Proton NMR Based Investigation of the Effects of Temperature and

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Proton NMR Based Investigation of the Effects of Temperature and NaCl on Micellar Properties of CHAPS Xianguo Qin,†,‡ Maili Liu,† Xu Zhang,*,† and Daiwen Yang*,§ †

Wuhan Center for Magnetic Resonance, State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan 430071, China ‡ School of Physics, Huazhong University of Science and Technology, Wuhan 430074, China § Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543

bS Supporting Information ABSTRACT: The effects of temperature and NaCl on the micellization of CHAPS, a zwitterionic detergent widely used in membrane protein studies, have been investigated by NMR spectroscopy. We found that the two apparent critical micelle concentration (cmc) values of CHAPS decrease with the increase of temperature, as well as the NaCl concentration. The thermodynamic parameters derived from the temperaturedependent cmc values show that the micellization process is spontaneous and exothermic, and the van der Waals interaction is likely to be the main factor for the micellization of CHAPS. The micellar hydrodynamic radii remain almost the same in a range of 100-600 mM NaCl, indicating that the aggregate states of CHAPS are not sensitive to the change of the surrounding conditions. In addition, the dependence of nuclear Overhauser effect (NOE) intensities on temperatures further demonstrates the existence of the unique staggered micellar structure of CHAPS at a concentration above the apparent second cmc, which was suggested in our previous work. Our results provide a basis for optimizing CHAPS concentration in the solubilization or stabilization of membrane proteins under nondenaturing conditions and may be helpful to understand its interaction with proteins.

1. INTRODUCTION As an efficient medium for a variety of chemical and biological analyses, detergents are widely used and have enjoyed considerable attention from researchers.1-5 Detergents that form micelles in aqueous solution (because of their amphiphilic nature) above a critical micelle concentration (cmc) are an interesting topic due to their important physicochemical characteristic.6-9 In general, the properties and functions of a micellar solution are tightly connected with the conformation of a detergent and external factors, such as temperature, pressure, and additives (e.g., cosolvents, cosurfactants, electrolytes, polar organics, nonpolar organics, etc.).10-19 Consequently, investigation of the external factors on cmc values, micellar size and shape, and thermodynamics properties of a given detergent is necessary. CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate) is one of the detergents designed for membrane biochemistry.20 As shown by many researchers, CHAPS has little effect on protein conformation and appears as a novel zwitterionic detergent candidate by combining efficiency of solubilization and purification for membrane proteins and receptors.21-27 In addition, it has been proposed that CHAPS exhibits selective solubilization of the disordered lipid phase at appropriate concentrations and higher CHAPS concentrations could improve the solubility and stability of difficult proteins.28-30 In the application of other detergents, it has been reported that protein conformations in micelles are strongly influenced by the aggregate states of the micelles,31 such as the dimension of the r 2011 American Chemical Society

micelles.32 This reveals that the dependence of protein solubility on CHAPS concentration may also be attributed to the concentration-dependent aggregate state of CHAPS. The extensive use of CHAPS in research creates a need to improve our understanding on its micellar properties. The possible aggregate number and morphology of CHAPS in aqueous solution have been studied.33-37 Recently, we have also shown that the micellization of CHAPS is concentration-dependent.38 However, the effect of the external factors on the aggregate state of CHAPS has not been considered. In this work, the micellization behavior of CHAPS at a molecular level in a range of temperatures and salt concentrations, especially at CHAPS concentrations larger than the second cmc (denoted as the micellar structure transition concentration), is examined by 1H NMR spectroscopy.

2. EXPERIMENTAL SECTION 2.1. Materials. The purities of both CHAPS (Sigma-Aldrich) and deuterium water (D2O, Arcos) were 98%. The internal reference Me3Si-CD2CD-CO2Na (TSP) was obtained from ISOTEC. Sodium chloride (analytical grade) was obtained Received: September 12, 2010 Revised: January 19, 2011 Published: February 15, 2011 1991

dx.doi.org/10.1021/jp108694v | J. Phys. Chem. B 2011, 115, 1991–1998

The Journal of Physical Chemistry B

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Figure 1. Critical micelle concentrations, standard Gibbs free energy (ΔG0), enthalpy (ΔH0), and entropy (ΔS0) of micelle formation for CHAPS in D2O solution at different temperatures.

