Contribution of Phosphate Groups to the Dipole Potential of

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Contribution of Phosphate Groups to the Dipole Potential of Dimyristoylphosphatidylcholine Membranes S. Diaz,† F. Lairio´n,‡ J. Arroyo,† A. C. Biondi de Lopez,§ and E. A. Disalvo*,‡ Laboratorio de Fisicoquı´mica de Membranas Lipı´dicas, Ca´ tedra de Quı´mica General e Inorga´ nica, Facultad de Farmacia y Bioquı´mica, Universidad de Buenos Aires, Junı´n 956, 2°P, 1113 Buenos Aires, Argentina, and Instituto de Fisicoquı´mica, Universidad Nacional de Tucuma´ n, Tucuma´ n, Argentina Received May 16, 2000. In Final Form: October 23, 2000 Phloretin, a molecule which is known to decrease the dipole potential of lipid membranes, has a little effect on the CdO frequencies of dimyristoylphosphatidylcholine bilayers (DMPC) in comparison to that observed on the phosphates. In the first case, the frequency is displaced very slightly to higher values, while a pronounced downward shift is observed on the asymmetric vibration frequencies of the phosphates. The effect of phloretin on the phosphate groups is correlated with a decrease in the monolayer potential of DMPC in the gel and in the liquid crystalline state. From the comparison with monolayers spread on water and on 0.15 M trehalose, it is concluded that the PdO bonds of the phosphates can contribute to the dipole potential of the membrane in addition to the contribution given by the orientation of the -P-N+ dipole.

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 The potential is caused by the orientation of constitutive and adsorbed dipoles within the lipid-water interface.2 There are several possible structural portions of the phospholipid molecules contributing to the dipole potential. One of them seems to arise from the glycerol-ester region of the lipids, since when ether-linked lipids are compared to ester-linked lipids, the magnitude of the dipole potential is to be less by approximately 120 mV.3 Another one has been ascribed to the orientation of the -P-N+ dipole with respect to the plane of the membrane.10,11 In addition to phospholipid structural moieties, water, which may penetrate to the level of the carbonyl oxygens, is another component of the dipole potential.4,5 Around 18-25 water molecules/lipid are associated to phosphatidylcholines in the fluid phase. Among these, molecules that are strongly oriented can account for the magnitude of the dipole potential. Part of this type of water appears to be linked to the carbonyl groups. Substitution of these water molecules by hydrogen-bonding compounds, such * Corresponding author. Phone: 54 11 49648249. Fax: 54 11 49648274. E-mail: [email protected]. † Universidad Nacional de Tucuma ´ n. ‡ Universidad de Buenos Aires. § Deceased on April 24, 2000. (1) Brockman, H. Chem. Phys. Lipids 1994, 73, 57. (2) Haydon, D. A.; Hladky, S. B. Q. Rev. Biophys. 1972, 5, 187. (3) Gawrisch, K.; Ruston, D.; Zimmerberg, J.; Parsegian, V. A.; Rand, R. P.; Fuller, N. Biophys. J. 1992, 61, 1213. (4) Simon, S. A.; McIntosh, T. J. Methods Enzymol. 1986, 127, 511. (5) White, S. H.; Wiener, M. C. In Permeability and Stability of Lipid Bilayers; Disalvo, Simon, Eds.; CRC Press: Boca Raton, FL, 1995; pp 1-20. (6) Diaz, S.; Amalfa, F.; Biondi de Lopez, A. C.; Disalvo, E. A. Langmuir 1999, 14, 5858. (7) Luzardo, M del C.; Amalfa, F.; Nun˜ez, A. M.; Dı´az, S.; Biondi de Lopez, A. C.; Disalvo, E. A. Biophys J. 2000, 78, 2452. (8) Franklin, J. C.; Cafiso, D. S. Biophys. J. 1993, 65, 289. (9) Pohl, P.; Rokitskaya, T. I.; Pohl, E. E.; Saparov, S. M. Biochim. Biophys. Acta 1997, 1323, 163. (10) Bechinger, B.; Seelig, J. Biochemistry 1991, 30, 3923. (11) Cseh, R.; Benz, R. Biophys. J. 1998, 77, 1477.

