Letter pubs.acs.org/Langmuir
Buffers Affect the Bending Rigidity of Model Lipid Membranes Hélène Bouvrais,*,† Lars Duelund, and John H. Ipsen Department of Physics, Chemistry and Pharmacy, MEMPHYS-Center for Biomembrane Physics, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark ABSTRACT: In biophysical and biochemical studies of lipid bilayers the influence of the used buffer is often ignored or assumed to be negligible on membrane structure, elasticity, or physical properties. However, we here present experimental evidence, through bending rigidity measurements performed on giant vesicles, of a more complex behavior, where the buffering molecules may considerably affect the bending rigidity of phosphatidylcholine bilayers. Furthermore, a synergistic effect on the bending modulus is observed in the presence of both salt and buffer molecules, which serves as a warning to experimentalists in the data interpretation of their studies, since typical lipid bilayer studies contain buffer and ion molecules.
■
INTRODUCTION Many biophysical or biochemical studies of model membrane systems and their interactions with other biomolecules are performed at physiologically relevant conditions (pH fixed at 7.4 and ionic strength of about 150 mM). It is there assumed that biological buffers do not interact with lipid bilayers, and this is usually accepted. This explains why the interactions of buffer substances with lipid membranes have not been the subject of many investigations. However, two studies have underlined that some buffers might affect the model lipid membranes.1,2 From the analysis of the repeat spacing distance between multilamellar vesicles (MLVs) in a study of 2009,1 it was concluded that common buffers (HEPES and PIPES) have a swelling effect in the bilayer stack: it was suggested that mechanical softening of the membrane as well as steric entropic repulsion are the main causes, while electrostatic repulsion plays a secondary effect. A subsequent X-ray study from 20112 showed buffer-induced swelling effect for HEPES, MOPS, and MES and came to the opposite conclusion, namely, the creation of an electrostatic repulsion by the charging effect of the buffer that explains the swelling. The understanding of this question is of key importance in interpreting experiments on membrane protein activity, where both membrane surface charge and membrane elasticity can play a role.3 In the present paper, we will show that buffers can induce changes in the bending rigidity of lipid membranes and we will underline a synergistic effect between the buffer molecules and the salts. Numerous biological buffers are available, and we have chosen to study some of the typical buffers used in biophysical studies: HEPES, histidine, MES, MOPS, PIPES and Tris. HEPES, MES, MOPS, and PIPES are some of the “Good’s buffers” developed in the 1960s to fulfill criteria deemed to be important for biochemical and cell biology assays.4 These criteria included a pKa between 6 and 8, a maximum solubility © 2013 American Chemical Society
in water compared to other substances, a weak ability to pass through biological membranes, a minimum salt effect, a minimum influence of buffer concentration, temperature and ionic composition of the medium on the dissociation of the buffer, a chemical and enzymatic stability, a minimum light absorption in the visible or ultraviolet regions of the spectrum, and an easy and inexpensive preparation and purification. The bending rigidity, which is the physical quantity measured in the present study, defines the energy necessary to bend a surface. This very small parameter, whose order of magnitude is the thermal energy, plays a role in shaping up cells and organelles. The bending elastic modulus, κ, is acknowledged to depend strongly on the organization of the lipid bilayers. Thus, the observed changes in the bending elastic modulus induced by additives or environmental modifications might give precious information on the nature of the interactions between lipids and additives.5−7 The sensitivity of the parameter κ will enable us to detect effects of buffer molecules that might not be seen with others physical constants.
