Langmuir 1998, 14, 4489-4494
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Conformation of Poly(L-glutamate) in Cationic Surfactant Solutions with Reference to Binding Behaviors Jun Liu, Noboru Takisawa, Hiroaki Kodama, and Keishiro Shirahama* Department of Chemistry and Applied Chemistry, Faculty of Science and Engineering, Saga University, Saga 840-8502, Japan Received January 5, 1998. In Final Form: April 7, 1998 The conformational changes of sodium poly(L-glutamate) (P(Glu)) in surfactant solutions were measured by circular dichroism (CD) as a function of the degree of surfactant binding. The influences of both surfactant headgroup and chain length on the formation of the secondary structure of P(Glu) are investigated. The effect of headgroup is studied by comparing three kinds of cationic surfactants carrying a common dodecyl chain. The observed overall binding affinity is in the order of dodecylammonium chloride (DoA) > dodecylpyridinium chloride (DoP) > dodecyltrimethylammonium chloride (DoTA). Typical R-helix spectra of P(Glu) were found in DoA solutions, but no ordered structure of P(Glu) could be induced in DoP or DoTA solutions. Surfactant hydrophobicity is another parameter that controls the conformation of polypeptide, and it is studied by using n-alkylammonium chlorides with different chain length (CnA, n ) 10, 12, 14). The conformational change of P(Glu) is proportional to the degree of binding in the solution of CnA with long alkyl chains but sigmoidal in the solution of short alkyl chains. The hydrophobic interaction couples with but is not necessarily compatible with the transition to the ordered conformation of the polypeptide.
Introduction Surfactants are widely used by biochemists in solubilization, purification, and characterization of proteins. Many studies have been devoted to the physical interaction between surfactants and polypeptides to understand the biological function and activity of proteins in these systems.1 The studies of polypeptide/surfactant systems are also expected to be the models for lipoprotein interaction. Ionic surfactants are well-known as effective denaturants of native proteins, inducing various conformational changes in polypeptides and proteins.2-11 Generally, the effect of surfactant on polypeptide conformation is very complex. Heretofore, most of the works have been performed with anionic surfactant and cationic polypeptide, while systems with cationic surfactant/anionic polypeptide pairs have been less studied. Circular dichroism (CD) provides a powerful and direct method to measure the conformational changes of polymer on binding with surfactants. The majority of the investigations was carried out by measuring CD with respect to the mixing concentration ratio of surfactant to polypeptide. However, we think it may be more suitable to employ the degree of surfactant binding derived from binding isotherm (1) Goddard, E. D. In Interactions of Surfactants with Polymers and Proteins; Goddard, E. D., Ananthapadmanbhan, K. P., Eds.; CRC Press: Boca Raton, FL, 1992; Chapter 8. (2) Satake, I.; Yang, J. Y. Biochem. Biophys. Res. Commun. 1973, 54, 930. (3) Shirahama, K.; Yang, J. T. Int. J. Peptide Protein Res. 1979, 341. (4) Mattice, W. L.; McCord, R. W.; Shippey, P. M. Biopolymer 1979, 18, 723. (5) Takeda, K.; Ibe, A.; Shirahama, K. Bull. Chem. Soc. Jpn. 1981, 54, 1793. (6) Takeda, K.; Ibe, A.; Shirahama, K. Bull. Chem. Soc. Jpn. 1982, 55, 985. (7) Maeda, H.; Kato, H.; Ikeda. S. Biopolymers 1984, 23, 1333. (8) Maeda, H.; Kimura, M.; Ikeda, S. Macromolecules 1985, 18, 2566. (9) Hayakawa, K.; Fujita, M.; Yokoi, S.; Satake, I. Bioact. Compat. Polym. 1991, 6, 36. (10) Satake, I.; Uchino, K.; Hayakawa, K. Bull. Chem. Soc. Jpn. 1992, 65, 1146. (11) Hayakawa, K.; Nagahama, T.; Satake, I. Bull. Chem. Soc. Jpn. 1994, 67, 1232.
