Langmuir 1998, 14, 679-682
679
Condensing and Expanding Effects of the Odorants (+)and (-)-Carvone on Phospholipid Monolayers Suram Pathirana,† William C. Neely,‡ and Vitaly Vodyanoy*,† Institute for Biological Detection Systems, Department of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine and Department of Chemistry, Auburn University, Auburn, Alabama 36849 Received June 24, 1997. In Final Form: October 28, 1997 Interactions of the odorants (+) and (-)-carvone (5-isopropenyl-2-methyl-2-cyclohexenone) with L-R1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (L-DPPC) monolayers are studied using surface pressure versus area isotherms measured in the temperature range 10-35 °C in the presence and absence of 5 mM odorants in the subphase. The data indicate that the molecular area occupied by the L-DPPC monolayer becomes larger when the monolayer is spread on the subphase containing (-)-carvone and that, in contrast, it becomes smaller when the monolayer is exposed to (+)-carvone. Both expanding and condensing effects are dependent on temperature. L-DPPC monolayers with (-)-carvone absorb twice as much heat as those with (+)-carvone when compressed at 30 °C. Under the same conditions, monolayers with (-)-carvone undergo a larger entropy change than monolayers with (+)-carvone. Variations in molecular areas and thermodynamic properties may contribute to membrane recognition of optical isomers.
Introduction The olfactory system exhibits an extremely sensitive and selective molecular recognition system for odorants. Humans and animals can discriminate between odorants with a high degree of molecular similarity, for example, between optical isomers.1-4 It is well established that the detection of odorants results from the association of odorous molecules with specific receptors on olfactory sensory neurons.5 Thus, the binding of odorant to the specific receptor is the important initial event in olfaction. Accepting this major sensory mechanism, we can pose a question of whether there are some other events in olfactory transduction, prior to or simultaneous with the odorant receptor interaction, which contribute to the specificity of the olfactory response. It has been suggested by Murphy6 that specificity of the response could be elicited by the specific distribution of the odorants in the olfactory mucus, modulating access to the receptor sites. The selective partitioning of an odorant into the receptor membrane itself can also cause changes in the physical environment of the olfactory receptor. The interaction of odorous molecules with membrane lipids can cause various physical changes such as changes in the order parameters of lipid bilayers, changes in molecular area (or volume), and alterations of surface potential and surface tension. This, in turn, would result in profound changes in membrane dynamic properties, such as membrane fluidity, viscosity, and electrostriction, which can substantially modulate the olfactory receptor macromolecules.6 It has been shown that lipid membrane components of several cell types are affected by the presence of odorant * To whom correspondence should be addressed. † College of Veterinary Medicine. ‡ Department of Chemistry. (1) Leitereg, T. J.; Guadagni, D. G.; Harris, J.; Mon, T. R.; Teranishi, R. Nature 1971, 230, 455-456. (2) Russel, G. F.; Hill, J. I. Science 1971, 17, 1043-1044. (3) Friedman, L.; Miller, J. G. Science 1971, 17, 1044-1046. (4) Delay, F.; Ohloff, G. Helv. Chim. Acta 1979, 62, 2168-2173. (5) Buck, L.; Axel, R. Cell 1991, 65, 175-187. (6) Murphy, R. B. In Molecular Neurobiology of the Olfactory System; Margolis, F. L., Getchell, T. V., Eds.; Plenum Press: New York, 1988; pp 121-142.
