Langmuir 2000, 16, 5467-5470
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Trehalose Interacts with Phospholipid Polar Heads in Langmuir Monolayers Cecilia Lambruschini,† Annalisa Relini,*,† Andrea Ridi,† Lorenzo Cordone,‡ and Alessandra Gliozzi† Istituto Nazionale per la Fisica della Materia and Dipartimento di Fisica, Universita` di Genova, Via Dodecaneso 33, I-16146 Genova, Italy, and Istituto Nazionale per la Fisica della Materia and Dipartimento di Scienze Fisiche e Astronomiche, Universita` di Palermo, Via Archirafi 36, I-90123, Palermo, Italy Received December 15, 1999. In Final Form: March 14, 2000 Surface pressure-area isotherms, surface potential-area isotherms and fluorescence microscopy were employed to study the behavior of phospholipid monolayers at the air/water interface when trehalose was added to the aqueous subphase. In the presence of this sugar, the critical area corresponding to the onset of surface potential increases, indicating that trehalose is participating in the network of hydrogen bonds between the phospholipid polar heads. In addition, it causes an expansion of the isotherm, hindering the formation of the liquid-condensed phase. The collapse area is significantly increased, indicating that trehalose takes part in the monolayer structure without being expelled even at high surface pressures. A quantitative comparison of the collapse areas and critical areas for surface potential in the presence and in the absence of the sugar shows that an almost fixed number of trehalose molecules interacts with the monolayer independently of the surface packing, thus indicating that the observed effects can be ascribed to a tight binding of trehalose to the polar heads in a defined ratio. No similar effects were observed in the presence of glucose. We rationalize the reported data in light of the water replacement hypothesis, developed to explain the preservation of biomembranes by trehalose; this hypothesis suggests that trehalose forms hydrogen bonds with the membrane polar headgroups, thus replacing the water of hydration at the membrane-fluid interface and maintaining the headgroups at their hydrated position.
Introduction Trehalose (R-D-glucopyranosyl-R-D-glucopyranoside) is a nonreducing disaccharide of glucose. It is well-known that this sugar is an effective protector against dehydration and/or very low or high temperatures. In fact, it is accumulated in large quantities in organisms able to survive in conditions of extremely low water content1 (such as yeast, spores of fungi, brine shrimp cysts, dry larvae and adults of several species of nematodes), high temperatures (such as some desert plants2), or low temperatures (such as gall fly larvae3). Protection against water stresses is also carried out by other sugars, as sucrose, although trehalose seems to be the most effective.4 Structure preservation against freezing or drying is also observed in artificial systems such as liposomes.5 The molecular mechanisms involved in the protective action of biostructures and biomimetic structures are still an object of study. It has been shown that not only is trehalose a good glass former, thus preventing liposome fusion during freeze-drying, but also it interacts directly with the phospholipid polar headgroups, lowering the melting temperature of the lipid in the dry state.6,7 * Corresponding author. † Universita ` di Genova. ‡ Universita ` di Palermo. (1) Crowe, J. H.; Crowe, L. M.; Chapman, D. Science 1984, 223, 701. (2) Bianchi, G.; Gamba, A.; Murelli, C.; Salamini, F.; Bartels, D. Plant J. 1991, 1, 355. (3) Storey, K. B.; Baust, J. G.; Storey, J. M. J. Comp. Physiol. 1981, 144, 183. (4) Crowe, L. M.; Reid, D. S.; Crowe, J. H. Biophys. J. 1996, 71, 2087. (5) Crowe, L. M.; Crowe, J. H. In Liposomes, New Systems and New Trends in Their Applications; Puisieux, F., Couvreur, P., Delattre, J., Devissaguet, J.-P., Eds.; Editions de Sante´: Paris, 1995; p 237. (6) Crowe, J. H.; Leslie, S. B.; Crowe, L. M. Cryobiology 1994, 31, 355.