as a reference parameter because the binding affinity and the onset of binding vary from one surfactant to another. It can be expected that the combination of binding isotherms and CD measurement will provide further information and contribute to a more detailed picture of the formation of secondary structure in polypeptides by surfactant binding. However, there are very few papers concerning the measurements of both binding isotherms and conformational changes of polypeptides.12,13 In the present study, sodium poly(L-glutamate) (P(Glu)) was selected as an anionic polypeptide bound by cationic surfactants. A surfactant-selective electrode was used to measure the binding quantitatively. The binding isotherms can therefore be built up. The conformational changes of P(Glu) induced by surfactants are investigated by CD measurement in reference to the binding isotherms. There are two dominant physical chemical parameters that apparently control the conformational change of polypeptide in surfactant solution, surfactant headgroup and chain length. The effects of these two parameters have been systematically examined with reference to the binding isotherms. Experimental Section Materials. n-Alkylammonium chlorides were prepared from the corresponding n-alkylamines (Tokyo Chemical Industry Co., Ltd.) according to a method described by Kolthoff and Strickes.14 The products were recrystallized three times from acetoneethanol mixture (75/25 in volume). The critical micelle concentrations (cmc) determined by electric conductivity method in aqueous solutions at 25 °C are 55.2 mM, 14.9 mM, and 3.4 mM for decylammonium chloride (DeA), dodecylammonium chloride (DoA), and tetradecylammonium chloride (TeA), respectively. n-Dodecylpyridinium chloride (DoP) was synthesized by treating 1-dodecylbromide with dried pyridine, followed by ion-exchanging the bromide in concentrated sodium chloride solution and recrystallizing three times from acetone. A triple recrystalli(12) Satake, I.; Gondo, T.; Kimizuka, H. Bull. Chem. Soc. Jpn. 1979, 52, 361. (13) Hayakawa, K.; Murata, H.; Satake, I. Colloid Polym. Sci. 1990, 268, 1044. (14) Kolthoff, I. M.; Stricks, W. J. Phys. Colloid Chem. 1948, 52, 915.
S0743-7463(98)00028-6 CCC: $15.00 © 1998 American Chemical Society Published on Web 07/11/1998
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Figure 1. EMF vs concentration (Cs) of surfactant with various headgroups (DoA, DoP, DoTA) in the absence and presence of P(Glu) at 25 °C. zation from acetone was also carried out for dodecyltrimethylammonium chloride (DoTA) (Tokyo Chemical Industry Co., Ltd.). The cmc of DoP and DoTA are 14.7 mM and 21.7 mM, respectively. All of these cmc values are in good agreement with the literature.15 Sodium poly(L-glutamate) was purchased from Sigma Chemical Co. The average molecular weight (M h w) determined by the manufacture is 36 200 (by viscosity). And the polydispersity h n) is evaluated as 1.15 by SEC-LALLS. The binding site (M h w/M number per polymer, being equal to the degree of polymerization (DP), is 240. The concentration of P(Glu), Cp, is determined by gravimetric method and expressed in residual molarity. To secure the complete dissociation of P(Glu), all measurements were carried out in a buffer solution of pH 8.0, which contains 4.32 mM sodium borate and 7.36 mM hydrochloric acid according to ref 12. Potentiometric Titration. The binding isotherms of cationic surfactants to P(Glu) were obtained by potentiometric titration using a surfactant-selective electrode. The electrode membrane was made from partially sulfonated PVC, polymerized plasticizer (Elvaloy 742, Du Pont) and tricresyl phosphate (Wako Pure Chemical) (3:4:2 in weight). A schematic diagram of the electrode cell assembly was described elsewhere.16 The electromotive force (emf) was picked up by a digital voltmeter (Advantest TR 6845). All the experiments were carried out at 25 °C. Circular Dichroism. The CD spectra were taken on a Jasco J-720 spectropolarimeter using a 1-mm path length cell. Solutions were incubated for 15-20 h before measurements were taken.
