J. Phys. Chem. 1996, 100, 11675-11680
11675
Thermotropic Structural Change of Disialoganglioside Micelles Studied by Using Synchrotron Radiation Small-Angle X-ray Scattering Mitsuhiro Hirai,*,† Toshiharu Takizawa,† Sadato Yabuki,† Toshihiro Hirai,‡ and Kouhei Hayashi† Department of Physics, Gunma UniVersity, Maebashi 371, Japan, and Faculty of Textile Science and Technology, Shinshu UniVersity, Ueda 386, Japan ReceiVed: January 24, 1996; In Final Form: March 29, 1996X
This work treats the thermotropic structural change of the disialoganglioside (GD1) micelles studied by using synchrotron radiation X-ray scattering. Scattering data and analyses show that thermotropic structural change around 20 °C accompanies the contraction of the whole micellar dimension and the internal structural change of the micelles. Double-shell modeling analyses reasonably describe the observed changes in the experimental scattering curves, distance distribution functions, and structural parameters. The modeling analyses show that the major changes of the micellar structure induced by the elevation of temperature are the contraction of the hydrophilic region and the increase of the average scattering density, suggesting the change of the oligosaccharide chain conformation from an extended one to a folded one. Simultaneously, the minor extension of the hydrophobic region occurs with a slight increase of the average scattering density, suggesting a minor rearrangement of the hydrocarbon chains. We assume that such a thermotropic structural change around room temperature will be a typical characteristic of glycolipids containing long oligosaccharide chains with sialic acid residues in hydrophilic heads, which would affect stability of membrane structure and relate to promotion of various physiological surface events in biomembranes.
Introduction Gangliosides, the most complex of the glycosphingolipids, have been attracting scientific concerns on those physiological functions and studied intensively by many investigators.1 Gangliosides are acidic glycolipids composed of a ceramide linked to oligosaccharide chain containing one or more Nacetylneuraminic acid (called sialic acid) residues, which are very hygroscopic compared to other membrane lipids. They are most abundant in the plasma membrane of nerve cells with 5-10% of the total lipid mass, while they are also found widely in most vertebrate cell types.2 Through a numerous variety of those structures in both oligosaccharide and lipid moieties, gangliosides are considered to be involved in the self-organization of tissues, immune response, and cell differentiation through molecular recognition.3-6 Many studies have been carried out to clarify physicochemical and thermotropic properties of gangliosides and of mixtures of gangliosides with phospholipids.7 Those studies showed the presence of gangliosides in phospholipid systems greatly affect thermotropic phase behaviors;8-11 however, there are sometimes confused and conflicting results with each other.10,11 It may be attributable not only to varieties in ganglioside characteristics depending on species but also to some ambiguity of the knowledge of the ganglioside structures. In spite of many studies, it seems that structural properties of ganglioside aggregates are still ambiguous and that there is little direct evidence of those structures based on appropriate experimental data and analyses, as far as we know. By using scattering techniques, calorimetry, and NMR spectroscopy, we have studied structures and functions of micellar and lamellar systems in connection with hydrodynamic properties and with phase behaviors, where we have treated other * Corresponding author. Fax: INT+81 272-20-7405. Phone: INT+81 272-20-7554. E-mail:
[email protected]. † Gunma University. ‡ Shinshu University. X Abstract published in AdVance ACS Abstracts, June 15, 1996.