■
MATERIAL AND METHODS Six different buffers have been studied, namely HEPES [4-(2hydroxyethyl)-1-piperazineethanesulfonic acid], histidine, MES [2-(N-morpholino)ethanesulfonic acid], MOPS [3-(Nmorpholino)propanesulfonic acid], PIPES [piperazine-N,N′bis(2-ethanesulfonic acid)], and Tris [tris(hydroxymethyl)aminomethane], obtained from Sigma-Aldrich (Copenhagen, Denmark). POPC (1,2-palmitoyloleyl-sn-glycero-3-phosphocholine) was purchased from Avanti Polar Lipids (Alabater, AL), while NaCl (sodium chloride, purity > 99.5%) and glucose (purity > 98%) were from Fluka (Sigma-Aldrich, Copenhagen, Received: September 18, 2013 Revised: December 23, 2013 Published: December 30, 2013 13
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Letter
higher than that obtained by X-ray scattering;11 however, it is consistent with a measure performed using the micropipet technique.12 In ref 13, J.F. Nagle argued that these observed differences among the different techniques (micropipet aspiration technique of GUVs, X-ray stacks technique, and GUVs shape analysis) might be attributed to (i) experimental conditions applied that are not completely identical (differences in temperature, sugar concentrations, and bilayer structures) and (ii) the length scales investigated that are different among the techniques (smaller length scales are captured with X-ray scattering and aspiration pipet techniques than the ones captured by the flickering technique). Three of the six buffers investigated, namely Tris, MOPS, and MES, lead to a large decrease of the bending rigidity at 10 mM from 39.52 kBT in pure water to 36−33 kBT. This softening effect induced by the buffer may indicate that the molecules interact with the headgroup region of the lipid. While 10 mM of HEPES and 10 mM of histidine did not significantly change the bending rigidity of the POPC GUVs, a small decrease in κ is observed in the presence of 10 mM PIPES. We notice that histidine is the buffer molecule with the lowest water solubility from the ones studied and thus might partition to the lipid bilayer, explaining the slight increase in κ observed. Our results for HEPES are in agreement with the absence of significant change in the d spacing of 98:2 DOPC/ DOPE1 or DLPC2 MLVs measured by SAXS in the presence of 10 mM of HEPES. For 10 mM PIPES, our observed small decrease in κ correlates well with the slight increase in the d spacing of 98:2 DOPC/DOPE.1 For 10 mM Tris, a major decrease in κ is measured, while a slight increase of the d spacing has been observed for 98:2 DOPC/DOPE MLVs.1 Similarly, for 10 mM MOPS or MES, a slight increase in d spacing of DLPC2 was found previously, while our measurements show a major decrease in κ. Table 2 shows the obtained bending rigidity values for POPC GUVs in pure water, in 10 mM Tris, in 100 mM NaCl, and in
Denmark). Ultrapure Milli-Q water (18.3 MΩ cm) was used in all steps involving water. POPC giant unilamellar vesicles (GUVs) of diameters in the range from 20 to 50 μm were formed by the electroswelling technique following the protocol presented in ref 8 from SUVs obtained by sonication (Misonix sonicator 3000, Misonix, NY). The SUVs suspension was diluted to 0.2 mg/mL of lipids and droplets of 2 μL of this suspension were carefully deposited on platinum electrodes. The lipid film was then partially dried overnight by inserting the electrodes in a desiccator. The electroswelling of the lipid film was done in various buffer solutions, and the parameters of the electric field were adjusted to their composition, especially to the salt concentration. It was necessary to apply a high frequency for the “swelling period” of 500 Hz for physiological salt conditions, while a frequency of 10 Hz was applied in absence of salt using a waveform generator (Agilent 33120A, Agilent, CA). Vesicles, after having been detached from the electrodes at the end of the electroformation protocol, were observed directly in the electroformation cuvette. A temperaturecontrolled chamber holder (T = 20 °C) was used to maintain constant temperature. The giant vesicles were visualized using a phase contrast microscope (Axiovert S100 Zeiss, Göttingen, Germany), equipped with a x40/0.60 objective (440865 LD Achroplan). The vesicle two-dimensional contour in the focal plane of the objective was thus obtained, and a CCD Camera (SONY SSC-DC50AP) was used to record series of 15000 pictures at a rate of 25 frames per second with a video integration time of 4 ms. The video image sequences of the GUV thermal fluctuations were analyzed using custom-made software to perform contour extraction, contour cleaning, and the fluctuation analysis procedures presented in refs 9 and 10. The bending rigidity was determined by a precise analysis of the statistical distribution of vesicle contours based on a Fourier decomposition of the angular correlation function developed for equilibrium fluctuating liposomes and introduced in ref 9. For a given system, the bending elastic modulus, κ, represented an average of measurements among a population of 7 to 12 vesicles, whose diameters were between 20 and 50 μm. These measurements were performed on different vesicle and buffer preparations.