Results Effect of Surfactant Headgroup. The binding of surfactants can be calculated from the emf responses in surfactant solutions, with and without P(Glu). Figure 1 shows the relationship between the emf and the logarithmic concentrations of dodecyl surfactants with different headgroups (DoA, DoP, and DoTA). The Nernst response was obtained in the absence of polypeptide. In the presence of 0.1 mM P(Glu), deviations from the linearity were observed suggesting that a part of surfactants are bound onto the polymer. By comparing the emf values with the calibration line, the equilibrium concentration of surfactant, Cf, and the degree of binding, β, can be obtained, where β ) (Cs - Cf)/Cp. The binding isotherms were built up as shown in Figure 2. The features of the cooperative binding are displayed with the sudden onset of binding and the saturation within a narrow surfactant (15) Mukerjee, P.; Mysels, K. J. Nat. Stand. Ref. Data Syst. (U.S. Nat. Bur. Stand.) 1971, 36. (16) Shirahama, K.; Kameyama, K.; Takagi, T. J. Phys. Chem. 1992, 96, 6817.
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Figure 2. Binding isotherms of DoA, DoP, and DoTA to P(Glu). Solid line indicates simulation by Satake-Yang equation. Table 1. Various Properties in Relationship with Surfactant Headgroup cmc/mM Kij uK conformation
DoA
DoP
DoTA
14.9 0.237 12.2 R-helix
14.7 3.25 2.9 random
21.7 1 1.6 random
concentration in the binding isotherms. The binding of DoA to P(Glu) is much stronger than that of the other two surfactants. These binding isotherms were analyzed by the Satake-Yang equation17
β ) 1/2{1 - (1 - s)/x(1 - s)2 + 4s/u}
(1)
where s is a reduced concentration, Cf/Cf(β)0.5). The other parameters are defined as
K ) [uCf(β)0.5)]-1 and xu/4 ) dβ/(d ln s) at β ) 0.5 (2) Physically, K stands for an intrinsic binding constant to an isolated binding site, and u stands for a cooperativity parameter for an interaction between neighboring bound surfactants. Simulations were carried out by choosing the parameters to fit the lower half of the binding isotherm. Table 1 lists the values of binding affinity uK which give the best fit of the calculated binding isotherm, the solid lines in Figure 2, to the experimental ones. The observed order of the overall binding affinity, uK, is DoA > DoP > DoTA. Note that the cmc’s of these three surfactants are very close to the same value, DoTA being slightly higher. In another experiment, we found the same order of these surfactants binding with a linear polymer, sodium poly(2-acrylamide-2-methylpropane sulfonate), in 10 mM NaCl.18 We measured the CD spectrum as a function of β under experimental conditions the same as those for the binding isotherm. Figure 3 shows the CD spectra of P(Glu) in DoA solutions. A spectrum in the absence of DoA (β ) 0) represents the state of a random coil. As the binding degree increases, there develops a typical R-helix of P(Glu) characterized by double minima at 208 nm and 222 nm, and a deep maximum at 196 nm. The magnitudes of the CD extremes of the R-helix increase with the increase in the surfactant binding degree. An isoellipticity point is (17) Satake, I.; Yang, J. T. Biopolymers 1976, 15, 2263. (18) Liu, J., unpublished work.
Poly(L-glutamate) in Cationic Surfactant Solutions
Figure 3. CD spectra of P(Glu) in DoA solutions with respect to β: (a) β ) 0; (b) β ) 0.1; (c) β ) 0.22; (d) β ) 0.31; (e) β ) 0.47; (f) β ) 0.62; (g) β ) 0.75.
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Figure 5. Dependence of CD intensity of P(Glu) at 222 nm on the degree of binding of DoA, DoP, and DoTA.
Figure 6. Binding isotherms of TeA, DoA and DeA to P(Glu). Solid line depicts simulation by Satake-Yang equation. Figure 4. CD spectra of P(Glu) in DoP solutions with respect to β: Bold solid line denotes β ) 0; dotted line denotes β ) 0.2; dashed line denotes β ) 0.5; solid lightface line denotes β ) 0.7.
observed around 205 nm, indicating that only a single conformational change from a random coil to an R-helix is involved. We did not find any distorted CD spectra as attributed to the aggregates of R-helices of P(Glu) by Maeda et al.,7 presumably because the present study was limited to the surfactant concentration above which the solution became turbid. We observed no CD spectrum ascribable to the ordered conformation of P(Glu) in the solution of DoP or DoTA. Since the spectra in these two surfactant solutions look quite similar, only the one for DoP is displayed in Figure 4. With the increasing degree of surfactant binding, the spectra of P(Glu) show a decreasing minimum band at 198 nm and a shallow broad negative band around 230 nm. Very similar spectra were found in poly(L-lysine) (PLL) film cast at pH 7.5.19 In that case, definite constraints in a dried film are imposed on the polymer chain, therefore, the structure of PLL is no longer the same as a random coil in constant thermal motion. Similarly, it is supposed that in the presence of DoP or DoTA, the conformation of P(Glu) is not truly that of a random coil but may be highly constrained by the bound surfactant. The effect of surfactant headgroup on inducing (19) Stevens, L.; Townend, R.; Timasheff, S. N.; Fasman, D.; Potter, J. Biochemistry 1968, 7, 3737.