Consequently, the transition between gel and liquid crystalline phase during rehydration is avoided and leakage due to packing defects in the membrane is inhibited. It has been proposed that the sugar directly interacts with the phospholipid polar headgroups, resulting in the replacement of bound water and therefore in structure preservation; this is the so-called water replacement hypothesis.8 Moreover, it has recently been reported that for carbon monoxy myoglobin embedded in a trehalose glass the protein specific motions, i.e., the nonharmonic motions arising from thermal fluctuation of a protein molecule among conformational substates,9 are strongly hindered;10,11 in particular, the hydrogen mean-square displacement and density of states result to be those of a perfectly harmonic solid even at high temperature.12 With the aim of investigating to which extent trehalose can affect the formation of hydrogen-bonded structures involving water and phospholipid headgroups, we have measured surface potential-area and surface pressurearea isotherms on phospholipid monolayers at the airwater interface in the absence and in the presence of trehalose. The former measurements yield information about the hydrogen-bonded water network connecting the (7) Crowe, J. H.; Hoekstra, F. A.; Nguyen, K. H. N.; Crowe, L. M. Biochim. Biophys. Acta 1996, 1280, 187. (8) Crowe, J. H.; Carpenter, J. F.; Crowe, L. M. Annu. Rev. Physiol. 1998, 60, 73. (9) Frauenfelder, H.; Parak, F.; Young. R. D. Annu. Rev. Biophys. Biophys. Chem. 1988, 17, 451 (10) Cordone, L.; Galajda, P.; Vitrano, E.; Gassman, A.; Ostermann, A.; Parak, F. Eur. Biophys. J. 1998, 27, 173. (11) Librizzi, F.; Vitrano, E.; Cordone, L. In Biological Physics: Third International Symposium on Biological Physics; Frauenfelder, H., Hummer, G., Garcia, R., Eds.; American Institute of Physics: Melville, NY, 1999; p 132. (12) Cordone, L.; Ferrand, M.; Vitrano, E.; Zaccai, G. Biophys. J. 1999, 76, 1043.
10.1021/la991641e CCC: $19.00 © 2000 American Chemical Society Published on Web 05/11/2000
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lipid polar heads. In fact, previous measurements on fatty acid and phospholipid monolayers13 (without sugars in the subphase) have shown that the onset of surface potential corresponds to the formation of such a network. Pressure-area isotherms provide information about changes in collapse area and phase transition, possibly induced by trehalose. Moreover, information on monolayer phase behavior was obtained by fluorescence microscopy. The effect of sugars on phospholipid monolayers had already been studied by only measuring pressure-area isotherms. These measurements pointed out the occurrence of monolayer expansion induced by the sugars.14,15 However, this effect was at least partly attributed to the presence of surfactant impurities.16 Being aware of this result, particular care was taken in the purification of the subphase and an accurate statistical analysis of the data was performed in order to establish whether the effects observed were significant from a quantitative point of view. It will be shown that the results obtained in the present study combining different techniques represent an experimental evidence at the molecular level in support of the role of trehalose in water replacement. Experimental Section Dipalmitoyl phosphatidylcholine (DPPC) and the fluorescent probe phosphatidylethanolamine-(lissamine rhodamine B sulfonyl) (Rh-PE) were purchased from Avanti Polar Lipids (Alabaster, AL). The lipids were dissolved in Aristar-grade chloroform (BDH, Milan, Italy). The DPPC concentration was 1 mg mL-1. Trehalose was obtained from Hayashibara Shoij (Okayama, Japan), while glucose and EDTA were purchased from Sigma Aldrich (Milan, Italy). All chemicals were at the purest grade available. Water was purified by means of a Millipore Milli-Q system including a terminal 0.22 µm filter. The specific resistivity of purified water was greater than 18.2 MΩ cm. Since evidence of the presence of surfactant impurities in the sugars as supplied from the chemical companies had been reported in the literature,16 particular care was taken to check the sugars employed and, when necessary, to remove the impurities. A cleaning procedure modified from Pincet et al.17 was employed. Sugar solutions were left for 1 h in the trough with barriers at the maximum expansion (A ) 323 cm2) in order to allow for surfactants to come to the surface. No change in surface pressure was observed as a function of time. However, upon reducing the surface area to the minimum (A ) 40 cm2), the surface pressure raised to approximately 20 mN/m in the case of subphases containing 0.1 M trehalose. Therefore, a small amount of solution was sucked from the surface until the surface pressure dropped to zero and the barriers were opened again. After another hour no surface pressure increase was observed either as a function of time, or reducing the area to the minimum, indicating that the amount of impurities had been reduced well below the sensitivity of the measurements. This purification procedure was always applied to trehalose solutions before monolayer spreading. On the other hand, no pressure increase was recorded applying the same procedure to subphases containing 0.2 M glucose. Glucose solutions were then used without further purification. Monolayers were formed in a circular Teflon multicompartment trough (RCM2-T Monofilmmeter, Mayer Feintechnik, Go¨ttingen, Germany), by spreading from 10 to 24 µL of lipid stock solution with the aid of a Hamilton microsyringe. The subphase contained 1 mM EDTA to avoid possible contamination by divalent ions. After monolayer spreading, 5 min were allowed (13) Leite, V. P.; Cavalli, A.; Oliveira, O. N. Phys. Rev. E 1998, 57, 6835. (14) Crowe, J. H.; Whittam, M. A.; Chapman, D.; Crowe, L. M. Biochim. Biophys. Acta 1984, 769, 151. (15) Johnston, D. S.; Coppard, E.; Valencia Parera, G.; Chapman, D. Biochemistry 1984, 23, 6912. (16) Arnett, E. M.; Harvey, N.; Johnson, E. A.; Johnston, D. S.; Chapman, D. Biochemistry 1986, 25, 5239. (17) Pincet, F.; Perez, E.; Wolfe, J. Cryobiology 1994, 31, 531.
