Langmuir 2004, 20, 9151-9155
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Effect of Phloretin on the Dipole Potential of Phosphatidylcholine, Phosphatidylethanolamine, and Phosphatidylglycerol Monolayers Fabiana Lairion and E. Anibal Disalvo* Laboratorio de Fisicoquı´mica de Membranas Lipı´dicas y Liposomas, Ca´ tedra de Quı´mica General e Inorga´ nica, Facultad de Farmacia y Bioquı´mica, Universidad de Buenos Aires, Junı´n 956 2° Piso (1113), Capital Federal, Argentina Received February 24, 2004. In Final Form: May 11, 2004 The effect of phloretin on the potential of phosphatidylcholine (PC), phosphatidylethanolamine (PE,) and phosphatidylglycerol (PG) monolayers below and above the phase transition in mixtures of different PC/PE ratios with and without cholesterol of ester and ether phospholipids have been determined. The effectiveness of phloretin to decrease the dipole potential of monolayers in the fluid state is lessened by the moieties esterified to the phosphate group in the sequence choline > ethanolamine > glycerol. These effects on the dipole potential of monolayers are independent of the presence of carbonyls. In addition, in the gel state phloretin does not affect the dipole potential on dimyristoylphosphatidylethanolamine, although it is very pronounced in dimyristoylphosphatidylcholine. The changes of the dipole potential induced by phloretin were correlated with the packing of the lipids and with the formation of intermolecular hydrogen bonds between adjacent phospholipid molecules. These results may be indicative of the different distribution of polarized water around the phosphate groups imposed by the surrounding environment.
Introduction The dipole potential of a lipid membrane is manifested between the hydrocarbon core of the membranes and the first few water molecules adjacent to the lipid headgroups.1 This potential is caused by the uniform orientation of the phosphocholine moiety, the carbonyl groups of the ester union, and, in some extension, by the presence of polarizable groups in the membrane hydrocarbon phase.1-4 The dipole potential has also been related to the water hydrating the polar headgroups that opposes the membrane-membrane contact during adhesion processes.5-7 It has been shown that it modulates the penetration of peptides to the membrane phase, the permeability of hydrophobic anions, and the permeation of water.4,8-10 In this context, the understanding of the origin of this potential is important to have a more precise picture of the distribution of water at the lipid interface and its participation in the mechanism of relevant membrane phenomena. In this regard, the effect of the insertion of molecules that may decrease or increase the dipole potential, such as phloretin and ketocholestanol, respectively, has been analyzed in lipid monolayers.8,9 It has been reported that the inclusion of phloretin in a 20% mole ratio in phosphatidylcholine bilayers and monolayers produces a decrease of about 200 mV in the dipole potential.4,8 * To whom correspondence may be addressed. Fax: 54 11 49648274. E-mail:
[email protected]. (1) Brockman, H. Chem. Phys. Lipids 1994, 73, 57. (2) Haydon, D. A.; Hladky, S. B. Q. Rev. Biophys. 1972, 5, 187. (3) Cseh, R.; Benz, R. Biophys. J. 1998, 74, 1399. (4) Cseh, R.; Benz, R. Biophys. J. 1999, 77, 1477. (5) Gawrish, K.; Ruston, D.; Zimmerberg, J.; Parsegian, V. A.; Rand, R. P.; Fuller, N. Biophys. J. 1992, 61, 1213. (6) Simon, S. A.; McIntosh, T. J. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 9263. (7) MacDonald, R. C.; Simon, S. A. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 4089. (8) Franklin, J. C.; Cafiso, D. S. Biophys. J. 1993, 65, 289. (9) Perkins, W. R.; Cafiso, D. S. J. Membr. Biol. 1987, 96, 165. (10) Flewelling, R. F.; Hubbell, W. L. Biophys. J. 1986, 49, 541.