S0022-3654(96)00235-3 CCC: $12.00
amphiphilic phospholipid systems,12-15 a surfactant/protein/ water systems16 and surfactant/protein/organic solvent system.17-18 In the neutron and X-ray scattering studies treating the ganglioside mixture (monosialogangliosides (GM) and disialogangliosides (GD1)) in aqueous solution, we report the ellipsoidal micellar structure of ganglioside aggregates with a strong repulsive interparticle interaction19 and the complexation process of gangliosides with proteins.20 In this paper we will show a thermotropic structural stability of disialoganglioside (GD1) aggregates in aqueous solution studied by using synchrotron radiation small-angle X-ray scattering. We have found a thermotropic structural change of the ganglioside micelles at relatively low temperature around 20 °C. By using shellmodeling analyses the structural changes in the hydrophilic and hydrophobic regions of the micelle will be discussed separately. The present findings on the thermotropic stability of gangliosides are very different from those of general phospholipids, which would relate to various physiological functions through modulation of structure and stability of biomembranes by gangliosides. Experimental Section Sample Preparation. As we have described elsewhere,21 the gangliosides from bovine brain were extracted and purified by the partition technique using Svennerholm’s method.22 The chemical structures and contents of various gangliosides (monosialoganglioside, disialoganglioside, and trisialoganglioside) were checked by using thin-layer chromatography. The present sample was the mixture of equimolecular amounts of two types of disialogangliosides (IV3NeuAc-, II3NeuAc-GgOse4-Cer, and II3NeuAc2-GgOse4-Cer abbreviated as GD1a and GD1b). The lyophilized powder of the mixture with 0.5% w/v was dissolved in 50 mM Hepes (N-(2-hydroxymethyl)piperazine-N′-(2-ethanesulfonic acid)) buffer adjusted to pH 6.8 and used for the scattering experiments. Small-Angle X-ray Scattering Measurements. Small-angle X-ray scattering experiments were performed by using the © 1996 American Chemical Society
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synchrotron radiation small-angle X-ray scattering spectrometer installed at BL10C line of the 2.5 GeV storage ring at the Photon Factory at the National Laboratory for High Energy Physics, Tsukuba, Japan. The details of the instruments are shown elsewhere.23 The wavelength used was 1.49 Å and the sampleto-detector distance was 87 cm. The sample was contained in a sample cell with a pair of mica windows, which were placed into a cell holder. Each exposure time for one measurement was 100 s and the integrated exposure time was 1200 s. The temperature of the sample was controlled from 6.0 to 60 °C by a thermostat within the precision below 0.5 deg. Methods of Scattering Data Analyses and Modeling. The scattering data were analyzed according to the following standard methods. The beginning of the scattering curve I(q) depends on the Guinier equation in the form
I(q) ) I(0) exp(-q2Rg2/3)
(1)
where I(0) designates the zero-angle scattering intensity, Rg the radius of gyration, and q the magnitude of scattering vector defined by q ) (4π/λ) sin(θ/2) (θ, the scattering angle; λ, the wavelength). Usually we determine both I(0) and Rg values by using the Guinier plot (ln(I(q)) vs q2). Here we used the data sets in the q interval of 0.020-0.025 Å-1 for this plot. The distance distribution function p(r) was calculated by the Fourier inversion of the scattering intensity I(q) as
p(r) ) (2/π)∫0 rqI(q) sin(rq) dq ∞
(2)
The p(r) function reflects the particle shape, the intraparticle scattering density distribution, and the interparticle correlation.24 The maximum dimension Dmax of the particle is estimated from the p(r) function satisfying the condition p(r) ) 0 for r > Dmax. For the calculation of the p(r) function, the extrapolation of the small-angle data sets by using the Guinier plot and the modification of the scattering intensity as
I′(q) ) I(q) exp(-kq2)
(3)
(k is the artificial damping factor) were employed to reduce the Fourier truncation effect. The indirect Fourier transform method (the Glatter’s method) was also applied to avoid inherent systematic artifacts on the estimation of Rg and Itotal that often occur in the use of the Guinier approximation.24,25 Rg and Itotal are given as
Itotal ) ∫0
Dmax
∫0D ) D 2∫0
max
2
Rg
p(r) dr
(4)
p(r)r2 dr
max
(5) p(r) dr
Equation 4 was used for normalization of the p(r) functions. The shell-modeling method was also used for fitting the experimental data to determine structural parameters of the objects. As we have shown elsewhere,16,26 based on the convolution theory the spherically averaged scattering function I(q) from of a particle composed of n shells with different average scattering densities is simply given as
I(q) ∝ 〈A(q)A*(q)〉 ) ∫0 [3{Fh1V1j1(qR1)/(qR1) + 1
n
(Fhi - Fhi-1)Vij1(qRi)/(qRi)}]2 dx ∑ i)2
(6)
Figure 1. Temperature dependence of the scattering curve I(q) and the distance distribution function p(r) of the disialogangliosides (GD1) in 50 mM Hepes buffer at pH 6.8 from 6.0 to 60 °C: (a) I(q), (b) p(r). The inserts show the difference between the profiles for 6.0 and 60 °C.