from Sinopharm Chemical Reagent Corp., Shanghai, China. These reagents were used without any further purification. 2.2. Sample Preparation. CHAPS samples were prepared by dissolving CHAPS in D2O solution with gentle agitation. Salt solutions were prepared by dissolving NaCl in aqueous CHAPS solutions. High-concentration CHAPS was prepared first and then diluted to desired low concentrations (ranging from 1 to 78 mM, total 18 samples). Finally, TSP (with a final concentration of 1 mM) was directly added into the samples as an internal reference to calibrate temperature-induced chemical shifts. In order to remove the dissolved paramagnetic oxygen and make the solutions homogeneous, sonication was done for 10 min for each sample. The solutions were then transferred to 5 mm NMR tubes and sealed with paraffin film immediately, and then stored at ambient temperature and normal pressure overnight before use. 2.3. Methods. All NMR experiments were performed on a Bruker AVANCE 500 MHz NMR spectrometer. 2.3.1. 1H NMR Spectra. The samples were allowed to equilibrate at a desired temperature for at least 15 min prior to measurements. One-dimensional (1D) 1H NMR experiments were accomplished by a single pulse scheme with a small flip angle of π/6, 32 scans, and an interscan delay of 3 s. 2.3.2. DOSY Spectra. The self-diffusion coefficients were obtained using the BPPLED (bipolar pulse field gradient with eddy current delay) pulse sequence.39,40 The eddy current delay and diffusion time were 5 and 150 ms, respectively. The duration of the pulse field gradients used for coding diffusion (δ, 2-4 ms) was optimized for each sample to obtain accurate measurements. A linear ramp of 16 steps was used for the increment of the diffusion gradient strength between 1 and 47.5 G/cm. 2.3.3. 1D Selective NOESY Spectra. The selective 1D NOESY (nuclear Overhauser effect spectroscopy) spectra were recorded at 60 mM CHAPS using a 80 ms gauss-shaped π pulse for

selective inversion of the resonance interested,41-43 a spectral width of 9600 Hz, an acquisition time of 3.3 s, 32 scans, and an interscan delay of 4 s.

3. RESULTS AND DISCUSSION 3.1. Effect of Temperature on Micellization. Because 1H

NMR chemical shift is sensitive to its local electronic environment, chemical shift variation as a function of the detergent concentration is an excellent approach to provide direct and strong evidence of micelle aggregation.44-46 In this study, the change of the proton chemical shift of CHAPS with the reciprocal of concentration at different temperatures was analyzed. Two apparent cmc values can be extracted from the chemical shift profiles for CHAPS, which correspond to the two break points in Supporting Information Figure S1. Similar to conventional detergents, the micellization of zwitterionic detergents can be described with the closed association model which assumes equilibrium between individually dissolved detergent molecules and micelles.47 According to the model, thermodynamic quantities such as the standard Gibbs free energy (ΔG0), enthalpy (ΔH0), and entropy (ΔS0) of micellization can be obtained from the cmc dependence on temperature. The obtained apparent cmc values and thermodynamic parameters as a function of temperature are shown in Figure 1. Both cmc1 and cmc2 values decrease with the increase of the temperature (Figure 1a), which suggest that it is easier for the formation of aggregation of CHAPS at higher temperatures. The effect of temperature on the cmc of detergents in aqueous solution is usually attributed to two competitive effects. First, an increase in temperature causes the destruction of the water structure surrounding the hydrophobic groups, which is beneficial for micellization. Second, the hydration degree of the hydrophilic groups decreases with the increase of temperature, 1992

dx.doi.org/10.1021/jp108694v |J. Phys. Chem. B 2011, 115, 1991–1998


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Figure 2. 1D selective NOESY spectra of 60 mM CHAPS at a series of temperatures. The spectra were recorded using an NOE mixing time of 60 ms when H31/H32 were selectively inverted.