Different mechanisms have been described to explain the phloretin effect on the dipole potential. The primary effect of phloretin is thought to occur by the alignment of phloretin dipoles in opposite direction to the lipid ones.11 It is also possible that phloretin may alter the structure of the interface changing the ordering of water or the orientation of the ester carbonyls. A decrease of the dipole potential of ether-phospholipids, i.e., those in which the fatty acid chains are bound to the glycerol by an ether union instead of an ester one, has also been observed with phloretin.12 This suggests that the effect of phloretin on the dipole potential is not related to carbonyl groups. In this regard, the FTIR measurements have shown that there is no effect of phloretin on the ester carbonyl vibration modes. Thus, the decrease in dipole potential produced by phloretin on ester lipids is not due to H-bond formation or dehydration of the carbonyl dipoles. Another explanation to the phloretin action also merges from FTIR measurements, which show that phloretin causes a pronounced downward shift of the frequency corresponding to the asymmetric vibration of the PdO groups of the phospholipids. This effect has been ascribed to the formation of hydrogen bonds between the OH groups of phloretin and the PdO. This interaction is concomitant with a decrease in the hydration of the lipids,12,13 from which it is inferred that the PdOsHO interaction is produced with water displacement. Thus, the action of phloretin on the dipole potential can be partly ascribed to the elimination of oriented water dipoles from the lipid interface hydrating the phosphate. To analyze further this point, it is of interest to correlate the phloretin action with the exposure of phosphate groups to the aqueous solution. In this regard, the headgroup (11) Cafiso, D. In Permeability and Stability of bilayers; DisalvoSimon, Ed.; CRC Press: Boca Raton, FL, 1995; Chapter 9, pp 179-195. (12) Diaz, S.; Lairion, F.; Arroyo, J.; Biondi de Lopez. A. C.; Disalvo, E. A. Langmuir 2001, 17, 852. (13) Disalvo, E. A. Lairion. F.; Diaz, S.; Arroyo, J. In Recent Research developments in Biophysical Chemistry; Condat-Baruzzi, Ed.; Research Signpost: Trivandrum, India, 2002; pp 181-197.
10.1021/la049515k CCC: $27.50 © 2004 American Chemical Society Published on Web 09/14/2004
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arrangement of phospholipids, such as, phosphatidylcholine, phosphatdylethanolamine, and phosphatidylglycerol is different, specially to the space requirement to orient the P-N dipoles. As a consequence, the hydration of the phosphate groups may change with the phase state of the lipids, the lateral surface pressure, and the intermolecular interactions between adjacent molecules.14,16 In phosphatidylcholines (PCs), the phosphate groups are linked to infinite zigzag ribbons by water molecules of hydration. In this case, the choline end of the --P-O-N+(CH3)3 group is laying toward the hydrocarbon phase, due to the hydrophobic character of the methylenes.16 In contrast, the ammonium groups in phosphatidylethanolamine (PE) link together with unesterified phosphate oxygen by very short bonds.17 The ethanolamine groups form linkage between adjacent phosphates producing a very compact, rigid headgroup network at the bilayer surface. The -N+ end of PEs seems to be oriented toward the water phase, forming hydrogen bonds between the phosphates and the amines of neighboring molecules, promoting a membrane packing.18 In phosphatidylglycerol (PG), the glycerol moiety appears to mimic the phosphate hydration.19 In addition, the internal hydrogen bonds between different types of phospholipids or the intercalation of molecules acting as spacers (cholesterol, trehalose) could alter the access of phloretin to the phosphate groups, and as a consequence, its effect on the dipole potential. For these reasons, we have determined the effect of phloretin on the potential of different monolayers composed of PC, PE, and PG, below and above the phase transition, in mixtures of different PC/PE ratios and cholesterol and with ether linked lipids (without carbonyls). The changes of the dipole potential induced by phloretin were correlated with the packing of the lipids and with the formation of intermolecular hydrogen bonds. Changes in the dipole potential of monolayers in which the phosphate groups are surrounded by different environments may