molecules.7-9 The implications for the role of the odorantmembrane nonspecific interactions in olfaction have been supported further by the observation that the addition of odorants to a suspension of olfactory epithelial cells induces changes in the membrane potential and fluidity.10 In a previous report,11 it has been shown that small odorant molecules, as exemplified by cyclohexanone, were able to modify the thermodynamic properties of phospholipid monolayers. Chiral recognition of the odorants (+)- and (-)-carvone by phosphlipid monolayers was demonstrated by analyzing the thermodynamic properties and the orientation of molecules within lipid assembles.12 Enantioselective binding was also reported for a poly(Lglutamic acid)-functionalized monolayer.13 In this work, we have examined the effects of two enantiomeric molecules of an odorant, carvone (Figure 1 A and B), on the steady-state properties of L-DPPC (Figure 1C) monolayers as an in vitro model for chiral recognition in membranes. In particular, we were interested in the effects of the odorants on the molecular area of the phospholipid monolayers. It is well-known that interactions within mixed monolayers can cause either positive or negative deviation from the mean molecular areas.14-16 The present work describes a peculiar expression of the chiral recognition in monolayers. Interactions of L-DPPC monolayers with (+)-carvone resulted in the decrease of the apparent molecular area of the monolayer. In contrast, under the same conditions, (-)-carvone caused the increase of the (7) Koyama, N.; Kurihara, K. Nature 1972, 236, 402. (8) Kashiwayanagi, M.; Kurihara, K. Brain Res. 1985, 359, 97-103 (9) Nomura, T.; Kurihara, K. Biochemistry 1987, 26, 6135. (10) Kashiwayangi, M.; Kurihara, K. J. Gen. Physiol. 1987, 89, 443457. (11) Ito, H.; Morton, T. H.; Vodyanoy, V. Thin Solid Films 1989, 180, 1-13. (12) Pathirana, S.; Neely, W. C.; Myers, L. J.; Vodyanoy, V. J. Am. Chem. Soc. 1992, 114, 1404-1405. (13) Higashi, N.; Saitou, M.; Mihara, T.; Niwa, M. J. Chem. Soc., Chem. Commun. 1995, 20, 2119-2120. (14) Harkins, W. D.; Florence, R. T. J. Chem. Phys. 1938, 6, 847855. (15) Phillips, M. C.; Ladbrooke, B. D.; Chapman, D. Biochim. Biophys. Acta 1970, 196, 35-44. (16) Gershfeld, N. L.; Pagano, R. E. J. Phys. Chem. 1972, 76, 12441249.
S0743-7463(97)00671-9 CCC: $15.00 © 1998 American Chemical Society Published on Web 01/10/1998
680 Langmuir, Vol. 14, No. 3, 1998
Pathirana et al.
Figure 1. Structures of (-)-carvone (A), (+)-carvone (B), and L-DPPC (C). The carbons which are marked by a star are chiral centers of molecules.
molecular area of the monolayer. Arnett and co-workers17 have clearly demonstrated that the chiral recognition in monolayers depends strongly on the ability of interacted molecules to bring their chiral centers into a favorable stereospecific interaction. Orientations of relatively large amphiphilic molecules in monolayers are restricted by strong interactions with a subphase, so that favorable orientations resulting in stereoselectivity cannot always be accomplished. L-DPPC monolayers do not usually exhibit molecular chirality because their chiral centers are hidden inside hydrocarbon tails,17,18 and only a strong structural perturbation can produce significant stereoselective interactions.19 To increase the orientational freedom of chiral molecules in monolayers, these experiments use relatively small semipolar molecules of carvone which are able to penetrate the phospholipid monolayer11,12,20 and interact with the chiral centers of L-DPPC. We speculate that small semipolar chiral molecules may be a better choice for expression of chiral recognition than amphiphilic molecules with restricted surface orientations. Materials and Methods Synthetic crystalline 1,2-dipalmitoyl-sn-glyceryl-3-phosphocholine (L-R-dipalmitoyllecithin) (L-DPPC) (purity >99%) was obtained from Avanti Polar Lipids, Inc. (S)-(+)- and (R)-(-)carvone (5-isopropenyl-2-methyl-2-cyclohexenone) were obtained from Fluka (purity >99%). The odorants were analyzed for purity by gas chromatography. The purity of the L-DPPC was examined by HPLC and IR spectroscopy. The subphase used in the experiments was a solution containing 55 mM KCl, 4 mM NaCl, 0.1 mM CaCl2, 1 mM MgCl2, and 2 mM 3-(N-morpholino)propanesulfonic acid (MOPS) made with deionized doubly distilled water (pH adjusted to 7.4 with 1 N KOH). For the experiments with the odorants, the carvones were dissolved in the above subphase. Measurements of surface pressure were performed on a KSV 2200 LB Langmuir-Blodgett film balance (17) Arnett, E. M.; Gold, J. M. J. Am. Chem. Soc. 1982, 104, 636639. (18) Arnett, E. M.; Gold, J.; Harvey, N.; Johnson, E. A.; Whitesell, L. G. In Biotechnological applications of lipid microstructures; Gaber, B. P., Schnur, J. M., Chapman, D., Eds.; Plenum Press: New York, 1988; pp 21-36. (19) Weis, R. M.; McConnel, H. M. Nature 1984, 310, 47-49. (20) De Boeck, H.; Zidovetzki, R. Biochim. Biophys. Acta 1988, 946, 244-252.