leading to an increase in repulsion between polar groups which is unfavorable to micellization. Therefore, the decrease of the cmc indicates that the first effect is predominant within the temperature range studied here. Thermodynamic measurement plays an important role in understanding the micellization process, which has been widely used to elucidate the mechanism of micelle formation.48-53 Because the variations of cmc dependence on temperature are small, the values of ΔG0 might be not accurate. For this reason, the thermodynamic parameters in Figure 1 must be considered only as an approximation. Even so, some information can be extracted from the present data. First, it is obviously that the values of ΔG10 and ΔG20 become more negative with the increase of temperature (Figure 1b), demonstrating that the tendency of micellization is a spontaneous process and increases as a consequence of the dehydration effect.54 In addition, the values of ΔH10 and ΔH20 are negative and become smaller as the temperature increases (Figure 1c). Negative values of micellization enthalpy indicate an exothermic nature of the micellization process. This suggests that the London dispersion interactions as an attractive force play a major role for micellization.55 For the entropy change, TΔS0 instead of ΔS0 is plotted in order to clarify the contribution of entropy to the free energy associated with the micelle formation. Both TΔS10 and TΔS20 values are positive, demonstrating the importance of the hydrophobic effect in the micellization of CHAPS in aqueous solution. Positive ΔS0 values are generally attributed to the release of structural water from the hydration layers around the hydrophobic parts of detergent molecules. In conventional detergent systems, once detergent molecules are dissolved at a low concentration in aqueous solution, highly ordered structural water molecules are created around the hydrophobic segment of each detergent molecule. When the detergent content becomes higher, the hydrophobic segments start to associate to form oligomers due to the highly hydrophobic nature. Consequently, ordered water molecules are expelled from the hydrophobic parts during the micellization process, resulting in an entropy increase of the system. The entropy gain in the water overcomes the entropy loss in the

assembly of detergent molecules. Therefore, the increase of dehydration of the hydrophobic segments with temperature enhances the hydrophobic interaction among the CHAPS monomers and drives the formation of micelles and finally leads to a further decrease in the value of TΔS0 as shown in Figure 1d. However, the contribution of the enthalpy to the micellization becomes increasingly important in contrast to the entropy in the aqueous solution (Figure 1c). This can be understood that the hydrogen bonds between water molecules (participating in the hydration layers) diminish as the temperature increases, and therefore less energy is required to break up the water cluster. This leads to less enthalpy required to release the water of hydration. Then, the enthalpy gain coming from the contact of hydrophobic segment of CHAPS would overcome the enthalpy loss due to the release of water molecules. Hence, ΔH10 and ΔH20 become more negative with increased temperature. Taken together, both enthalpy and entropy are favorable for the micellization of CHAPS, although enthalpy is the main factor to drive hydrophobic interactions in the micellization of CHAPS in aqueous solution within this temperature range. It is reported that temperature plays a significant role in micellar morphology transition that is common in a detergent system.11-15 Since the structure of aggregates influences the properties of detergent solutions like solubilization capacity, it is necessary to examine the structural properties of CHAPS micelles at various temperature. CHAPS may exist in different aggregate states. One type aggregate state is the normal monolayer micelles similar to that for most other detergents. Another type of aggregate state at a concentration above the suggested second cmc is most probably the staggered micelles (in our previous report,38 the “staggered micelle” was named as “bilayer micelle”, but the name of “staggered micelle” is more appropriate since staggered micelles may contain two or more staggered layers). The staggered micelle structure is supported by the NOEs between the protons in the hydrophilic segment and the protons in the hydrophobic segment.38 After the second cmc, no plateaus were observed in the dependence of chemical shifts on CHAPS concentration 1993



Figure 3. (a) Chemical structure of CHAPS; the molecule is divided into two parts: a hydrophobic segment [with a convex side (R-plane or back) and a concave side (β-plane or face) on which there are a few hydrophilic hydroxyl groups], represented by the filled ovals, and the hydrophilic segment with both positive and negative electric charges. (b) Schematic representation of the dynamic behavior of staggered CHAPS micelles during the heating process.

(Supporting Information Figures S1 and S2), indicating that the staggered micelles grow with CHAPS concentration. To examine if the staggered micelle structure is sensitive to temperature, 1D selective NOESY was employed to obtain information of conformational changes. In the selective 1D NOESY experiment, the spin-diffusion effect was suppressed by the application of a π pulse in the middle of the NOE mixing time.42 1D selective NOESY spectra of 60 mM CHAPS at different temperatures are shown in Figure 2. As analyzed in our previous report,38 the NOEs between H31/H32 in the hydrophilic part and their proximal protons (H26, H27, H28, and H29) might originate from intramolecular dipoledipole interactions, whereas the NOEs between H31/H32 and their non-neighboring hydrophobic groups (H18, H19, H21, H7, and H12) might come from intermolecular interactions (refer to the chemical structure of CHAPS shown in Figure 3a). On the basis of the NOE data in Figure 2, the staggered micelle structure always exists in the temperature range of 10-40 °C, because other aggregation states, such as the classical growth micelle (cylinder-shape-like),56 are not possible to give rise to the NOEs between the protons in the hydrophilic segment and the protons in the hydrophobic segment in the absence of spin diffusion. Interestingly, with the increase of temperature, the signal intensities of the intramolecular NOEs mentioned above (NOEs between H31/H32 and H26-H29) initially decrease and then increase gradually; moreover, the sign of the NOEs changed from negative to positive, suggesting that some of the groups involved in those NOEs exposed to the solvent become more flexible with the increase of the temperature (Figure 3b). On the other hand, the signal intensities of the potential intermolecular NOEs [between H31/H32 and hydrophobic groups (H18, H19, H21, H7, and H12)] decrease slightly with the increase of temperature, while the sign of the NOEs remains the same in the temperature range of 10-40 °C. This result indicates that those protons involved in the intermolecular NOEs