Table 2. Synergistic Effect Observed in the Bending Rigidity Between Tris Buffer and NaCla system
■
pure water 10 mM Tris 100 mM NaCl 10 mM Tris, 100 mM NaCl, 2 mM EDTA 10 mM Tris, 100 mM NaCl
RESULTS Table 1 gives the bending rigidity measured in the presence of 10 mM of the various buffers, all at pH = 7.4. The studied concentration is in the low range of the ones commonly used (i.e., 10 to 100 mM). The bending modulus of POPC bilayers in pure water is measured at (39.52 ± 0.77) kBT. This value is considerably
a
buffer mM mM mM mM mM
HEPES Histidine MES MOPS PIPES
κ (kBT)
number of vesicles
± ± ± ± ±
12 8 12 10 7
39.09 40.61 33.07 34.70 37.98
0.70 0.44 0.43 0.74 1.00
number of vesicles
± ± ± ±
0.77 0.46 0.82 0.32
9 10 9 19
24.62 ± 1.28
4
39.52 35.79 31.01 24.00
The errors indicated are the standard errors.
the presence of both Tris and NaCl. Both Tris and NaCl, taken individually, give rise to significant reduction of κ. However, the combined effect of NaCl and Tris on the κ decrease appears too much stronger than the effect of each of them, giving rise to a 40% reduction in κ. This is thus a major warning, since it is common to use a mixture of NaCl or KCl with a buffering molecule.
Table 1. Weighted Average of the Bending Rigidity Measurements for POPC Giant Vesicles in the Presence of 10 mM of Various Buffersa 10 10 10 10 10
κ (kBT)
■
DISCUSSION In this paper, we have demonstrated that some common buffers affect the mechanical property of the lipid bilayer. Major differences between the six investigated buffers have been observed and this observation thus raises some questions.
The bending rigidity of POPC bilayers in pure water is (39.52 ± 0.77) kBT, and the errors indicated are the standard errors. a
14
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Letter
MLVs with 100 mM PIPES,1 while we find that NaCl gives an additional reduction in κ).
One of the parameters that could explain the observed differences is the concentration of charged compounds. As shown in Figure 1, the highest concentrations of charged
■
CONCLUSION The mechanical properties of the membrane play a role in a multitude of membrane processes going from endocytosis and vesicle transport to shape changes. So, our work underlines the importance of careful considerations in the choice of the buffer and salt contents in the solution for experiments involving lipid bilayers, like studies of enzymatic behavior of membrane proteins. Similarly, previous observations described that buffers can become toxic to biological matter at high concentrations: MES at 20−40 mM in growth of mammalian cell lines,15 HEPES for neuronal and glial cells16 as well as endothelial cells,17 and MOPS for the endothelial barrier.18 We hope that the here presented data will lead to greater care taken when using buffers and salts for biochemical and biophysical studies of membranes. Evidently, further investigations of the impacts of buffers and salts on biological membranes are required.
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Figure 1. The determined bending rigidity of the investigated buffers (whose bending moduli at 10 mM are displayed in Table 1) as a function of the calculated concentration of charged compounds for each buffer (using the pKa values of the buffering agents). Main form of the buffers: (●) zwitterionic form, (□) negatively or positively charged form in equilibrium with the neutral form, and (○) negatively charged form in equilibrium with the zwitterionic form, while the dotted line represents the bending rigidity of POPC lipid bilayers in pure water.
AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Present Address †
Institut de Génétique et de Développement de Rennes, Université de Rennes 1, Faculté de Médecine, 2 Avenue du professeur Léon Bernard, CS34317, 35043 Rennes Cedex, France. Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval of the final version of the manuscript.
compounds seem to lead to the largest decreases in the bending modulus. The two buffers with the smallest effect on the bending rigidity, histidine and HEPES, are the ones mainly present in their zwitterionic form. The three buffers leading to a major decrease in the bending rigidity, MES, MOPS, and Tris, are mainly composed of the positively or negatively charged form of the buffer molecule in equilibrium with the neutral form. The last buffer, PIPES, is at pH 7.4 mainly found in its negatively charged form in equilibrium with the zwitterionic form and leads to a small decrease of the bending rigidity. Since electrolyte solutions induce softening of the PC lipid bilayer as observed with the measure of the bending modulus at 100 mM of NaCl in Table 2,14 it is suggestive to attribute these decreases in the bending rigidity observed in the presence of the buffers to be related to their charged form. We cannot exclude that some other parameters influence the bending rigidity, such as the solubility in the membrane as well as the structure of the molecule. Another conspicuous finding of our work is the synergistic effect observed on the bending rigidity in the presence of both salt and buffer. The reduction in bending rigidity is not an additive effect between salt and buffer contents. The previously mentioned X-ray studies of the effects of buffers on the d spacing of MLVs pointed at either mechanical softening1 or changes in electrostatic and van der Waals interactions2 as the main causes of the swelling effects. Our study confirms that there can be major changes in the membrane bending rigidity by the presence of a buffer in the solution. However, we find it not likely that it can fully explain the changes found in the interbilayer interactions, since there is no consistency between the changes in κ and d spacing (e.g., NaCl is found to decrease the d spacing of 98:2 DOPC:DOPE
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS MEMPHYS - Center for Biomembrane Physics is supported by the Danish National Research Foundation. ABBREVIATIONS DLPC, 1,2-dilauroyl-sn-glycero-3-phosphocholine; DOPC, 1,2dioleoyl-sn-glycero-3-phosphocholine; DOPE, 1,2-dioleoyl-snglycero-3-phosphoethanolamine; GUV, giant unilamellar vesicle; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; MES, 2-(N-morpholino)ethanesulfonic acid; MLV, multilamellar vesicle; MOPS, 3-(N-morpholino)propanesulfonic acid; PIPES, piperazine-N,N′-bis(2-ethanesulfonic acid); POPC, 1,2-palmitoyloleyl-sn-glycero-3-phosphocholine; SAXS, small-angle X-ray scattering; Tris, tris(hydroxymethyl)aminomethane
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REFERENCES
(1) Peiró-Salvador, T.; Ces, O.; Templer, R. H.; Seddon, A. M. Buffers May Adversely Affect Model Lipid Membranes: A Cautionary Tale. Biochemistry 2009, 48, 11149−11151. (2) Koerner, M. M.; Palacio, L. A; Wright, J. W.; Schweitzer, K. S.; Ray, B. D.; Petrache, H. I. Electrodynamics of lipid membrane interactions in the presence of zwitterionic buffers. Biophys. J. 2011, 101, 362−369. (3) Lee, A. G. How lipids affect the activities of integral membrane proteins. Biochim. Biophys. Acta 2004, 1666, 62−87. 15