where 〈 〉 means the spherical average of the scattering intensity I(q) defined by A(q)A*(q) (the structural factor A(q)), Fhi is the average excess scattering density (so-called contrast) of ith shell with a shape with an ellipsoid of rotation, and j1 is the spherical Bessel function of the first rank. Ri is defined as
Ri ) ri(1 + x2(νi2 - 1))1/2
(7)
where ri and νi are the semiaxis and its ratio of ith ellipsoidal shell, respectively. Here we assumed a simplified model, namely, a double-shell ellipsoidal structure to fit the experimental scattering data by using eq 6. Fhi, ri and νi were used as fitting parameters. Although this modeling method cannot avoid a systematic disagreement with experimental data at high angle region due to the simplified model representation as a set of ellipsoids, eq 6 is very useful to sort out various models for a globular particle with a center-symmetrical internal scattering density heterogeneity such as micellar structures.15,17-19 Results and Discussion Temperature Dependence of Scattering Curve and Distance Distribution Function. The temperature dependence of the scattering curve of the GD1 aggregates is shown in Figure 1a. Every scattering curve has a minimum at q ∼ 0.065 Å-1 and a rounded peak at q ∼ 0.1 Å-1, evidently reflecting the micellar structures. The simply saturating tendencies below q ∼ 0.05 Å-1 and the evident minimums in the scattering curves suggest a high monodispersity of the present dispersion system
Thermotropic Structural Change of Disialoganglioside Micelles in the whole temperature range from 6 to 60 °C. As will be shown in the following modeling analyses based on the assumption of a monodispersity, the simulated scattering profiles, distance distribution functions, and structural parameters agree very well with those experimental ones, also strongly suggesting high monodispersity. If there is some evident polydispersity in shape and dimension, the scattering curve would be rather smeared and the modeling fitting would show poor agreement with the experimental data. The major change is seen in the q region below ∼0.04 Å-1 and from 0.06 to 0.1 Å-1, which accompanies the shift of the rounded peak position from 0.092 to 0.10 Å-1. The change of the scattering curve passes through mostly three different steps, namely, the gradual change at 6-15 °C followed by the rather drastic change at 15-25 °C, and the moderate change at 25-60 °C, which suggests the most evidential structural transition of the micelles occurs at the temperature range from 15 to 25 °C. The difference between the scattering curves at the initial and final temperatures is shown in the insert of Figure 1a. Figure 1b shows the distance distribution functions p(r) obtained by applying eq 2 to the scattering curves in Figure 1a. Every p(r) profile is characterized by the shoulder around 30 Å followed by the peak at ∼75 Å, evidently showing the presence of a heterogeneity of the internal scattering density distribution of the solute particle. Such a profile indicates the solute particle has a higher scattering density region outside and a lower one inside.26 The GD1 molecule is composed of a ceramide (hydrocarbon tail) and of a oligosaccharide chain with two sialic acids (hydrophilic head), which have lower and higher scattering densities, respectively. The p(r) profile reasonably reflects the micellar structure of GD1 aggregates.19 Similarly to the case of the scattering curve, the change of the p(r) function also passes through mostly three different steps. At 6-15 °C the increase of the peak height at ∼75 Å in the p(r) gradually occurs. The change at 15-25 °C is most drastic, that is, the rapid increase of the peak height with accompanying the evident shifts of the peak position and the intercept (p(r) ) 0 for r > 0, relating to the maximum dimension of the solute particle) to short distance side. At 25-60 °C the shoulder around 30 Å moves slightly to the short distance side along with changing its shape from a shoulder to a minor peak at ∼25 Å, and the gully around 38 Å becomes gradually deeper. In general, the p(r) function depends both on the particle geometry and on the internal scattering density distribution of the particle, and therefore, the above systematic change of the p(r) profile evidently shows the thermotropic changes both in the micellar dimension and in the internal structure of the micelle. The major changes are the increase of the scattering density of the hydrophilic region and the contraction of the maximum dimension. A minor change of the hydrophobic region also occurs. Temperature Dependence of Structural Parameters. The structural parameters determined from the experimental data are shown in Figure 2, a and b. Figure 2a shows the gyration radii Rg estimated by both the Guinier and Glatter methods using eqs 1 and 4. Compared with the use of the latter method, the determination of Rg using the Guinier method is liable to accompany an apparent systematic error governed by several factors such as the Gaussian approximation of the scattering curve, the selection of the interval of q, the shape, and the internal scattering density distribution of the object, the scattering data statistic, an attractive or repulsive interparticle interaction, and other experimental conditions.24,25 In the present case the accuracy of Rg from the Guinier method is relatively
J. Phys. Chem., Vol. 100, No. 28, 1996 11677
Figure 2. Dependence of the experimental structural parameters on temperature: (a) the gyration radius determined by the Guinier and Glatter methods, (0) the Guinier method, (O) the Glatter method; (b) the peak position pmax and the maximum diameter Dmax determined by the p(r) function, (0) pmax, (O) Dmax. In (a) × marks represent the gyration radii obtained from the shell-modeling analysis.