be important to understand the mechanisms taking place in different membrane phenomena such as permeability, adhesion, and peptide penetration, in which dehydration-hydration steps appear to be involved. Materials and Methods Lipids. Dimyristoylphosphatidylcholine (DMPC), 1,2-di-Otetradecyl-sn-glycero-3-phosphocholine (etherDMPC), dimyristoylphosphatidylethanolamine (DMPE), 1,2-di-O-tetradecyl-snglycero-3-phosphoethanolamine(etherDMPE), egg phosphatidyl ethanol amine (eggPE), and dimyristoylphosphatidylglycerol (DMPG) were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL), and used as received. The purity of lipids was checked by thin-layer chromatography using a chloroform/methanol/water mixture as running solvent. Cholesterol was obtained from Sigma Chemical Co. (St. Louis, MO). Chloroform and KCl were analytical grade. Water was MilliQ quality. Determination of Dipole Potential in Monolayers. The values of interfacial potential (Vsurf) were determined through a circuit of high impedance by means of an ionizing electrode on the monolayer and a reference electrode in the aqueous subphase using the expression
Lairion and Disalvo Vsurf ) VAg/AgCl - Vgrd ) Vsolution - Vgrd where VAg/AgCl is the potential of the reference electrode and Vgrd is the potential of the shield covering the ionizing electrode. Temperature was set at the values indicated in each assay (mostly, 8, 18, and 28 °C) and measured with a calibrated thermocouple immersed in the subphase and maintained within (0.5 °C. Two kinds of experiments were done in order to obtain relevant results of the effect of phloretin on the dipole potential of lipid monolayers. In one of them, aliquots of a chloroform solution of lipids and phloretin at different ratios were added to the interface of a water solution 1 mM KCl, exhaustively cleaned by suction, until a constant surface pressure was reached (see next section).20,21 The dipole potential of the monolayer (∆Vm) was evaluated as
∆Vm ) Vsurf - Vlip where Vsurf is the potential of the clean surface (without lipids) and Vlip is the potential after the monolayer was formed. In another set of experiments, a monolayer with each of the pure lipids was formed at the interphase to reach a constant surface pressure (see next section). In this condition, aliquots of a chloroform/ethanol solution of phloretin were added to the preformed monolayer from the air side and allowed to stabilize. In this case, the changes in the dipole potential were evaluated as the dipole potential difference, ∆VT, between the dipole potential of the lipid monolayer (∆Vm) and the monolayer after the addtion of phloretin (∆Vflo)
∆VT ) ∆Vflo - ∆Vm This value is negative since it reflects the decrease of the monolayer potential by the addition of phloretin. The differences obtained by employing both methods were lower than (30 mV. Previous reports have shown that at pH 5-6, more than 95% of phloretin is in the monolayer due to the scarce solubility of the uncharged form in water.4 In this regard, the addition of phloretin from the air side to a preformed monolayer or the formation of a mixed monolayer at the air-water interface minimizes the partition of phloretin into the aqueous phase at this working pH. These procedures avoid the addition of solvents into the aqueous phase that are difficult to eliminate and allow work with the noncharged form of the phloretin without changing the buffer solution and ionic strength of the subphase. Formation of Lipid Monolayers. Measure of Surface Pressure. The saturation point of monolayers with and without phloretin was monitored by measurements of the surface pressure of diverse lipid monolayers in a Langmuir trough (Kibron MicroTrough S), at constant temperature and area. Aliquots of a chloroform solution of lipids with different phloretin ratios were spread on a clean aqueous surface and allowed to reach constant surface pressures, until no changes were observed with further additions of lipids (saturation). All dipole potential measurements were assayed at this saturation condition. Results of surface pressure were expressed in mN/m. In the second procedure, lipids without phloretin were allowed to reach the saturation point and then phloretin was added from the air-lipid side. The changes in the surface pressure were monitored and found to be between 1 and 2 mN/m at the phloretin ratios used in this work. In this condition, the dipole potential values were determined.