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may be in the highly packed micellar core, whose mobility is not sensitive to temperature and remains stable within the temperature range studied here (Figure 3b). The slight decrease in intensities of intermolecular NOE is caused mainly by the decrease of the overall correlation time of micelles (or decrease of viscosity) with temperature. The conclusion drawn here is in good agreement with our previous staggered micelle model—the CHn (n = 1, 2, or 3) groups in the hydrophilic segment in the inner layer of the micelle are in close contacts with the hydrophobic groups in the outer layer through hydrophobic interactions.38 At a CHAPS concentration larger than the second cmc (or micellar structure transition concentration), monomer, monolayer micelles, and staggered micelles may coexist. Because the observed intermolecular NOEs are only from the staggered micelles, the NOESY data indicate that a significant amount of staggered micelles exist at 60 mM CHAPS and the population does not change significantly in the temperature range investigated here. 1 H NMR spectra of 60 mM CHAPS in D2O solution at various temperatures were acquired and are shown in Figure 4. Significant upfield shift of the protons H25, H27, H28, and H31/H32 in the hydrophilic segment reveals that the micellar exterior experiences a certain extent of dehydration with the increase of temperature. Moreover, the chemical shifts of the methyl protons H18, H19, and H21 (belonging to the convex side of the hydrophobic segment, or back side) remain almost the same over the entire temperature range studied, indicating that the local environment of the micellar core is almost unchanged, and this side of the hydrophobic segments of CHAPS molecules still keeps in close interaction with each other. The slight upfield shift of H7 and H12 (belonging to the concave side of the hydrophobic segment, or the face side) may be attributed to the direct contact with the aqueous solvent. The results obviously support the back-to-back self-association model.35 3.2. Effect of NaCl on Micellization. To investigate the effect of inorganic electrolyte NaCl on the cmc of CHAPS, the chemical shift changes of H31/H32 in CHAPS at four different NaCl concentrations were recorded (Figure S2 in the Supporting Information). The obtained cmc1, cmc2, ΔG1, and ΔG2 values are presented in Figure 5, parts a and b, respectively. The increase of NaCl concentration results in the decreases of cmc1 and cmc2 values (Figure 5a). This seems to be ascribed to the salt-out effect of the hydrophobic moiety of CHAPS, which is quite similar to the effect of temperature on the hydration around the hydrophobic segments of CHAPS. The particular distribution of the hydroxyl groups in the hydrophobic segment of CHAPS provides one hydrophilic and one lipophilic side (Figure 3a). It is known that the interaction between the hydroxyl groups of an amphiphile and water should be affected by both the anion and cation of an electrolyte.57 An increase in the electrolyte concentrations causes partial destruction of the hydration shell around the detergent to form aggregates by the salting-out effect (amplification of the hydrophobic interaction). On the other hand, the hydrophilic group SO3- is on the surface, in close contact with water, whereas the less hydrophilic quaternary ammonium ion is far from the water and located in near the hydrocarbon-like interior. The presence of both positively and negatively charged hydrophilic groups in the same molecule makes CHAPS electrically neutral and leads to the hydrophilicity lie between the conventional ionic and nonionic detergents.58 With the increase of NaCl concentration, the shielding of CHAPS charges increases and the electrostatic repulsion among 1994



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Figure 4. Selected regions of 1H NMR spectra of 60 mM CHAPS in D2O solution at various temperatures.

Figure 5. Critical micelle concentrations and standard Gibbs free energy (ΔG0) of CHAPS in D2O solution at different NaCl concentrations at 298 K.

CHAPS molecules decreases. Consequently, the hydrophilic groups of CHAPS are packed more tightly, resulting in micelle formation at a lower detergent concentration.