high, which also suggests a monodispersity of the present dispersion. To confirm the accuracy of Rg values, we have used both the Guinier and the Glatter methods. As shown by the error bars in Figure 2a, the accuracies of Rg obtained by the latter method are higher than those by the former method. In Figure 2a the Rg value decreases gradually from 49.9 to 49.3 Å at 6.0-15 °C, significantly from 49.3 to 46.5 Å at 15-25 °C and remains mostly constant above 25 °C. In Figure 2b the evident changes in the highest peak position pmax and in the maximum dimension Dmax occur at 15-25 °C, that is, the changes of the pmax and Dmax values from 74.1 to 72.2 Å and from 124 to 111 Å, respectively. Apparently, the above changes suggest that the major structural transition of the micelle occurs around 20 °C with the contraction of the dimension accompanying the change of the internal scattering density distribution. Modeling Analyses of Scattering Curves and Distance Distribution Functions. To discuss the above thermotropic structural transition in detail, we applied the shell-modeling analysis to the experimental data on the assumption of monodispersity for the present study. As we have shown in other reports,17-19 the shell-modeling analysis is very applicable to the scattering data from globular particles having an evident internal scattering density heterogeneity with a center symmetry such as micellar structures. The hydrophilic head (the oligosaccharide chain with two sialic acids) and the hydrophobic tail (the ceramide with two hydrocarbon chains) have evidently different scattering densities from each other, and accordingly the GD1 micellar structure can be described by a double-shell
11678 J. Phys. Chem., Vol. 100, No. 28, 1996
Figure 3. Scattering curves I(q) and distance distribution functions p(r) from the model structures optimized to fit to the experimental data: (a) I(q), (b) p(r). In (a) and (b) × a and O are experimental I(q) and p(r) at 6.0 and 60 °C. Full lines are I(q) and p(r) for the optimized double-shell ellipsoid models for 6.0 and 60 °C. Dotted line, hard sphere model; broken line with a dot, hard ellipsoid model for 6.0 °C.
ellipsoidal structure consisting of a core (hydrophobic region) surround by a shell (hydrophilic region). In Figure 3 the experimental scattering curves and distance distribution functions are simulated very well by using the double-shell ellipsoid of rotation. Obviously other models without considering an internal structure, such as a hard-sphere and a hard-ellipsoid, cannot describe the experimental data, especially the scattering curve at the high q region in Figure 3a and the modulation of the p(r) profile in Figure 3b. Fitting parameters used are the outer radii of the shell and core, the axial ratios, and the average scattering densities relative to that of the solvent. The optimized model for 6 °C has the core and shell radii of 24.5 and 48.5 Å, the axial ratios of 1.38 and 1.43, and the average scattering densities of 0.58 and 1.42. The optimized model for 60 °C has the core and shell radii of 26.6 and 44.3 Å, the axial ratios of 1.33 and 1.34, and the average scattering densities of 0.60 and 1.51. The Rg values for the above two models are 50.0 and 46.7 Å, respectively, which agree fairly well with those experimental ones of 49.9 and 46.5 Å. All scattering curves and p(r) functions obtained by the double-shell ellipsoid fittings are shown in Figure 4. Rg values are estimated by using the Glatter method under the same conditions used for the experimental data. As shown in Figure 2a, the simulated Rg values agree very well with the experimental ones. The reliability factor R for the present optimized models, as defined by R ) ∑|Iobs(q) - Imodel(q)|/∑/obs(q), is in
Hirai et al.