Results and Discussion (14) Hu¨bner, W.; Blume, A. Chem. Phys. Lipids 1998, 96, 99. (15) Bush, F. S.; Adams, R. G.; Levin, I. W. Biochemistry 1980, 19, 4429. (16) Seelig, J.; MacDonald, P. M.; Scherer, P. G. Biochemistry 1987, 26, 7535. (17) Hauser, R.; Pascher, I.; Pearson, R. H.; Sundell, S. Biochim. Biophys. Acta 1981, 650, 21. (18) Hitchcock, P. B.; Mason, R.; Thomas, K. M.; Shipley, G. G. Proc. Natl. Acad. Sci. U.S.A. 1974, 71, 3036. (19) Peng Zhang, Y.; Ruthven, N. A.; Lewis, H.; McElhaney, R. N. Biophys. J. 1997, 72, 779.
The variations in the surface pressure of a water solution by the spreading of increasing aliquots of phospholipids at different temperatures are shown in Figure 1 for DMPC and DMPE. In all cases, at constant area, it is observed (20) Luzardo, M del C.; Peltzer, G.; Disalvo, E. A. Langmuir 1998, 14, 5858. (21) Diaz, S.; Amalfa, F.; Biondi de Lopez, A. C.; Disalvo, E. A. Langmuir 1999, 15, 5179.
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Langmuir, Vol. 20, No. 21, 2004 9153 Table 1. Surface Pressure and Dipole Potential of Different Monolayers Containing Phloretin
Figure 1. Formation of monolayers of phospholipids with and without phloretin as measured by the changes in surface pressure as a function of lipids added to the air-water interphase. (A) DMPC: pure, 18 °C (]); plus phloretin 1:1 ratio, 18 °C ([); pure, 28 °C (O); plus phloretin 1:1 ratio 28 °C (b). (B) DMPE, 28 °C: pure (4), plus phloretin 1:1 ratio (2)
Figure 2. Effect of phloretin on the dipole potential of DMPC monolayers at 8 °C (]), 18 °C ([), and 28 °C (b).
that a surface pressure limit of around 48 mN/m is reached at around 4 nmol of DMPC and 44 mN/m is reached at around 5 nmol of DMPE. Under the conditions at which the saturation of the surface pressure was reached, the dipole potential was determined. In Figure 2, the dipole potential of lipid monolayers containing increasing amounts of phloretin, spread at the saturation condition obtained in Figure 1, are shown. The dipole potential of pure PC monolayers in the gel state (surface pressure at saturation ) 48.2 mN/m) is around 66 mV higher than that in the fluid state (for which the
lipid
surface pressure (mN/m)
dipole potential (mV)
DMPC (18 °C) DMPC:phlo (1:1) (18 °C) DMPC (28 °C) DMPC:phlo (1:1) (28 °C) DMPE (28 °C) DMPE:phlo (1:1) (28 °C) DMPG (28 °C) DMPG:phlo (1:1) (28 °C)
48.2 ((0.8) 49.7 ((1.2) 47.5 ((0.6) 45.7 ((1.2) 40.6 ((0.6) 43.5 ((1.5) 35.5 ((2.5) 39.3 ((1.5)
515.5 ((13.6) 270.7 ((10.4) 449.1 ((23.9) 297.5 ((11.8) 543.0 ((4.5) 550.0 ((16.2) 301.8 ((7.2) 243.5 ((18)
surface pressure at saturation is 47.5 mN/m). The increase of phloretin ratio produces a sharper decrease of the dipole potential at 8 and 18 °C than at 28 °C. However, above 0.6 phloretin/PC ratio, the dipole potential at all temperatures is around the same value (Figure 2). At a 1:1 phloretin/PC ratio, the monolayer surface pressures are around 49.7 mN/m at 18 °C and 45.7 mN/m at 28 °C. The dipole potential at 28 °C of a monolayer composed by a 1:1 phloretin/DMPC ratio is 152 mV lower than that corresponding to a pure DMPC monolayer with a small decrease in the surface pressure. Table 1 summarizes the results of surface pressure and dipole potential for the different conditions assayed. The phase transition is concomitant to the increase in the hydration per lipid from 8 to 18 water molecules. Thus, the decrease by around 66 mV of the dipole potential in the absence of phloretin can be rationalized due to the increase in the area per molecule by water insertion at the gel-fluid phase transition. The inclusion of phloretin in gel DMPC monolayers increases the surface pressure in 1.5 mN/m. However, in this case, the dipole potential decreases in nearly 200 mV. Thus, phloretin action to decrease the dipole potential, at constant area, operates inversely to that shown at the phase transition. Previous FTIR measurements,12 showed that phloretin promotes a pronounced downward shift of the frequency of the asymmetric vibrations of the phosphate groups of phosphatidylcholines. According to Raman and NMR results, the