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(4) Good, N. E.; Winget, G. D.; Winter, W.; Connolly, T. N.; Izawa, S.; Singh, R. M. M. Hydrogen ion buffers for biological research. Biochemistry 1966, 5, 467−477. (5) Gracià, R. S.; Bezlyepkina, N.; Knorr, R. L.; Lipowsky, R.; Dimova, R. Effect of cholesterol on the rigidity of saturated and unsaturated membranes: Fluctuation and electrodeformation analysis of giant vesicles. Soft Matter 2010, 6, 1472−1482. (6) Méléard, P.; Gerbeaud, C.; Pott, T.; Fernandez-Puente, L.; Bivas, I.; Mitov, M. D. Bending elasticities of model membranes: Influences of temperature and sterol content. Biophys. J. 1997, 72, 2616−2629. (7) Méléard, P.; Gerbeaud, C.; Bardusco, P.; Jeandaine, N.; Mitov, M. D.; Fernandez-Puente, L. Mechanical properties of model membranes studied from shape transformations of giant vesicles. Biochimie 1998, 80, 401−413. (8) Pott, T.; Bouvrais, H.; Méléard, P. Giant unilamellar vesicle formation under physiologically relevant conditions. Chem. Phys. Lipids 2008, 154, 115−120. (9) Méléard, P.; Pott, T.; Bouvrais, H.; Ipsen, J. H. Advantages of statistical analysis of giant vesicle flickering for bending elasticity measurements. Eur. Phys. J. E: Soft Matter Biol. Phys. 2011, 34, 116− 130. (10) Mitov, M. D.; Faucon, J. F.; Méléard, P.; Bothorel, P. Thermal fluctuations of membranes. Adv. Supramol. Chem. 1992, 2, 93−139. (11) Kučerka, N.; Trsitam-Nagle, S.; Nagle, J. F. Structure of fully hydrated fluid phase lipid bilayers with monosaturated chains. J. Membr. Biol. 2005, 208, 193−202. (12) Nagle, J. Introductory Lecture. Basic quantities in model biomembranes. Faraday Discuss. 2013, 161, 11−29. (13) Henriksen, J. R.; Ipsen, J. H. Measurement of membrane elasticity by micro-pipette aspiration. Eur. Phys. J. E: Soft Matter Biol. Phys. 2004, 14, 149−167. (14) Bouvrais, H.; Garvick, O. S.; Pott, T.; Méléard, P.; Ipsen, J. H. Mechanics of POPC bilayers in presence of alkali salts. Biophys. J. 2010, 98, 272−272. (15) Eagle, H. Buffer combinations for mammalian cell culture. Science 1971, 174, 500−503. (16) Cowan, A. I.; Martin, R. L. Ionic basis of the membrane potential responses of rat dorsal vagal motoneurones to HEPES buffer. Brain Res. 1996, 717, 69−75. (17) Bowman, C. M.; Berger, E. M.; Toth, K. M. Repine, J. E. HEPES may stimulate cultured endothelial cells to make growth-retarding oxygen metabolites. In Vitro Cell. Dev. Biol. 1985, 21, 140−142. (18) van Haaren, P. M. A.; VanBavel, E.; Vink, H.; Spaan, J. A. E. Charge modification of the endothelial surface layer modulates the permeability barrier of isolated rat mesenteric small arteries. Am. J. Physiol.: Heart Circ. Physiol. 2005, 289, H2503−H2507.
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Buffers Affect the Bending Rigidity of Model Lipid Membranes Hélène Bouvrais,*,† Lars Duelund, and John H. Ipsen Department of Physics, Chemistry and Pharmacy, MEMPHYS-Center for Biomembrane Physics, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark ABSTRACT: In biophysical and biochemical studies of lipid bilayers the influence of the used buffer is often ignored or assumed to be negligible on membrane structure, elasticity, or physical properties. However, we here present experimental evidence, through bending rigidity measurements performed on giant vesicles, of a more complex behavior, where the buffering molecules may considerably affect the bending rigidity of phosphatidylcholine bilayers. Furthermore, a synergistic effect on the bending modulus is observed in the presence of both salt and buffer molecules, which serves as a warning to experimentalists in the data interpretation of their studies, since typical lipid bilayer studies contain buffer and ion molecules.
