; Scaramuzza, L. Trans. Faraday Soc. 1994, 90, 1523. (9) Bottari, E.; D’Archivio, A. A.; Festa, M. R.; Galantini, L.; Giglio, E. Langmuir 1999, 15, 2996 and references therein. (10) Funasaki, N.; Fukuba, M.; Kitagawa, T.; Nomura, M.; Ishikawa, S.; Hirota, S.; Neya, S. J. Phys. Chem. B 2004, 108, 438 and references therein. (11) Galantini, L.; Giglio, E.; Pavel, N. V. J. Phys. Chem. B 2005, 109, 9849. (12) Kessler, H.; Seip, S. NMR of Peptides. In The Nuclear OVerhauser Effect in Structural and Conformational Analysis, 2nd ed.; Neuhaus, D., Williamson, M. P., Eds.; Wiley-VCH: New York, 2000; Chapter 5. (13) Croasmun, W. R., Carlson, R. M. K. Two-Dimensional NMR Spectroscopy, 2nd ed.; Wiley-VCH: New York, 1994; Chapter 9. (14) Coello, A.; Meijide, F.; Rodrı´guez Nunez, E.; Va´zquez Tato, J. J. Phys. Chem. 1993, 97, 10186. (15) Coello, A.; Meijide, F.; Rodrı´guez Nunez, E.; Va´zquez Tato, J. J. Pharm. Sci. 1996, 85, 9. (16) Lindman, B.; Kamenka, N.; Brun, B. J. Colloid Interface Sci. 1976, 56, 328. (17) Ryu, K.; Lowery, J. M.; Evans, D. F.; Cussler, E. L. J. Phys. Chem. 1983, 87, 5015. (18) Funasaki, N.; Hada, S.; Neya, S. J. Phys. Chem. 1999, 103, 169. (19) Connors, K. A. Binding Constants; John Wiley and Sons: New York, 1987; Chapter 13. (20) Funasaki, N. AdV. Colloid Interface Sci. 1986, 26, 131. (21) Chang, Y.; Cardinals, J. R. J. Pharm. Sci. 1976, 67, 174. (22) Duane, W. C.; Gilboe, D. P. Anal. Biochem. 1995, 229, 997.