conformation of the PO42- group is determined by the first four or five water molecules.16,21 In addition, it has been shown that the decrease of hydration shifts the antisymmetric stretching frequencies to higher values when tightly bound molecules are displaced.14,15 The comparison of the effect of phloretin with other compounds that may displace water from the lipid interface, such as trehalose, suggests that the dipole potential decrease may be due to the displacement of water around the phosphates.21,22 That is, water from the phosphate shell can be eliminated by phloretin. Thus, the increase in surface pressure could be related to the strong hydrogen bonding of phloretin groups with the phosphates,12 at the expense of the release of water dipoles contributing to the dipole potential. The correlation of the FTIR measurements with the dipole potential decrease indicates that the PdO bonds of the phosphates contribute to the dipole potential. The addition of phloretin to a preformed monolayer of DMPC, at 28 °C in a 1:1 phloretin/lipid ratio, causes a potential decrease of about 120 mV (Figure 3). In the same figure, it is also observed that phloretin also decreases the dipole potential of fluid phosphatidylethanolamine (eggPE) and phosphatidylglycerol (DMPG) at 28 °C, (22) Luzardo, M. C.; Amalfa, F.; Nu´n˜ez, A.; Diaz, S.; Biondi de Lopez, A. C.; Disalvo, E. A. Biophys. J. 2000, 78, 2452.
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Figure 3. Effect of phloretin on PC, PE, and PG monolayers the fluid state (28 °C): (4) DMPC; (2) eggPE; (0) DMPG.
although in a more attenuated way, compared with the drastic decrease observed with DMPC. In fluid DMPG, the glycerol moiety mimics the solvation water of the phosphate group.19 This internal hydrogen bonding would make the hydrogen bonding between the phloretin and the phosphate less favorable than when the phosphate is linked to cholines. In other words, phosphate would be shielded by the glycerol moiety in PG for compounds that may form hydrogen bonds at the expense of dehydration. In contrast, the addition of phloretin to DMPE in the gel state (surface pressure at 28 °C ) 40.6 mN/m) did not affect the dipole potential in the range of the molar ratio tested, although the surface pressure changes to 43.5 mN/m (Table 1). The effect of phloretin on the dipole potential of gel DMPE increases correspondingly with the PC ratio in the mixed monolayer (Figure 4). In the case of DMPE, in which the saturation surface pressure is somehow higher in the presence of phloretin, the effect on the dipole potential is absent (Figure 1 and Figure 4). The -N+ end of phosphatidylethanolamines (PEs) seems to form hydrogen bonds between the adjacent phosphates and the amines of neighboring molecules, promoting a membrane packing produced by a very compact, rigid headgroup network at the bilayer surface.17,18 In this condition, there would be no chance for phloretin to insert and bind to the phosphates. As shown in Figure 2, the effect of phloretin is not directly related with the phase state of the lipids. In PCs in the gel state, which has a lower hydration than the fluid state, the effect of phloretin is more significant than that in the fluid one. However, in gel PE the effect is absent in contrast to the decrease observed in fluid PE. The small decrease in dipole potential observed in eggPE may be related to the fact that there would be an intermediate insertion of phloretin, because some internal hydrogen bonding may be concerted between the phospholipids, blocking, at least partially, the access to the phosphate. In this regard, the action of phloretin on dipole potential of DMPE in the gel state is enhanced by the inclusion of cholesterol in monolayers (Figure 5). In contrast, when cholesterol was present in the same ratio in fluid PE, the effect of phloretin is lower than that in pure PE (Figure
Lairion and Disalvo
Figure 4. Effect of phloretin on the dipole potential of monolayers of increasing DMPC/DMPE ratios at 28 °C: pure PC (9); 75:25 PC/PE (0); 50:50 PC/PE (2); 25:75 PC/PE (4); pure PE (b).