as trehalose at a 0.15 M concentration, results in a decrease of the dipole potential of DMPC in the gel state of about 80 mV.6,7 However, when carbonyls of the ester region are replaced by ether-linked lipids or trehalose is present in the aqueous solution, the remaining value of the monolayer potential is still significant. This suggests that, in addition to carbonyls and their hydration water, other structural groups at the lipid/water interface may be contributing to the dipole potential. It may be possible that the -P-N+ dipole can contribute to the dipole potential depending on the angle that this group may have with respect to the plane of the membrane.11 However, the normal component of the -P-N+ group appears to be rather low since the dipole is laying nearly parallel to the membrane plane. 2H NMR experiments have suggested that the choline end of the -P-N+ group is laying toward the hydrocarbon phase, due to the hydrophobic character of the methylenes. In contrast, the -N+ end in phosphoethanolamines seems to be oriented toward the water phase.18 Despite this conformational differences, the dipole potential of phosphatidylcholines and phosphatidylethanolamines monolayers are comparable,8,16 thus suggesting that choline and amine groups have little effects on the dipole potential. 31P NMR results have shown that phloretin, a molecule that decreases dramatically the dipole potential of fluid PC vesicle membranes,8 affects the anisotropy shift of the phosphate, in a way in which the choline group moves closer to the hydrocarbon layer.10 This rotation would create a larger dipole potential since the negative part of the -P-N+ group is more exposed to water. If this is the (12) MacDonald, R. C.; Simon, S. A. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 4089. (13) Luzardo, M del C.; Peltzer, G.; Disalvo, E. A. Langmuir 1998, 14, 5858 (14) Hu¨bner, W.; Blume, A. Chem. Phys. Lipids 1998, 96, 99. (15) Bach, D.; Sela, B.; Miller, I. R. Chem Phys. Lipids 1982, 31, 381. (16) Cafiso, D. S. In Permeability and Stability of Lipid Bilayers; Disalvo, Simon, Eds.; CRC Press: Boca Raton, FL, 1995; Chapter 9, p 197. (17) Seelig, J. Biochim. Biophys. Acta 1978, 515, 105. (18) Cseh, R.; Benz, R. Biophys. J. 1999, 77, 1477.

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Figure 2. Effect of phloretin on the dipole potential of DMPC monolayers in the Lβ′ and the Pβ′ phases: DMPC at 9 °C (b); DMPC at 20 °C (4).

Figure 1. Probable location of phloretin at the lipid-water interface.

case, the physical effect of including phloretin into the lipid headgroups would increase the dipole potential since the -P-N+ dipole normal component would be oriented in parallel to the carbonyls. Thus, the effectiveness of phloretin to decrease the dipole potential depends on its ability to adsorb in an oriented manner, in a way that its dipole moment would oppose, in an antiparallel array, the carbonyl and -P-N+ dipole components at the membrane interface (Figure 1). As phloretin does not affect the hydrocarbon region at low molar ratios10 but only the headgroups, it has been suggested that this molecule lies in this region. Studies correlating the changes in the electrical potential with the structural modifications at specific groups at the interface are lacking. The action of phloretin was ascribed to the hydrogen bonding of this compound to the phosphate group.19 In contrast, phloretin does not affect the carbonyls, thus suggesting that the action of this compound to decrease the dipole potential is not due to a direct hydrogen binding to these groups.6 Thus, it is not clear if phloretin action is a consequence of a direct chemical interaction with phosphates, to changes in the water polarized by this group, or to an electrical neutralization of the CdO and -P-N+ dipoles at the interface. The purpose of this paper is to study the effect of phloretin on the phosphates of DMPC membranes by FTIR spectrometry in correlation to changes in the dipole potential in monolayers. Infrared spectrometry can give information about the chemical interaction of a functional group in a structural fragment within a complex molecule. Similar chemical groups with different degrees of hydration are possible to be distinguished using this methodology.14 The comparison of the electrical measurements with structural ones would allow one to ascribe the changes to precise chemical bonds within the subgroups of the polar head and possibly to their state of hydration. Thus, in this paper we have compared the effect of phloretin on dipole potential in relation to its changes on the carbonyl and phosphates groups with that promoted by trehalose that (19) Wong, P. T. T.; Mantsch, H. H. Chem. Phys. Lipids 1988, 46, 213.

decreases the dipole potential by binding to carbonyls and displacing water. By comparison with measurements done in the presence and the absence of trehalose the possible contribution of phosphate to the dipole potential may be inferred. Materials and Methods. Dimyristoylphosphatidylcholine (DMPC) was obtained from Avanti Polar Lipids, Inc. (Birmingham, AL), and used as received. The purity of lipids was checked by thin-layer chromatography using a chloroform-methanol-water (65:25:4) mixture as running solvent. Phloretin was obtained from Sigma Chemical Co (St. Louis, MO). Trehalose was from Fluka, and water was MilliQ quality. Dipole Potential in Monolayers. The dipole potential was determined in monolayers formed on an air-water interface by spreading a chloroform solution of DMPC with different ratios of phloretin as described before.12,13 Aliquots of the chloroform solution of lipids were added to the air-water interface, exhaustively cleaned by suction, until a constant potential was reached above 20 nmol of lipids. Temperature was measured with a calibrated thermocouple and maintained within (0.5 °C. The values of dipole potential (Ψ) were determined by using the following expression:

Ψ ) VAg/AgCl - Vgrd ) Vsolution - Vgrd where VAg/AgCl is the potential of the reference electrode and Vgrd the potential of the shield covering the ionizing electrode. The values of monolayer potentials were taken within an experimental error of (20 mV. Infrared Spectroscopy by Fourier Transform (FTIR). FTIR measurements were done on multilamellar dispersion of DMPC/phloretin dispersed in D2O. A chloroformic solution of different ratios of phloretin/DMPC was dried under nitrogen stream and vacuum. The dried films were dispersed above the transition temperature in 10 mM Tris-HCl buffer solution in D2O at pH 7 by vortexing during 1 h at 45 °C. Spectra were obtained at temperatures below and above the phase transition, in a Perkin-Elmer 1600 spectrometer using a cell with AgCl windows, thermostated with a temperature control within (0.1 °C. The resolution of the equipment in the conditions employed in this work was 2 cm-1. For each condition a total of 1024 scans were done. The spectra were analyzed using the mathematical software Grams. The deconvolution algorithm was applied in order to define the contours of overlapped bands. The bands in the mixtures were assigned to the carbonyl and phosphates by comparison with pure lipids dispersed in D2O. The displacement of the corresponding frequencies were calculated from the spectra for the stereochemical groups. The mean

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Figure 3. Absorption bands of the carbonyls and the phosphates of DMPC bilayers containing phloretin: (A) carbonyl band corresponding to DMPC (a) and to DMPC with 20% phloretin (b) at 9 °C; (B) phosphate asymmetric band of DMPC (a) and DMPC with 20% phloretin (b) at 9 °C. Note the different scales in parts A and B. values were obtained from spectra in these conditions from a total of four different batches of samples. The standard deviation of the frequency shift calculated from pool of data was about (1.5 in all the conditions assayed.

Results The potential of a DMPC monolayer spread on water decreases from 500 to 300 mV with phloretin both at 9 °C (Lβ′ phase) and at 20 °C (Pβ′ phase) (Figure 2). At 0.4 phloretin/DMPC ratio, the dipole potential reaches a constant value. Under this condition, the infrared spectra of DMPC at 9 °C shows that the carbonyl bands at 1725 and at 1740 cm-1 remain unchanged within the experimental error (Figure 3 A). An increase in the intensity of the band at 1725 cm-1 is observed, denoting the increase of this carbonyl population. In contrast, a clear shift in the phosphate band from 1220 to 1200 cm-1 is noticed, with no changes in the intensities (Figure 3B). The major changes observed on the DMPC infrared spectra as a function of phloretin content at 20 °C correspond to the asymmetric vibration of the phosphate groups (Figure 4). Up to 20% phloretin, the effect on the carbonyls is very small near the experimental error ((2 cm-1) and only slight shifts to lower frequencies are observed on the symmetric phosphate frequencies. However, the asymmetric phosphate vibration is decreased by more than 30 cm-1. The effect of phloretin on the electric potential was determined on DMPC monolayers in the fluid state (27 °C) in the absence and in the presence of trehalose 0.15 M in the subphase. This sugar is known to decrease the dipole potential by displacing water from the carbonyls in the ester unions.7 The data of Figure 5 show a consistent decrease of the dipole potential of about 50 mV, caused by trehalose for all the phloretin/DMPC ratios assayed. Discussion The inspection of Figures 2 and 4 indicates that phloretin decreases the potential of DMPC monolayers in conditions in which a significant decrease in the asymmetric phosphate vibration is observed in the Pβ′ (20 °C) and in the Lβ′ (9 °C) phases.

Figure 4. Frequency shifts for the carbonyl and phosphate bands of DMPC as a function of phloretin at 20 °C: (O, b) CdO bands; (2) PO2 antisym; (4) PO2 sym.

Phloretin affects the carbonyl frequencies very slightly near the experimental error. The small displacement to higher values suggests that carbonyls are being dehydrated. However, this dehydration would not be caused by a direct hydrogen binding of phloretin to the carbonyls, since in that case a decrease of the frequency would be expected. Thus, part of the decrease of dipole potential produced by phloretin should be related to a partial depolarization of bound water and to an electrical neutralization of the CdO groups without a chemical direct interaction. The changes in the intensity corresponding to the population of carbonyls at 1725 cm-1 support the conclusion that, for a given mass of DMPC, phloretin is inserting its carbonyls at the membrane interface. The orientation of these additional dipoles is irrelevant for FTIR measurements. Moreover, it should be noticed that the intensity increase corresponds to the population of carbonyls with the higher hydration, while the population of lower hydration is not affected.14,15

Dipole Potential of DMPC Membranes

Figure 5. Effect of phloretin on the dipole potential of DMPC AT 27 °C in the presence (b) and in the absence (2) of trehalose in the subphase.