■
INTRODUCTION Many biophysical or biochemical studies of model membrane systems and their interactions with other biomolecules are performed at physiologically relevant conditions (pH fixed at 7.4 and ionic strength of about 150 mM). It is there assumed that biological buffers do not interact with lipid bilayers, and this is usually accepted. This explains why the interactions of buffer substances with lipid membranes have not been the subject of many investigations. However, two studies have underlined that some buffers might affect the model lipid membranes.1,2 From the analysis of the repeat spacing distance between multilamellar vesicles (MLVs) in a study of 2009,1 it was concluded that common buffers (HEPES and PIPES) have a swelling effect in the bilayer stack: it was suggested that mechanical softening of the membrane as well as steric entropic repulsion are the main causes, while electrostatic repulsion plays a secondary effect. A subsequent X-ray study from 20112 showed buffer-induced swelling effect for HEPES, MOPS, and MES and came to the opposite conclusion, namely, the creation of an electrostatic repulsion by the charging effect of the buffer that explains the swelling. The understanding of this question is of key importance in interpreting experiments on membrane protein activity, where both membrane surface charge and membrane elasticity can play a role.3 In the present paper, we will show that buffers can induce changes in the bending rigidity of lipid membranes and we will underline a synergistic effect between the buffer molecules and the salts. Numerous biological buffers are available, and we have chosen to study some of the typical buffers used in biophysical studies: HEPES, histidine, MES, MOPS, PIPES and Tris. HEPES, MES, MOPS, and PIPES are some of the “Good’s buffers” developed in the 1960s to fulfill criteria deemed to be important for biochemical and cell biology assays.4 These criteria included a pKa between 6 and 8, a maximum solubility © 2013 American Chemical Society
in water compared to other substances, a weak ability to pass through biological membranes, a minimum salt effect, a minimum influence of buffer concentration, temperature and ionic composition of the medium on the dissociation of the buffer, a chemical and enzymatic stability, a minimum light absorption in the visible or ultraviolet regions of the spectrum, and an easy and inexpensive preparation and purification. The bending rigidity, which is the physical quantity measured in the present study, defines the energy necessary to bend a surface. This very small parameter, whose order of magnitude is the thermal energy, plays a role in shaping up cells and organelles. The bending elastic modulus, κ, is acknowledged to depend strongly on the organization of the lipid bilayers. Thus, the observed changes in the bending elastic modulus induced by additives or environmental modifications might give precious information on the nature of the interactions between lipids and additives.5−7 The sensitivity of the parameter κ will enable us to detect effects of buffer molecules that might not be seen with others physical constants.
■
MATERIAL AND METHODS Six different buffers have been studied, namely HEPES [4-(2hydroxyethyl)-1-piperazineethanesulfonic acid], histidine, MES [2-(N-morpholino)ethanesulfonic acid], MOPS [3-(Nmorpholino)propanesulfonic acid], PIPES [piperazine-N,N′bis(2-ethanesulfonic acid)], and Tris [tris(hydroxymethyl)aminomethane], obtained from Sigma-Aldrich (Copenhagen, Denmark). POPC (1,2-palmitoyloleyl-sn-glycero-3-phosphocholine) was purchased from Avanti Polar Lipids (Alabater, AL), while NaCl (sodium chloride, purity > 99.5%) and glucose (purity > 98%) were from Fluka (Sigma-Aldrich, Copenhagen, Received: September 18, 2013 Revised: December 23, 2013 Published: December 30, 2013 13
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higher than that obtained by X-ray scattering;11 however, it is consistent with a measure performed using the micropipet technique.12 In ref 13, J.F. Nagle argued that these observed differences among the different techniques (micropipet aspiration technique of GUVs, X-ray stacks technique, and GUVs shape analysis) might be attributed to (i) experimental conditions applied that are not completely identical (differences in temperature, sugar concentrations, and bilayer structures) and (ii) the length scales investigated that are different among the techniques (smaller length scales are captured with X-ray scattering and aspiration pipet techniques than the ones captured by the flickering technique). Three of the six buffers investigated, namely Tris, MOPS, and MES, lead to a large decrease of the bending rigidity at 10 mM from 39.52 kBT in pure water to 36−33 kBT. This softening effect induced by the buffer may indicate that the molecules interact with the headgroup region of the lipid. While 10 mM of HEPES and 10 mM of histidine did not significantly change the bending rigidity of the POPC GUVs, a small decrease in κ is observed in the presence of 10 mM PIPES. We notice that histidine is the buffer