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Figure 1. Pressure-area isotherms of Langmuir monolayers of DPPC in the absence (dashed line) and in the presence (solid line) of 0.1 M trehalose. In both cases the subphase also contained 1 mM EDTA. T ) 22 °C. for the spreading solvent to evaporate. The monolayer was then compressed at a barrier speed from 0.2 to 0.5 Å2 molecule-1 s-1; pressure-area and surface potential-area isotherms were simultaneously recorded. Surface potential was measured by the ionizing electrode method, employing a differential electrometer (604, Keithley Instruments) connected to a 241Am electrode and to an Ag/AgCl reference electrode. For fluorescence microscopy, monolayers were formed in a rectangular trough (R&K, Wiesbaden, Germany) having dimensions of 24.9 × 2.5 cm2. The spreading solution contained 0.1 mol % of the fluorescent probe Rh-PE. The fluorescent probe can diffuse freely in the liquid-expanded phase, which therefore appears bright, while it is expelled from the liquid-condensed domains, which appear dark. The trough was placed under an Olympus BH-2 microscope connected to a Hamamatsu Argus 2000 camera.
Results and Discussion To ensure complete reproducibility of the experimental results, it was necessary to follow the subphase-cleaning procedure described in the Experimental Section; indeed, this procedure made the differences observed in pressure and surface potential isotherms, in the presence and in the absence of trehalose, statistically significant and reproducible. Figure 1 (dashed line) shows the pressurearea isotherm of DPPC on ultrapure water (containing 1 mM EDTA) with the transition from liquid-expanded to liquid-condensed phase at Π ) (8.8 ( 0.4) mN/m and area per molecule Atr ) (77 ( 2) Å2/molecule. Monolayer collapse occurs at (40 ( 2) Å2/molecule. The DPPC isotherm shows a different behavior when measured on 0.1 M trehalose and 1 mM EDTA (Figure 1, solid line). A sizable expansion of the isotherm is observed in the presence of the sugar, while the liquid-expanded/liquid-condensed transition almost disappears; only an inflection in the curve is visible at Π ) (7.0 ( 0.6) mN/m and area per molecule (100 ( 10) Å2/molecule. The collapse area is (46 ( 3) Å2/molecule, significantly larger than the collapse area in the absence of trehalose; this indicates that even at high surface pressures the sugar is not expelled from the lipid polar heads. We stress that the observed effects cannot be interpreted as an artifact due to the presence of surfactant impurities since, as mentioned above, the subphase was carefully purified before the measurement. An epifluorescence microscopy analysis of monolayer morphology shows the occurrence of liquid-condensed domains both in the presence and in the absence of trehalose. However, from the comparison of images under the same conditions of temperature and pressure, it turns out that trehalose hinders the formation of the liquid-
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Figure 3. Surface potential-area isotherms of Langmuir monolayers of DPPC in the absence (dashed line) and in the presence (solid line) of 0.1 M trehalose. In both cases the subphase also contained 1 mM EDTA. T ) 22 °C.
Figure 2. Epifluorescence microscopy of Langmuir monolayers of DPPC in the coexistence region betweeen liquid-expanded (bright) and liquid-condensed phase (dark): (a) monolayer in the absence of trehalose at Π ) 8 mN/m; (b) and (c) monolayers in the presence of 0.1 M trehalose at Π ) 11 and 16 mN/m, respectively. T ) 22 °C.
condensed phase. This finding is in agreement with the expanding behavior observed in the pressure-area isotherms. Representative pictures are shown in Figure 2. In the presence of the sugar, at 11 mN/m (Figure 2b), the domain density is lower and the domain size is generally smaller than at 8 mN/m in the absence of trehalose (Figure 2a). To obtain a domain density comparable to that reported in Figure 2a it is necessary to increase the pressure to 16 mN/m, although domain size is still smaller (Figure 2c). It has been shown that sugars can increase the melting temperature in hydrated bilayers.5 This fact has been interpreted as due to preferential exclusion of the sugars from the bilayer surface, in analogy with the exclusion of stabilizing solutes from the hydration shell of proteins.5 That effect is apparently backward from what one would predict from the monolayer results. The most likely explanation for the difference is that in the latter case the initial condition is an expanded gas phase and not a closely packed lamellar phase, thus allowing for different interaction patterns. Surface potential-area isotherms in the presence of trehalose also show marked differences from those on ultrapure water subphases (Figure 3). The first rise of the surface potential has been associated to the onset of monolayer structuring18 via hydrogen bond at the inter(18) Oliveira, O. N.; Bonardi, C. Langmuir 1997, 13, 5920.