Table 2. Various Properties in Relationship with Surfactant Chain Length cmc/mM uK u h σh conformation
TeA
DoA
DeA
3.4 74.4 2117 1.4 4.5 × 10-2 R-helix
14.9 12.2 1587 1.4 3.85 × 10-3 R-helix
55.2 1.2 257 3.3 8.75 × 10-6 R-helix
an R-helix can be seen more clearly in Figure 5. The residual CD intensity of P(Glu) at 222 nm, [θ]222, which refers to the helical content, is plotted against the degree of binding. Neither DoP nor DoTA is effective on inducing the R-helix of P(Glu), but DoA can induce the helical structure significantly. Effect of Surfactant Chain Length. To investigate the influence of surfactant chain length on the induction of R-helix of P(Glu), n-alkylammonium chlorides (CnA, n ) 10, 12, 14) were employed in the study. Figure 6 gives the binding isotherms of these surfactants to P(Glu). The binding isotherms were analyzed in the same way as mentioned above, and the binding parameters derived are listed in Table 2. The binding affinity uK increases dramatically for every addition of two CH2 groups, and the cooperativity parameter u also increases when the surfactant chain becomes longer, in line with the results found in other polyelectrolyte-surfactant systems.20 All of the three ammonium surfactants, DeA, DoA, and TeA, can induce R-helix conformation of P(Glu) on binding.
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Discussion
Figure 7. CD spectra of P(Glu) in DeA solutions with respect to β: (a) β ) 0; (b) β ) 0.1; (c) β ) 0.32; (d) β ) 0.4; (e) β ) 0.5; (f) 0.62; (g) β ) 0.72; (h) β ) 0.8.
Figure 8. Dependence of CD intensity of P(Glu) at 222 nm on the degree of binding of TeA, DoA, and DeA.
In DoA or TeA solution, only one isoellipticity point is observed around 205 nm. However, the transition in DeA solution, as seen in Figure 7, seems to have the following two steps: an isoellipticity point around 207 nm when β is less than 0.5, and another isoellipticity point around 203 nm when β becomes larger. The typical R-helix spectra appears only in the latter step. The dependence of residual CD at 222 nm on the degree of binding is shown in Figure 8. In the case of TeA, the conformational change occurs soon after the addition of the surfactant, and the helical content increases nearly linearly with β. As for DoA, there is a small kink at the early binding stage and the inducing power seems a little bit stronger than that of TeA in the higher degree of binding. In the case of DeA, it is interesting to find that the conformational change displays a reverse sigmoid curve on β: the conformation of P(Glu) seems to change reluctantly in lower binding degree, but the magnitude of [θ]222 suddenly increases near the half saturation (β ) 0.5), and P(Glu) assumes even more helical contents than in DoA or TeA solution thereafter. It is noted that the abrupt increase in [θ]222 of P(Glu) in DeA solution has also been observed by Satake and coworkers.12 (20) Hayakawa, K.; Kwak, J. C. T. In Cationic Surfactants: Physical Chemistry; Rubingh, D., Holland, P. M., Eds.; Marcel Dekker: New York, 1991; Vol. 37, Chapter 5.