molecule with the lowest water solubility from the ones studied and thus might partition to the lipid bilayer, explaining the slight increase in κ observed. Our results for HEPES are in agreement with the absence of significant change in the d spacing of 98:2 DOPC/ DOPE1 or DLPC2 MLVs measured by SAXS in the presence of 10 mM of HEPES. For 10 mM PIPES, our observed small decrease in κ correlates well with the slight increase in the d spacing of 98:2 DOPC/DOPE.1 For 10 mM Tris, a major decrease in κ is measured, while a slight increase of the d spacing has been observed for 98:2 DOPC/DOPE MLVs.1 Similarly, for 10 mM MOPS or MES, a slight increase in d spacing of DLPC2 was found previously, while our measurements show a major decrease in κ. Table 2 shows the obtained bending rigidity values for POPC GUVs in pure water, in 10 mM Tris, in 100 mM NaCl, and in
Denmark). Ultrapure Milli-Q water (18.3 MΩ cm) was used in all steps involving water. POPC giant unilamellar vesicles (GUVs) of diameters in the range from 20 to 50 μm were formed by the electroswelling technique following the protocol presented in ref 8 from SUVs obtained by sonication (Misonix sonicator 3000, Misonix, NY). The SUVs suspension was diluted to 0.2 mg/mL of lipids and droplets of 2 μL of this suspension were carefully deposited on platinum electrodes. The lipid film was then partially dried overnight by inserting the electrodes in a desiccator. The electroswelling of the lipid film was done in various buffer solutions, and the parameters of the electric field were adjusted to their composition, especially to the salt concentration. It was necessary to apply a high frequency for the “swelling period” of 500 Hz for physiological salt conditions, while a frequency of 10 Hz was applied in absence of salt using a waveform generator (Agilent 33120A, Agilent, CA). Vesicles, after having been detached from the electrodes at the end of the electroformation protocol, were observed directly in the electroformation cuvette. A temperaturecontrolled chamber holder (T = 20 °C) was used to maintain constant temperature. The giant vesicles were visualized using a phase contrast microscope (Axiovert S100 Zeiss, Göttingen, Germany), equipped with a x40/0.60 objective (440865 LD Achroplan). The vesicle two-dimensional contour in the focal plane of the objective was thus obtained, and a CCD Camera (SONY SSC-DC50AP) was used to record series of 15000 pictures at a rate of 25 frames per second with a video integration time of 4 ms. The video image sequences of the GUV thermal fluctuations were analyzed using custom-made software to perform contour extraction, contour cleaning, and the fluctuation analysis procedures presented in refs 9 and 10. The bending rigidity was determined by a precise analysis of the statistical distribution of vesicle contours based on a Fourier decomposition of the angular correlation function developed for equilibrium fluctuating liposomes and introduced in ref 9. For a given system, the bending elastic modulus, κ, represented an average of measurements among a population of 7 to 12 vesicles, whose diameters were between 20 and 50 μm. These measurements were performed on different vesicle and buffer preparations.
Table 2. Synergistic Effect Observed in the Bending Rigidity Between Tris Buffer and NaCla system
■
pure water 10 mM Tris 100 mM NaCl 10 mM Tris, 100 mM NaCl, 2 mM EDTA 10 mM Tris, 100 mM NaCl
RESULTS Table 1 gives the bending rigidity measured in the presence of 10 mM of the various buffers, all at pH = 7.4. The studied concentration is in the low range of the ones commonly used (i.e., 10 to 100 mM). The bending modulus of POPC bilayers in pure water is measured at (39.52 ± 0.77) kBT. This value is considerably
a
buffer mM mM mM mM mM
HEPES Histidine MES MOPS PIPES
κ (kBT)
number of vesicles
± ± ± ± ±
12 8 12 10 7
39.09 40.61 33.07 34.70 37.98
0.70 0.44 0.43 0.74 1.00
number of vesicles
± ± ± ±
0.77 0.46 0.82 0.32
9 10 9 19
24.62 ± 1.28
4
39.52 35.79 31.01 24.00
The errors indicated are the standard errors.
the presence of both Tris and NaCl. Both Tris and NaCl, taken individually, give rise to significant reduction of κ. However, the combined effect of NaCl and Tris on the κ decrease appears too much stronger than the effect of each of them, giving rise to a 40% reduction in κ. This is thus a major warning, since it is common to use a mixture of NaCl or KCl with a buffering molecule.
Table 1. Weighted Average of the Bending Rigidity Measurements for POPC Giant Vesicles in the Presence of 10 mM of Various Buffersa 10 10 10 10 10
κ (kBT)
■
DISCUSSION In this paper, we have demonstrated that some common buffers affect the mechanical property of the lipid bilayer. Major differences between the six investigated buffers have been observed and this observation thus raises some questions.
The bending rigidity of POPC bilayers in pure water is (39.52 ± 0.77) kBT, and the errors indicated are the standard errors. a
14
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Letter
MLVs with 100 mM PIPES,1 while we find that NaCl gives an additional reduction in κ).