Figure 4. Temperature dependence of the scattering curve I(q) and the distance distribution function p(r) for the optimized double-shell ellipsoid model structure: (a) I(q), (b) p(r). The inserts show the difference between the profiles for 6.0 and 60 °C.
the range from 0.02 to 0.04. Good agreements with experimental data show the reasonability and accuracy of the present modeling analyses based on the assumption of high monodispersity. The alkyl chains of gangliosides extracted from bovine brain have been reported to contain mainly C18 fatty acid and C20 sphingosine.8,27 By using the well-known empirical expressions for a hydrocarbon chain volume and for a critical chain length28 and by considering the apparent atomic volumes of the basic chemical elements,29 we may tentatively estimate the average scattering densities of the hydrophilic head and hydrophobic tail of the GD1 molecule to be 12.6 × 1010 cm-2 and 8.7 × 1010 cm-2 (for saturated hydrocarbon chains C18/C20 ) 1/1, 8.0 × 1010 cm-2), respectively. The critical chain length is 24.3 Å for C18 and 26.8 Å for C20, and the average scattering density of water solvent is 9.4 × 1010 cm-2. The values obtained by the modeling analyses agree well in the same orders with the empirical values. Simulated Structural Parameters Depending on Temperature. The structural parameters of the optimized models are plotted as a function of temperature in Figure 5. The uncertainties of these parameters are about 2-4% as the same order of the R factors. At 15-25 °C the shell radius (the micellar radius) decreases most significantly from 48.0 to 45.3 Å and the shell width (hydrophilic shell width) decreases from 23.3 to 19.4 Å. On the contrary, the core radius (hydrophobic core radius) increases from 24.8 to 26.1 Å at 20-30 °C. Below and above these temperature ranges, both radii change only slightly. The
Thermotropic Structural Change of Disialoganglioside Micelles
J. Phys. Chem., Vol. 100, No. 28, 1996 11679
Figure 6. Schematic picture of the structural change of the ganglioside micelles suggested by the modeling analysis. The hydrophobic core and hydrophilic shell radii are shown. The changes of the core and shell scattering densities are also displayed as those of the contrasts.
Figure 5. Plots of the shell and core radii and of those relative scattering densities, as a function of temperature: (a) the shell and core radii; (b) the relative scattering densities to that of the solvent; (O) shell, (0) core.
change of the shell accompanies an evident increase of the scattering density and that of the core does a minor one. Thus, the elevation of temperature induces the contraction of the micellar hydrophilic shell accompanied by the increase of the average scattering density, indicating the oligosaccharide chain conformation changes from an extended one to a folded one to take a more compact packing. On the other hand, same as other hydrated phospholipid membranes,30-33 it is generally considered that micellar hydrocarbon cores are virtually devoid of internal water and that the hydrocarbon chain configurations in micelles contain many gauche bonds.34-37 In addition, the previous fluorescent probe and calorimetric studies on ganglioside dispersions suggest that the packing of hydrocarbon chains depends on the changes in the head group region.38,39 Therefore, the minor extension of the hydrophobic core and the slight increase of the average scattering density would be attributable to a minor rearrangement of the hydrocarbon chains. Conclusion The present results, as schematically shown in Figure 6, elucidate an evident characteristic of the thermotropic structural change of the GD1 micelle that shows the contraction of the whole dimension and the change of the internal structure by elevating temperature. The structural change occurred most evidently in the temperature range from 15 to 25 °C, which agrees with the previous results of the calorimetric studies on ganglioside dispersions showing rather broad thermotropic phase transitions.39,40 As we have also observed similar thermotropic structural changes in other ganglioside systems, the presented