Surfactant Headgroup on Cooperative Binding. The length of the hydrocarbon tail of the surfactant is a crucial parameter for the interaction with a polyelectrolyte. Kwak’s group and other researchers have carried out extensive studies on this aspect and reached several general conclusions.21-24 Therefore, it is not the main interest of the present study. The free energy of binding for alkylammonium ions to P(Glu) is estimated as 1.03 kT per CH2 group in this work. It is not doubted that the surfactant headgroup also plays a role on the binding of ionic surfactants to the oppositely charged polymer, although few studies have been performed to investigate such effect. We found that the binding affinity of alkylammonium ions is much stronger than that of alkylpyridinium and alkyltrimethylammonium ions of the same chain length. We carried out computer simulation by using HyperChem software for the triad surfactants. It is found that the headgroups of DoTA and DoP have nearly the same volumes and surface areas, but the DoA headgroup is much more compact. The volume and surface area are only 50-60% of the trimethylammonium or pyridinium headgroup. A surfactant with a small headgroup may favor the electrostatic interaction by approaching the headgroup closer to the ionic binding sites on the polymer. Since the electrostatic interaction is recognized as one of the main driving forces in the binding of ionic surfactant to the oppositely charged polymer, it is not surprising that the enhancement of electrostatic attraction will also make the binding more enhanced. That explains why DoA can be bound most strongly on P(Glu). On the other hand, the dipole moment of DoP is the smallest in the series. It indicates high hydrophobicity of the pyridinium salt. The previous works in our research group have determined that the electrode selectivity Kij of the surfactants, which may mainly decided by the lipophilicity, is on the order of DoA < DoTA < DoP. It seems that the lipophilic interactions between neighboring bound headgroups may also contribute to the binding in addition to the headgroup size. For ammonium salt, the headgroup size effect overweighs the lipophilicity effect, which makes DoA the strongest binder in the series. Because the steric effects are nearly the same for DoP and DoTA, the contribution from the headgroup lipophilicity becomes the primary determinant factor in the interaction. As a result, the pyridinium surfactants are bound to the polymer slightly more strongly than trimethylammonium ions at equivalent chain length. Surfactant Headgroup and Chain Length on Conformational Change. The formation of the second structure of polypeptide by the addition of surfactant ions showed a marked dependence on the kind of surfactant headgroup. It is noteworthy that both alkylpyridium and alkyltrimethylammonium ions with bulky headgroups are ineffective in inducing the ordered structure of P(Glu), but alkylammonium ions with small headgroups can induce R-helical dramatically. Therefore, the steric hindrance of surfactant headgroup may mainly inhibit the induction of ordered conformation of polypeptide. The geometrical arrangement of DoP and DoTA required for (21) Malovikava, A.; Hayakawa, K.; Kwak, J. C. T. ACS Symposium Series; American Chemical Society: Washington, DC, 1984; Vol. 253, p 225. (22) Hayakawa, K.; Santerre, J. P.; Kwak, J. C. T. Macromolecules 1983, 16, 1642. (23) Malovikava, A.; Hayakawa, K.; Kwak, J. C. T. J. Phys. Chem. 1984, 88, 1930. (24) Thalberg, K.; Lindman, B. J. Phys. Chem. 1989, 93, 1478.
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Scheme 1. Helix Formation of P(Glu) Induced by TeA and DeA
hydrophobic interaction may not necessarily be compatible with the helical conformation. In addition, the electrostatic interaction between a carboxylate group of P(Glu) and a surfactant headgroup is also expected to participate in the formation of R-helix. The conformation induction by surfactant is much enhanced if surfactant headgroup is bound on the polypeptide more strongly. Another plausible explanation, as postulated by Maeda et al.,25 is related to the proton transfer from surfactant headgroups to carboxylate group. They suggested that the protonation leads to annihilation of polymer charges which contributes to the stability of the R-helix even more than the contribution from the electrostatic shielding. Therefore, even a small amount of protonation will greatly stabilize the helical structure. According to this idea, the fact that no R-helix is induced in DoP and DoTA solution may be the result of no protonation contribution from the headgroups of these surfactants. Hence, R-helix formation may be critically influenced by the difference in surfactant headgroup bulkiness, the electrostatic interaction, and/ or the proton transfer from the headgroups to the binding sites. With respect to the effect of surfactant chain length on conformational change of P(Glu), we found that the induction of an R-helix is linear with the degree of binding in the solution of long-chain ammonium surfactants but sigmoidal in the solution of short-chain ammonium surfactants. Such phenomenon may result from the following two tendencies existing in the surfactant and P(Glu) mixture: one is the tendency to form a surfactant cluster and the other is to form an ordered polypeptide structure. In the case of a long-chain alkyl surfactant such as TeA, the tendency to form a cluster is comparably strong because of the strong hydrophobic interaction between the alkyl chains. Hence, even at a very low degree of binding, the hydrophobic environment is sufficient to induce an R-helix. But on the other hand, this strong cohering tendency of the surfactant may twist the growth of helical conformation and finally result in an interrupted helix, as illustrated in Scheme 1. For short-chain alkyl surfactants, the cohering tendency is comparably weak due to the comparatively weak hydrophobic interaction between the tails, so that the R-helix could not be induced unless the degree of binding is large enough. But so long as the helix is concerned, the conformation change is highly cooperative, and tends to form a continuous helix. In other words, a short-chain surfactant has a greater tendency to form R-helix of P(Glu). The hydrophobic interaction couples with, but is not necessarily compatible with the transition to ordered structure. (25) Maeda, H.; Fujio, K.; Ikeda, S. Colloid Polymer Sci. 1988, 266, 248.