Figure 5. Effect of phloretin on the dipole potential of PE monolayers in the fluid and the gel state with and without cholesterol: gel DMPE without (b) and with cholesterol (O); eggPE without (4) and with cholesterol (2).
5). Thus, the effect of phloretin appears correlated with the cohesion changes induced by cholesterol in fluid or gel membranes. The intercalation of cholesterol or the presence of choline groups in gel DMPE would hinder the strong hydrogen bonding between the phospholipids. Thus, as a consequence of the decrease in membrane cohesion, the insertion of phloretin is enhanced. In contrast, the increase of membrane cohesion due to the inclusion of cholesterol in fluid eggPE monolayers reduces the phloretin action. In Figure 6, it is observed that the action of phloretin on PC and PE monolayers is independent of the presence of carbonyls. The decrease in the monolayer potential induced by phloretin was the same in DMPC (ester linked)
Effect of Phloretin on Monlayers
Figure 6. Effect of phloretin on the dipole potential of PC and PE lipids lacking of carbonyls: DMPC (9); etherDMPC (0); DMPE (b); etherDMPE (O).
and etherDMPC (ether linked, i.e., without carbonyls). In addition, no effect of phloretin was found in DMPE (ester and ether linked). These results show that phloretin does not interact with the carbonyl groups. The decrease induced by phloretin on DMPC is around 150 mV. In Figure 6 it can be seen that the decrease obtained when the ester carbonyls are absent, in the pure phospholipids, is around 100 mV. That is, the effect of phloretin cannot be ascribed solely to the neutralization of the carbonyl dipoles. As shown previously,12,13,23 phloretin action is related to its interaction with the phosphate groups of the phospholipids and the dehydration of the interface. Conclusion As the same changes are found on ester and on ether derivatives, it is concluded that the effect of the phloretin
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action is independent of the carbonyl groups. Therefore, the decrease of the dipole potential by phloretin involves the neutralization or reorientation of other dipole moieties at the interface in addition to the carbonyls. The present results demonstrate that the action of phloretin to decrease the dipole potential is modulated by the moieties linked to the phosphate groups. The phosphate lateral interactions and its consequences on its hydration would modulate the interaction with phloretin and hence the effect on dipole potential. The better insertion of phloretin would be related to the steric barriers to reach the phosphate region. The values of dipole potential are congruent with the type of interaction that the phosphate has with its neighbors. Therefore, the dipole potential is strongly affected by the degrees of freedom that the phosphate may have. This could be affected by lateral interaction and hydration. In this context, the insertion of phloretin may compete with the hydration layer, decreasing the dipole potential to a higher extent when the phosphate is bound to water. The modulation of the penetration of peptides to the membrane phase, the permeability of hydrophobic anions, and the permeation of water8,10 have been related to the dipole potential, mainly ascribed to water hydrating the polar headgroups.6,7 The present results show that water polarized at the phosphates appears to be an important contribution to the dipole potential, confirming previous reports. In addition, they demonstrate that the phosphate is a region sensitive to hydration changes modulated by the chemical environment. Hence, membrane processes involving mechanisms of water exchange with the surface could be modulated by how the phosphate is exposed to water and might be related to the lipid specificity of several interactions of biological relevance. This should be related directly to the phospholipid composition and cholesterol levels of the biological membranes. Acknowledgment. This work was supported with funds from Agencia Nacional de Promocio´n Cientı´fica y Tecnolo´gica, Grant PICT 06047, CONICET (PIP 836), and UBACyT. E.A.D. is a member of the Research career of CONICET (National Research Council, Argentina). F.L. is recipient of a fellowship from UBACyT (Universidad de Buenos Aires, Argentina). LA049515K (23) Bechinger, B.; Seelig, J. Biochemistry 1991, 30, 3923.