thermotropic behavior around room temperature would be one of the general characteristics of gangliosides. The present shellmodeling analysis describes very well the changes in the experimental scattering curves and structural parameters. The major change is the contraction of the hydrophilic region with the increase of the average scattering density, suggesting the conformational change of the oligosaccharide chain from an extended form to a compact one. Accompanied by the above change, the minor extension of the hydrophobic region occurs with a slight increase of the average scattering density. According to the previous theoretical studies on hydrocarbon chain conformations in micelles34-36 and to the experimental results on the dependence of hydrocarbon chain packing on oligosaccharide chains of gangliosides,38,39 the minor change of the hydrophobic region is attributable to the rearrangements of the hydrocarbon chains, which would be greatly affected by the conformational changes of the oligosaccharide chains. In general, hydration of a macromolecule can induce an expansion of the apparent dimension and a change of the average scattering density. The expanded oligosaccharide chains in the ganglioside micelle would be very accessible to water and rather hydrated. Hence, the compact packing of the oligosaccharide chains would extrude some boundary water around them and induce the increase of the scattering density of the hydrophilic shell. Alternatively, such an extrusion of water may be understood to accompany a dehydration of the hydrophilic shell. Moreover, a highly hydrophilic property of a large head of ganglioside may allow some water to penetrate deeply up to the hydrophobic core interface. In such a case the expansion of the hydrophobic region can be explained as an extrusion of water from the interfacial region between hydrophilic and hydrophobic regions. The slight difference between the transition temperatures in the shell and core regions might suggest a presence of different hydration levels in the hydrophilic region depending on the distance from the hydrophobic core interface. The endothermal phase transitions of ganglioside dispersions39,40 are reported to be very different from those of phospholipid lamellar systems which are well studied and explained as a so-called hydrocarbon chain melting depending on chain lengths. Those reports show a great dependency of transition manner on oligosaccharide chain characteristics. In addition, the theoretical studies suggest a great difference between hydrocarbon chain conformations in lamellar and micellar phases; therefore, the present thermotropic structural transition of ganglioside micelles would be attributable not only
11680 J. Phys. Chem., Vol. 100, No. 28, 1996 to a simple chain melting but also to a conformational change of oligosaccharide chains that might accompany dehydration. Acknowledgment. We thank Drs. Y. Amemiya and K. Kobayashi of the Photon Factory at the National Laboratory for High Energy Physics for their help with the small-angle scattering instrumentation. This work was performed under the approval of the Photon Factory Programme Advisory Committee (Proposal No. 92-069 and 93G047). References and Notes (1) Hakomori, S.; Igarashi, Y. AdV. Lipid Res. 1993, 25, 147. (2) Mikata, A.; Taniguchi, N. In Glycosphingolipid; Weigandt, H., Ed.; Elsevier: New York, 1985; p 59. (3) Hansson, H. A.; Holmgren, J.; Svennerholm, L. Proc. Natl. Acad. Sci. U.S.A. 1977, 74, 3782. (4) Hakomori, S. Sci. Am. 1986, 254, 32. (5) Hannun, Y. A.; Bell, R. M. Science 1989, 243, 500. (6) Svennerholm, L., Asbury, A. K., Reisfeld, R. A., Sandhoff, K., Suzuki, K., Tettamanti, G., Toffano, G., Eds. Biological Function of Gangliosides; Elsevier: Amsterdam, 1994. (7) Sonnion, S.; Cantu`, L.; Corti, M.; Acquotti, D.; Venerando, B. Chem. Phys. Lipids 1994, 71, 21. (8) Maggio, B.; Ariga, T.; Sturtevant, J. M.; Yu, R. K. Biochim. Biophys. Acta 1985, 818, 1. (9) Kojima, H.; Yoshikawa, K. H.; Katagiri, A.; Tamai, Y. J. Biochemistry 1988, 103, 126. (10) Ollmann, M.; Galla, H. J. Biochim. Biophys. Acta 1988, 941, 1. (11) Tsao, Y.-S.; Freire, E.; Huang, L. Biochim. Biophys. Acta 1987, 900, 79. (12) Takizawa, T.; Hayashi, K.; Mitomo, H. Thermochim. Acta 1988, 123, 247. (13) Takizawa, T.; Mitomo, H.; Hayashi, K. Thermochim. Acta 1990, 163, 133. (14) Takizawa, T.; Hayashi, K.; Mitomo, H. Thermochim. Acta 1991, 183, 313. (15) Hirai, M.; Takizawa, T.; Yabuki, S.; Hirai, T.; Hayashi, K. J. Phys. Chem. 1995, 99, 17456. (16) Hirai, M.; Kawai-Hirai, R.; Hirai, T.; Ueki, T. Eur. J. Biochem. 1993, 215, 55. (17) Hirai, M.; Takizawa, T.; Yabuki, S.; Kawai-Hirai, R.; Nakamura, K.; Kobayashi, K.; Amemiya, Y.; Oya, M. J. Chem. Soc., Faraday Trans. 1995, 91, 1081.