In addition, as shown in Figure 5b, the standard Gibbs free energies of micelle formation become more negative with the increase of NaCl concentration, suggesting that the hydrophobicity of CHAPS increases with salt and micelles form more easily. The effect of NaCl on ΔG20 is less pronounced than on ΔG10. This may be due to the decrease in the relative stability of the micellar structure above the second cmc. In general, the micellar size increases with the concentration of salts, and the micellar structure may be affected by the ionic strength of the medium as well.16-19 In order to obtain some useful micelle properties in NaCl solution above the second cmc, the salt-dependent behavior of 60 mM CHAPS in aqueous solution was carried out by 1H NMR spectroscopy. The chemical shift of the residual HDO signal in CHAPS solution displays a large shielding effect with the addition of salt (Figure 6). The upfield shift of HDO signal is correlated with the breakdown of hydrogen-bonding structure in water, indicating that the hydrogen bonds between water molecules are progressively weakened by the addition of salt. The ions strongly polarize the surrounding water and lower its free energy, which causes the removal of water from the micellar hydration shell where the water is in a high free energy state. The competition between the micelle and salt solution ions for the water of hydration significantly weakens the interaction of the micelle with water and finally causes the decrease of cmc of CHAPS with the increase of NaCl concentration. This is consistent with previous studies of conventional micelle systems. In contrast, the chemical shift of the hydrophilic group protons (H31/H32) increases slowly with the increase of NaCl concentration, indicating that the protons are in a more polar environment. The additional electrolytes may screen the electrostatic attractions between the opposite charges within the zwitterionic moiety and the neighboring intramicellar hydrophilic groups and allow the hydrophilic groups to interact with water more strongly. In addition, Cl- is heavily hydrated in aqueous solution and the charge is partially screened by the surrounding 1995



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Figure 6. Selected regions of 1H NMR spectra of 60 mM CHAPS in D2O solution at different NaCl concentrations.

Table 1. NaCl Concentration Dependences of the Self-Diffusion Coefficient D and the Corresponding Hydrodynamic Radius RH of 60 mM CHAPS in D2O Solutiona NaCl concn (mM)

DW (10-9 m2/s)

DM (10-10 m2/s)

RH (nm)

0

1.85

1.16

1.93

100

1.85

1.15

1.95

200

1.82

1.12

1.97

400

1.79

1.09

1.99

600

1.78

1.01

2.13

Deviations in RH values are (0.02, and in DW, DM they are (0.03, respectively. a

polar water molecules.59 The hydrated Cl- is not permeable to the micellar core to reduce the positive charge of the quaternary ammonium ion of CHAPS. Thus, the chemical shifts of the protons in the hydrophilic segment (e.g., H31/H32) increase with NaCl concentration. By contrast, the 1H chemical shifts of the hydrophobic groups (H7, H12, H19, and H18) hardly change over the entire salt concentrations used here, suggesting that the hydrophobic segments always remain in the micellar core. It has been reported that the NMR signal line width could be used to investigate morphological transitions in micelles.60 In fact, no significant line width changes were observed for all signals as shown in Figure 6, implying that no morphological transition of the CHAPS micelle occurs in a range of 100-600 mM NaCl. It is worth mentioning that no significant change in the line width of the residual HDO indicates no significantly appreciable variation in viscosity of the solutions studied here. The change of the micellar size and shape of CHAPS in the presence of NaCl can be inferred from self-diffusion coefficient measurements. As shown in Table 1, the apparent self-diffusion coefficient of CHAPS micelles, DM, slightly decreases with the increase of NaCl concentration. On the other hand, the apparent

self-diffusion coefficients of the residual HDO, Dw, show no substantial variations with the NaCl concentration, which is consistent with the result deduced from the HDO line width and proves that the contribution of the viscosity in the measured diffusion coefficients is small. The apparent hydrodynamic radius (RH) of the micelles can also be estimated from DM by the method mentioned in our previous report.38 It should be mentioned that the apparent diffusion coefficients observed experimentally are the weighted average of the diffusion coefficient of CHAPS in all possible aggregate states and depend on the equilibrium among the aggregate states of CHAPS, which is analogous to the case of chemical shift. Herein, the slight increase in the apparent micellar size is probably related to the ionic effects on the micellar surface or the tiny equilibrium change between the monomer and micelle states of CHAPS. Thus, it is reasonable to believe that salt affects only on the water activity of the hydration layer but has no significant effect on the size and shape of CHAPS micelle. Although the apparent self-diffusion coefficients cannot give us the size of the staggered micelles, qualitative conclusions drawn here should not be affected.