Figure 2. Surface pressure-area (A-C) and elasticity-area (D-F) isotherms of L-DPPC on buffered subphase solutions without odorants (solid lines), with 5 mM (+)-carvone (dashed lines), and with 5 mM (-)-carvone (dotted lines) at the indicated temperatures. Each isotherm was obtained by averaging three or four runs. Standard deviations of the surface pressure were determined at 11 equally spaced points along the curves at each temperature. The mean standard deviations at l0, 25, and 35 °C are as follows: 0.053, 0.121, 0.481 mN/m (L-DPPC); 0.311, 0.338, 0.307 mN/m (L-DPPC/(+)carvone); 0.249, 0.357, 0.199 mN/m (L-DPPC/(-)carvone). The surface elasticity E ) -A(∂Π/ ∂A)T was calculated directly from the isotherms.21 (KSV Chemicals, Finland) with a Teflon trough (45 cm × 15 cm). The apparatus for measurements of surface pressure isotherms, the methods of monolayer deposition, and the data analysis were as described in detail elsewhere.11,12,21 L-DPPC monolayers were spread from 50 µL of 1 mg/mL hexane solutions. The exact lipid concentration in the spreading solution was determined by a phosphorus assay.22 Each monolayer was allowed to equilibrate and to stabilize for 10 min before data collection. Surface pressure-surface area (Π-A) isotherms were measured at 10, 15, 20, 25, 30, and 35 °C. Using the minimum dispersion of mean molecular area as a criterion of the optimal rate of compression,21 0.5 cm2 s-1 was chosen for these experiments. Each isotherm was replicated 3-5 times. Thermodynamic values of free energy (∆G), entropy (∆S), enthalpy (∆H), and elasticity (E) for isothermal compression were calculated for bare L-DPPC monolayers and monolayers spread on subphases containing 5 mM (+)- or (-)-carvone (L-DPPC/(+)carvone and L-DPPC/(-)carvone monolayers).
Results Figure 2A-C represents the surface pressure-area isotherms obtained at 10, 25, and 35 °C, respectively, for L-DPPC monolayers spread on an odorant-free subphase and for L-DPPC monolayers spread on subphases containing 5 mM (+)- or (-)-carvone. The presence of odorants causes a significant difference in the shape and position (21) Vodyanoy, V.; Bluestone, G. L.; Longmuir, K. J. Biochim. Biophys. Acta 1989, 1047, 284-289. (22) Bartlett, G. R. J. Biol. Chem. 1959, 234, 466-468.
Effects of the Odorants (+)- and (-)-Carvone
of the isotherms. This indicates that these odorants interact with L-DPPC monolayers appreciably. The most outstanding feature of the monolayers exposed to odorants is that two inverse enantiomers shift L-DPPC isotherms in opposite directions. Thus, isotherms with the enantiomeric odorants show a clear chiral discrimination effect which is temperature dependent. At 25 °C, L-DPPC/()carvone monolayers are more expanded than bare L-DPPC monolayers, and L-DPPC/(+)carvone monolayers are more condensed compared to L-DPPC monolayers spread on the odorless subphase. Similar features are expressed by elasticity isotherms (Figure 2D-F), which are obtained from the respective surface area isotherms. All the functions are characterized by the attainment of maximum elasticity at high pressures.21,23 At 25 and 35 °C, the maxima of elasticity have been achieved at highest molecular areas for monolayers with (-)-carvone, and the maxima have been attained at lowest areas for the monolayers with (+)-carvone. Thus, elasticity isotherms of monolayers modified by different isomers appear to be shifted in opposite directions. The isotherms at 10 °C show only liquid-condensed (LC) states.24 The extrapolated “zero-pressure” areas (Ao) for L-DPPC, L-DPPC/(+)-carvone, and L-DPPC/(-)-carvone monolayers at 10 °C are 46.67 ( 0.67, 48.06 ( 0.20, and 48.34 ( 0.22 (% SD), respectively. Thus, addition of odorants to subphases at 10 °C significantly (p < 0.05; p < 0.02) increases the apparent molecular area of L-DPPC monolayers exposed to both isomers. The temperature increase results in the profound decrease of molecular areas of L-DPPC/(+)carvone monolayers compared to those of plain L-DPPC monolayers (Figure 3). That is to say, at higher temperatures (+)-carvone causes an apparent condensing effect. In contrast, under the same conditions (-)-carvone elicits an expanding effect, so that the apparent