Figure 9. Dependence of the relative helical fraction on the binding degree of TeA, DoA, and DeA. Simulation line: dotted line (TeA); dashed line (DoA); solid line (DeA).
This situation may be analyzed by using the method proposed by Satake et al.26 to get an insight into the conformational change. Assuming that P(Glu) adopts a complete helical conformation at β )1 in DeA solution,27 the helical fraction in each system (δ) can be estimated from the CD data and then δ is plotted as a function of β in Figure 9. Suppose that the coil to helix transition occurs only in bound surfactant clusters, and the average cluster size (m) of the bound surfactant ions is given by17,26
m ) 2β(u - 1)/[(4β(1 - β)(u - 1) + 1)1/2 - 1] (3) then, the value of δ can be evaluated from the helical fraction (f) of the polypeptide composing m residues, as δ ) fβ. According to Zimm and Bragg,28 the value of f is given by
f ) [h/(m - 3)][P0(R0(m - 2) + T0) P1(R1(m - 2) + T1)]/(P0-P1) - hW/(m - 3) (4) where
Pi ) λim-2(λi - h) Ti ) (∂λi/∂h - 1)/(λi - h) Ri ) (∂ ln λi)/∂h
(i ) 0, 1) (i ) 0, 1) (i ) 0, 1)
(5) (6) (7)
W ) (∂ ln(λ0 - λ1))/∂h
(8)
λ0 ) [1 + h + ((1 - h)2 + 4σh)1/2]/2
(9)
λ1 ) [1 + h - ((1 - h)2 + 4σh)1/2]/2
(10)
Here, h and σh refer to the statistical weight of the helical segment that follows a helical segment and a random segment, respectively. The values of h and σh that fit the data best are listed in Table 2. The dependence of f on the cluster size (m) is shown in Figure 10. The cluster sizes required for the onset of R-helix formation of P(Glu) are about 4, 8, and 11 for TeA, DoA, and DeA, (26) Satake, I.; Hayakawa, K. Chem. Lett. 1990, 1051. (27) The magnitude of [θ]222 in DeA solution at β )1 is around 30 mdeg/mmol. Actually, the same value can be also found in other systems when an R-helix of a polypeptide is induced by surfactant ions at saturation (see refs 7, 12, 13, and 25). (28) Zimm, B. H.; Bragg, J. K. J. Chem. Phys. 1959, 31, 526.
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ion, the degree of binding has to exceed 0.3 to form the cluster with m ) 11, 12. On the other hand, DeA can induce an R-helix almost completely when the cluster size is large enough. However, even when binding becomes saturated, the helical fractions are ca. 80% and 90% for TeA and DoA, respectively. That is, long-chain alkyl surfactant cannot induce an R-helix of P(Glu) perfectly. Stronger interaction between the surfactants may not necessarily be compatible with the conformation change. From these discussions, the specificity of surfactant chain length on the induction of R-helix can be well understood.
Figure 10. Dependence of f on cluster size m.
respectively. Such cluster size can be obtained even when β is less than 0.1 in the case of TeA or DoA. But for DeA
Acknowledgment. We thank Dr. K. Yoshizuka (Saga University) for helping us calculate the surfactant molecular parameters by HyperChem Release 4. We also thank Dr. H. Maeda (Kyushu University), Dr. K. Hiramatsu and Mr. T. Morisaki (Gifu University) for their helpful discussion. LA980028C