Hirai et al. (18) Hirai, M.; Kawai-Hirai, R.; Takizawa, T.; Yabuki, S.; Hirai, T.; Kobayashi, K.; Amemiya, Y.; Oya, M. J. Phys. Chem. 1995, 99, 6652. (19) Hirai, M.; Yabuki, S.; Takizawa, T.; Nakata, Y.; Mitomo, H.; Hirai, T.; Shimizu, S.; Kobayashi, K.; Furusaka, M.; Hayashi, K. Physica B 1995 213 and 214, 748. (20) Hirai, M.; Takizawa, T.; Yabuki, S.; Nakata, Y.; Mitomo, H.; Hirai, T.; Shimizu, S.; Kobayashi, K.; Furusaka, M.; Hayashi, K. Physica B 1995 213 and 214, 751. (21) Hayashi, K.; Katagiri, A. Biochim. Biophys. Acta 1974, 337, 107. (22) Svennerholm, L. In Methods in Carbohydrate Chemistry, Whister, R. L., BeMiller, J. N., Eds., Academic Press: New York, 1972; Vol. III, p 464. (23) Ueki, T.; Hiragi, Y.; Kataoka, M.; Inoko, Y.; Amemiya, Y.; Izumi, Y.; Tagawa, H.; Muroga, Y. Biophys. Chem. 1985, 23, 115. (24) Glatter, O. In Small Angle X-ray Scattering; Glatter, O., Kratky, O., Eds.; Academic Press: London, 1982. (25) Feifin, L. A.; Svergun, D. I. Structure Analysis by Small-Angle X-ray and Neutron Scattering; Plenum Press: New York, 1987. (26) Hirai, M.; Hirai, T.; Ueki, T. Macromolecules 1994, 27, 1003. (27) Corti, M.; Cantu´, L. AdV. Colloid Interface Sci. 1990, 32, 151. (28) Israelachvili, J. N.; Mitchell, D. J.; Ninham, B. W. J. Chem. Soc., Farady Trans 2 1976, 72, 1525. (29) Zamyatin, A. A. Prog. Biophys. Mol. Biol. 1972, 24, 107. (30) Levine, Y. K.; Bailey, A. I.; Wilkins, M. H. F. Nature 1968, 220, 577. (31) Franks, N. J. Mol. Biol. 1976, 100, 345. (32) Worcester, D. L.; Franks, N. P. J. Mol. Biol. 1976, 100, 359. (33) Bu¨ldt, G.; Gally, H. U.; Seelig, A.; Seelig, J.; Zaccai, G. Nature 1978, 271, 182. (34) Gruen, D. W. R. J. Colloid Interface Sci. 1981, 84, 281. (35) Gruen, D. W. R.; de Lacey, E. H. B. In Surfactants in Solution; Mittel, K. L., Lindman, B., Eds.; Plenum Press: New York, 1984; Vol. 1, p 279. (36) Dill, K. A.; Koppel, D. E.; Cantor, R. S.; Dill, J. dD.; Bendedouch, D.; Chen, S.-H. Nature 1984, 309, 42. (37) Israelachvili, J. N. Intermolecular and Surface Forces; Academic Press: London, 1985. (38) Uchida, T.; Nagai, Y.; Kawasaki, Y.; Wakayama, N. Biochemistry 1981, 20, 162. (39) Bach, D.; Miller, I. R.; Sela, B.-A. Biochim. Biophys. Acta 1982, 686, 233. (40) Maggio, B.; Ariga, T.; Sturtvant, J. M.; Yu, R. K. Biochemistry 1985, 24, 1084.
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