molecular area of L-DPPC/(-)carvone monolayers is considerably larger than the area of L-DPPC monolayers (Figure 3). Table 1 shows values of free energy, enthalpy, and entropy changes for the compression between 0 and 30 mN/m for the L-DPPC monolayers with (+) and (-)carvone, respectively. As the table indicates, at 27.5 °C L-DPPC monolayers with (-)-carvone absorb twice as much heat as monolayers with (+)-carvone. Under the same conditions, L-DPPC monolayers with (-)-carvone undergo a larger negative entropy change than monolayers with (+)-carvone. Discussion Π-A and surface elasticity (E) isotherms (Figure 2) for L-DPPC monolayers show a significant condensing effect16
when the monolayers are spread on the subphase containing (+)-carvone, and in contrast, a profound expanding effect14,15 is evident for the monolayers with (-)-carvone. The specific molecular areas (Ao) of L-DPPC, L-DPPC/(+)carvone, and L-DPPC/(-)carvone monolayers were calculated from Π-A isotherms in the temperature range 10-25 °C. Figure 3 provides a comparison of unmodified L-DPPC monolayers with monolayers spread on a subphase containing the odorants. The values of the molecular surface area for L-DPPC monolayers compare well with published X-ray diffraction data for a fully hydrated multilamellar L-DPPC system (Table 2).25 (23) Blank, M.; Soo, L.; Abbott, R. E. J. Membr. Biol. 1979, 47, 185193. (24) Gaines, G. L., Jr. Insoluble Monolayers at Liquid-Gas Interfaces; Interscience: New York, 1966; Chapter 4. (25) Marsh, D. CRC Handbook of Lipid Bilayers; CRC Press: Boston, 1990; pp 163-194.
Langmuir, Vol. 14, No. 3, 1998 681
Figure 3. Temperature dependence of the extrapolated “zeropressure” area24 for L-DPPC (open circles), L-DPPC/(+)carvone (filled circles), and L-DPPC/(-)carvone (triangles). The areas are obtained from each experimental isotherm, and the mean values and standard deviations are calculated at 10, 15, 20, and 25 °C. The mean standard deviations are as follows: 0.33 Å2/molecule (L-DPPC); 0.30 Å2/molecule (L-DPPC/(+)carvone); 0.22 Å2/molecule (L-DPPC/(-)carvone). Points represent experimental data, while curves are cubic spline interpolations. The LC portions of the Π-A isotherms at 30 and 35 °C were very limited and were not suitable for the reliable extrapolation of “zero-pressure” areas. Upper inset: The layer-parallel arrangement of the phosphorylcholine group of phospholipid monolayers (requires 47-54 Å2/molecule). Lower inset: The tilted arrangement of the phosphorylcholine group of phospholipid monolayers (space-saving orientation, requires less than 47 Å2/molecule).28 Table 1. Values of Thermodynamic Properties for the Compression of the Monolayers at the Air-Water Interface material
∆G (kcal mol-1)
-∆H (kcal mol-1)
-∆S (cal mol-1 K-1)
L-DPPCa L-DPPC/(+)carvonea L-DPPC/(-)carvonea L-DPPCb L-DPPCc palmitic acidd
3.499 ( 0.029 3.120 ( 0.039 3.582 ( 0.055 3.600 ( 0.020 3.43 2.31 ( 0.014
15.53 ( 2.56 14.25 ( 4.00 29.40 ( 5.00 11.90 ( 1.20 12.57 14.49 ( 0.25
62.3 ( 8.5 57.8 ( 13.4 109.6 ( 17.0 52.3 ( 3.3 52.8 56.0 ( 2.0
a L-DPPC monolayers with no odorants; with 5 mM (+)- or (-)carvone in subphases. Free energy (∆G), enthalpy (∆H), and entropy (∆S) at the surface pressure change ∆Π ) 0-30 mN/m, ∆T ) 2530 °C, and Tm ) 27.5 °C (mean temperature for the interval) were computed as described in refs 11, 21, 30, 33. Each value represents a mean of three or four measurements ( SD. b Values for L-DPPC on a subphase of 100 mM NaCl, 5 mM KCl, 5 mM Tris-HCl, pH 7.4; at ∆Π ) 0-40 mN/m, ∆T ) 20-30 °C, Tm ) 25 °C.21 c Values for L-DPPC on a subphase of pure water at ∆Π ) 0-40 mN/m, ∆T ) 20-30 °C, and Tm ) 25 °C.33 d Helmholtz free energy, enthalpy, and entropy for the compression of the liquid-condensed monolayer of palmitic acid on a subphase of water at pH 2, ∆T ) 15-25 °C, and Tm ) 20 °C.30
The addition of 5 mM of (-)-carvone to a subphase at 10 °C caused a significant (P < 0.02) increase in A0 from 46.67 to 48.34 Å2 molecule-1. A further temperature increase resulted in both an additional increase of the apparent molecular area of L-DPPC/(-)carvone monolayers and a further considerable positive departure from
682 Langmuir, Vol. 14, No. 3, 1998
Pathirana et al.