Figure 4. (a, b) Histograms of the area Ap corresponding to the first potential rise in the absence and in the presence of trehalose, respectively. For each histogram, at least 10 measurements obtained on different monolayers in the same conditions were considered. The distribution is shifted toward larger areas in the presence of the sugar.
face.13 In the presence of trehalose, this rise occurs at a significantly larger area per molecule than on ultrapure water. In the almost linear region of the isotherm, the molecular orientation does not change and therefore the normal component of molecular dipole moment is constant.19 Then the changes in surface potential are only related to molecular density. In the presence of trehalose this region is reached at larger areas (∼135 Å2/molecule) and therefore at lower molecular surface densities than in the absence of the sugar, resulting in lower potential values. The histograms of the critical area Ap corresponding to the first rise of the surface potential in the presence and in the absence of trehalose are plotted in parts a and b of Figure 4, respectively. For each histogram, at least 10 measurements obtained on different monolayers in the same conditions were considered. The area Ap has been (19) Mo¨hwald, H. Rep. Prog. Phys. 1993, 56, 653.
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calculated extrapolating to zero the linear portion of the first-rise curve. In the presence of trehalose Ap is shifted from (122 ( 3) to (142 ( 6) Å2/molecule. The set of surface potential-area isotherms recorded in the presence of trehalose can be scaled in such a way as to obtain the same value of Ap measured in the absence of the sugar. The required scaling factor ranges between 0.81 and 0.95. A similar scaling can be performed on the pressure-area isotherms in order to obtain the same collapse area. In this case the scaling factor turns out to be in the range between 0.74 and 0.95. It is remarkable that the values of the scaling factors obtained in the two cases are in good agreement, suggesting that an almost fixed number of trehalose molecules interacts with the monolayer both in the very expanded and in the highly compressed phase. Oliveira et al.13 suggested that the onset of surface potential corresponds to monolayer structuring due to the extensive formation of hydrogen bonds between monolayer headgroups and adjacent water molecules, giving rise to a bond network; this suggestion is supported by measurements of monolayer lateral conduction,20 showing that in correspondence with the onset of surface potential, a mechanism of proton conduction takes places across the postulated network, likely via a hop and turn mechanism. Our results of surface potential-area isotherms can be interpreted on the basis of the above hydrogen bond network formation; in fact, trehalose bound to the phospholipid polar headgroups can promote the structuring of the monolayer-water interface at larger areas per molecule than those observed in the absence of the sugar, bringing about larger values of the critical area for the first rise of the surface potential. Moreover, the agreement between the scaling factors for Ap and for the collapse area suggests that trehalose is inserted in the hydrogenbonded network at a almost fixed stoichiometry, independent of the surface pressure. Since trehalose is a dimer of glucose, to compare the effects of dimers and monomers, the same measurements were repeated at a glucose concentration (0.2 M) which was twice that of trehalose. A slight expansion of the isotherms is observed (Figure 5a,b). However, the collapse area in the pressure-area isotherm turns out to be the same both in the presence and in the absence of the sugar. In addition, also the critical area Ap obtained by a series of 10 measurements (123 ( 4 Å2/molecule) turns out to be compatible, within statistical errors, with the value in the absence of glucose. Therefore, the effect of the (20) Morgan, H.; Taylor, D. M.; Oliveira, O. N. Biochim. Biophys. Acta 1991, 1062, 149.
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Figure 5. (a) Pressure-area and (b) surface potential-area isotherms of Langmuir monolayers of DPPC in the absence (dashed line) and in the presence (solid line) of 0.2 M glucose. In both cases the subphase also contained 1 mM EDTA. T ) 22 °C.
interaction of glucose with DPPC monolayers is much weaker; in particular, at high pressures the sugar does not seem to be entrapped within the monolayer anymore. To the best of our knowledge, this is the first experimental evidence at the molecular level of a direct involvement of the sugar in water replacement at the surroundings of the phospholipid polar head interface in dilute aqueous solutions of trehalose. Acknowledgment. We are indebted to R. Rolandi for many fruitful discussions and helpful suggestions. We also express our thanks to H. Haas, F. Librizzi, M. Pretti, and E. Vitrano for helpful discussions. This work is part of a Project cofinanced by the European Community (European Funds for Regional Development). It has also been partially supported by the Italian Ministry of Scientific Research (MURST). LA991641E