The decrease induced by the addition of trehalose to the subphase of a DMPC monolayer containing phloretin is independent of the phloretin/lipid ratio (Figure 5). This denotes that phloretin does not modify the sites of insertion of the trehalose, i.e the carbonyls in the ester unions. Therefore, this suggests that the PC carbonyl exposure is not affected by the phloretin inclusion. This is in agreement with the fact that at the phloretin/lipid concentration employed in this study (1:5 phloretin-DMPC) no effect on the phase state of the membrane was produced (data not shown). The effects of trehalose and phloretin in decreasing the potential are not cooperative. Thus, it is concluded that phloretin does not bind to the PC carbonyls and does not alter significantly its orientation but is adding more of these groups to the interface, which are hydrated and opposed to the constitutive dipoles of the membrane. However, the action of phloretin seems not to be restrained to carbonyl insertion. The displacement of the asymmetric frequency of the phosphates to lower values is indicative of strong hydrogen bonds between phloretin and those groups. It may be possible that this binding may perturb the orientation of the -P-N+ group. Dipole potential arises from charge separation at the interface. This can be partially due to the charges of PC and PE, which are known to have large dipole potentials. However, the normal component is low since the -P-N+ group is laying nearly parallel to the membrane plane. In addition, due to the different hydrophobicities of cholines and ethanolamines, the choline group is oriented toward the hydrocarbon phase and the ethanolamine toward the aqueous phase. The negligible differences reported between PC and PE monolayers suggest that, first, the choline or ethanolamine groups has no influence “per se” on the dipole potential and, second, the changes in the orientation due to choline or ethanolamine linked to the phosphate seem to be of low magnitude. It has been proposed that the insertion of phloretin may change the orientation of the -P-N+ in phosphatidylcholine membranes.18 Thus, the interaction of phloretin with the phosphates may change the orientation of the -P-N+ group. This phenomenom has been demonstrated

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by 2H NMR, in which the choline groups appear to move into the hydrocarbon phase.10,17 However, this orientation has, as a consequence, an increase of the dipole potential, and therefore, phloretin would have to counteract the normal component of the -P-N+ plus those of the carbonyls. Although this may be a possibility, the PdO or P-Odipoles of the phosphate group can be as relevant as the normal component of the -P-N+ dipole. Those moieties appear to be oriented in a way in which the oxygen end is exposed to water, as derived from FTIR measurements.14 The displacement of the frequency of the asymmetric vibration of the P-O bonds to lower frequencies has been related to the states of hydration of the group. In consequence, this group has been suggested to act as a sensor of the hydration level of the interface.14,15 Thus, water hydrating the PdO group may be polarized, contributing to the dipole potential as well. The hydration of the anhydrous lipids displaces the band of the asymmetric phosphate vibration to lower frequencies. The same trend is observed when phloretin is added to hydrated DMPC bilayers, thus suggesting that the phloretin/phosphate interaction is stronger than the water-phosphate interaction. It may be suggested that this interaction would cause a displacement of polarized water from the phosphate. The exposure of the P-O- or PdO bonds normal to the water phase appears relevant since an electrostatic contribution to the phloretin adsorption and the concomitant changes of dipole potential at different pHs have been suggested elsewhere.11,18 The strong frequency shift of the asymmetric vibration denotes a change in the dipole moment of the bond. Therefore, the contribution of the PdO dipole per se, and the water polarized by it, cannot be discarded, in addition to the contribution that this group could have as part of the -P-N+ dipole. The effect of phloretin on lipid membranes has been discussed in terms of a static contribution to the dipole potential given by the hydrocarbon methylenes and the ester carbonyls and a dynamic contribution ascribed to the P-N rotation with respect to the membrane plane.11 The comparison of the effects of trehalose and phloretin on the electrical and structural properties of the interface allow one to suggest that the contributions to the dipole potential can be summarized as a static component due to carbonyls and hydrocarbon methylenes and a dynamical component due to the orientation of the -P-N+ group given by the rotation of the N+ end of the phosphocholine and the P-O- bonds which might be also part of the static component. The static component (CdO) and its hydration state is not affected by phloretin, as suggested by the similar decrease of dipole potential induced by the different ratios of phloretin in the absence and in the presence of trehalose. However, water molecules hydrating each of these components might have different degrees of mobilities. Acknowledgment. This work was supported with funds from CONICET (Argentina) (PIP 836/98), UBACyT, U. Buenos Aires Argentina (TB26), and CIUNT, University of Tucuma´n, Argentina. E.A.D. is a member of the Research Career of CONICET. F.L. is a fellow of the University of Buenos Aires. The authors are grateful to Dr. Delia L. Bernik for the reading of the manuscript. LA000683W