One of the parameters that could explain the observed differences is the concentration of charged compounds. As shown in Figure 1, the highest concentrations of charged
■
CONCLUSION The mechanical properties of the membrane play a role in a multitude of membrane processes going from endocytosis and vesicle transport to shape changes. So, our work underlines the importance of careful considerations in the choice of the buffer and salt contents in the solution for experiments involving lipid bilayers, like studies of enzymatic behavior of membrane proteins. Similarly, previous observations described that buffers can become toxic to biological matter at high concentrations: MES at 20−40 mM in growth of mammalian cell lines,15 HEPES for neuronal and glial cells16 as well as endothelial cells,17 and MOPS for the endothelial barrier.18 We hope that the here presented data will lead to greater care taken when using buffers and salts for biochemical and biophysical studies of membranes. Evidently, further investigations of the impacts of buffers and salts on biological membranes are required.
■
Figure 1. The determined bending rigidity of the investigated buffers (whose bending moduli at 10 mM are displayed in Table 1) as a function of the calculated concentration of charged compounds for each buffer (using the pKa values of the buffering agents). Main form of the buffers: (●) zwitterionic form, (□) negatively or positively charged form in equilibrium with the neutral form, and (○) negatively charged form in equilibrium with the zwitterionic form, while the dotted line represents the bending rigidity of POPC lipid bilayers in pure water.
AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Present Address †
Institut de Génétique et de Développement de Rennes, Université de Rennes 1, Faculté de Médecine, 2 Avenue du professeur Léon Bernard, CS34317, 35043 Rennes Cedex, France. Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval of the final version of the manuscript.
compounds seem to lead to the largest decreases in the bending modulus. The two buffers with the smallest effect on the bending rigidity, histidine and HEPES, are the ones mainly present in their zwitterionic form. The three buffers leading to a major decrease in the bending rigidity, MES, MOPS, and Tris, are mainly composed of the positively or negatively charged form of the buffer molecule in equilibrium with the neutral form. The last buffer, PIPES, is at pH 7.4 mainly found in its negatively charged form in equilibrium with the zwitterionic form and leads to a small decrease of the bending rigidity. Since electrolyte solutions induce softening of the PC lipid bilayer as observed with the measure of the bending modulus at 100 mM of NaCl in Table 2,14 it is suggestive to attribute these decreases in the bending rigidity observed in the presence of the buffers to be related to their charged form. We cannot exclude that some other parameters influence the bending rigidity, such as the solubility in the membrane as well as the structure of the molecule. Another conspicuous finding of our work is the synergistic effect observed on the bending rigidity in the presence of both salt and buffer. The reduction in bending rigidity is not an additive effect between salt and buffer contents. The previously mentioned X-ray studies of the effects of buffers on the d spacing of MLVs pointed at either mechanical softening1 or changes in electrostatic and van der Waals interactions2 as the main causes of the swelling effects. Our study confirms that there can be major changes in the membrane bending rigidity by the presence of a buffer in the solution. However, we find it not likely that it can fully explain the changes found in the interbilayer interactions, since there is no consistency between the changes in κ and d spacing (e.g., NaCl is found to decrease the d spacing of 98:2 DOPC:DOPE
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS MEMPHYS - Center for Biomembrane Physics is supported by the Danish National Research Foundation. ABBREVIATIONS DLPC, 1,2-dilauroyl-sn-glycero-3-phosphocholine; DOPC, 1,2dioleoyl-sn-glycero-3-phosphocholine; DOPE, 1,2-dioleoyl-snglycero-3-phosphoethanolamine; GUV, giant unilamellar vesicle; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; MES, 2-(N-morpholino)ethanesulfonic acid; MLV, multilamellar vesicle; MOPS, 3-(N-morpholino)propanesulfonic acid; PIPES, piperazine-N,N′-bis(2-ethanesulfonic acid); POPC, 1,2-palmitoyloleyl-sn-glycero-3-phosphocholine; SAXS, small-angle X-ray scattering; Tris, tris(hydroxymethyl)aminomethane
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