4. CONCLUSIONS In this study, the effects of temperature and salt on CHAPS micellization were investigated by NMR spectroscopy. Our data show that the two apparent cmc values of CHAPS decrease with the increase of temperature as well as NaCl concentration, and the apparent size of the micelles formed above cmc2 is nearly independent of NaCl concentration. The experimental results further demonstrate the existence of the staggered micellar structure of CHAPS at a concentration above the apparent second cmc, which was suggested in our previous work. The thermodynamic parameters obtained here reveal that the micellization process is spontaneous and exothermic, and the van der Waals interaction is likely to be the dominant factor of micelle 1996


The Journal of Physical Chemistry B formation. These characteristics provide new insights into the ability of CHAPS to solubilize membrane proteins under conditions that preserve their structure and function.

’ ASSOCIATED CONTENT

bS

Supporting Information. Proton chemical shifts versus the reciprocal CHAPS concentration (Figures S1 and S2) and thermodynamic equations used in the paper. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected] (X.Z.); [email protected] (D.Y.). Phone: þ86-27-87197056, þ86-13618666591(X.Z.); 65-65161014 (D.Y.). Fax: þ86-27-871992921 (X.Z.); 6567792486 (D.Y.).

’ ACKNOWLEDGMENT This research is supported by Grants from the National Natural Science Foundation of China (Nos. 20875098, 20635040, 20975111) and National Major Basic Research Program of China (No. 2009CB918603) to M.L. and X.Z. and by a Grant from the Ministry of Education of Singapore (R154000453112) to D.Y. ’ REFERENCES (1) Rigaud, J. L.; Chami, M.; Lambert, O.; Levy, D.; Ranck, L. L. Biochim. Biophys. Acta 2000, 1508, 112–128. (2) McGregor, C. L.; Chen, L.; Pomroy, N. C.; Hwang, P.; Go, S.; Chakrabartty, A.; Prive, G. G. Nat. Biotechnol. 2003, 21, 171–176. (3) Sanders, C. R.; S€onnichsen, F. Magn. Reson. Chem. 2006, 44 24–40. (4) Prive, G. G. Methods 2007, 41, 388–397. (5) Speers, A. E.; Wu, C. C. Chem. Rev. 2007, 107, 3687–3714. (6) Maeda, H.; Muroi, S. C.; Kakehashi, R. J. Phys. Chem. B 1997, 101, 7378–7382. (7) Chen, L. J.; Lin, S. Y.; Huang, C. C.; Chen, E. M. Colloids Surf., A 1998, 135, 175–181. (8) Mehta, S. K.; Bhasin, K. K.; Chauhan, R.; Dham, S. Colloids Surf., A 2005, 255, 153–157. (9) Palladino, P.; Rossi, F.; Ragone, R. J. Fluoresc. 2010, 20, 191–196. (10) Zhao, J.; Fung, M. B. Langmuir 1993, 9, 1228–1231. (11) Streletzky, K.; Phillies, G. D. J. Langmuir 1995, 11, 42–47. (12) Perez, A. G.; Czapkiewicz, J.; Prieto, G.; Rodríguez, J. R. Colloid Polym. Sci. 2004, 282, 1169–1173. (13) Kumar, A.; Dubin, P. L.; Hernon, M. J.; Li, Y. J.; Jaeger, W. J. Phys. Chem. B 2007, 111, 8468–8476. (14) Silva, B. F. B.; Marques, E. F.; Olsson, U. Langmuir 2008, 24, 10746–10754. (15) Perez, A. G.; Ruso, J. M. Colloids Surf., A 2010, 356, 84–88. (16) Bales, B. L.; Messina, L.; Vidal, A.; Peric, M.; Nascimento, O. R. J. Phys. Chem. B 1998, 102, 10347–10358. (17) Hassan, P. A.; Raghavan, S. R.; Kaler, E. W. Langmuir 2002, 18, 2543–2548. (18) Thongngam, M.; McClements, D. J. Langmuir 2005, 21, 79–86. (19) Rodríguez, A.; Graciani, M. M.; Angulo, M.; Moya, M. L. Langmuir 2007, 23, 11496–11505. (20) Hjelmeland, L. M. Proc. Natl. Acad. Sci. U.S.A. 1980, 77, 6368– 6370. (21) Freeman, K. S.; Tang, T. T.; Shah, R. D. E.; Kiserow, D. J.; McGown, L. B. J. Phys. Chem. B 2000, 104, 9312–9316.

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Proton NMR Based Investigation of the Effects of Temperature and

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