Table 2. Average Area (Å2/molecule) Available to One Hydrophilic Group on the Lipid-Water Interface t, °C
L-DPPC monolayersa
10 15 20 25
46.67 ( 0.72 47.76 ( 0.21 48.49 ( 0.20 51.29 ( 0.21
L-DPPC
fully hydrated multilamellar phaseb
phaseb
45.8
Lc′
48.6 52.3
Lβ′ Lβ′
a Data obtained in the present work as extrapolated “zeropressure” areas of Π-A isotherms. b X-ray diffraction data.25
the area of plain L-DPPC monolayers (Figure 3). These findings are very similar to those reported earlier11 for another odorant, cyclohexanone, and they are in general accord with X-ray and 2H nuclear magnetic resonance (NMR) data26,27 on dimyristoylphosphatidylcholine bilayers, which show that n-alkanes and n-alcohols increase the area per lipid molecule. The values of the molecular area found in the present work for L-DPPC monolayers (Table 2) are in good agreement with that expected from a layer-parallel arrangement28 of the phosphorylcholine group (Figure 3, upper inset), which requires an area of 47-54 Å.2 This area is significantly larger than the crosssection of two palmitoyl chains in the condensed crystalline state.28 Addition of (-)-carvone, however, causes an expansion of the molecular area above 55 Å2 (Figure 3) when an appropriate two-dimensional headgroup contact cannot exist, and this expansion can be accomplished only in the presence of guest molecules which act as spacers and form lateral bridges between the host phospholipid molecules.28 We suggest that (-)-carvone molecules have a stereospecific advantage over (+)-carvone molecules when they interact with L-DPPC monolayers. Thus, the (-)-carvone imposes greater restrictions on the motion of hydrocarbon chains, decreasing configuration freedom, with a consequent decrease in entropy.11,20 The addition of 5 mM (+)-carvone to a subphase at 10 °C caused a significant (P < 0.05) increase in A0 from 46.67 to 48.06 Å2 molecule-1. A further temperature increase resulted in changes which were completely opposite to the changes of L-DPPC/(-)carvone monolayers. At higher temperatures, the apparent molecular area of L-DPPC/(+)carvone monolayers decreased (Figure 3), showing a negative departure from the area of plain L-DPPC monolayers. This condensing effect may be similar to that found in dipalmitoyllecithin/cholesterol mixed monolayers.16 The decrease of apparent molecular areas under 47 Å2 molecule-1 cannot be described by the layer-parallel arrangement of phosphorylcholine groups (Figure 3, upper inset) and requires a more space-saving arrangement in which the headgroups are inclined (Figure 3, lower insert) and/or alternately displaced.28 The apparent condensing effect elicited by (+)-carvone can be explained by another space-filling model. Compression of L-DPPC/(+)carvone monolayers can lead to a loss of surface lipid molecules by vertical displacement into the multilamellar phase, and obviously, the apparent molec(26) Pope, J. M.; Walker, L. W.; Dubro, D. W. Chem. Phys. Lipids 1986, 35, 259-277. (27) Pope, J. M.; Dubro, D. W. Biochim. Biophys. Acta 1986, 858, 243-253. (28) Hauser, H.; Pascher, L.; Pearson, R. H.; Sundell, S. Biochim. Biophys. Acta 1981, 650, 21-51.
ular area decreases. A similar phenomenon has been demonstrated for monolayers prepared from lamellar bodies containing a high level of unsaturated fatty acids.21 On the basis of the present data we are not able to discriminate between an “inclined”28 and a “squeezedout” space-filling model.21 Values of thermodynamic properties for the compression of the monolayers are given in Table 1. These values show good agreement with those reported previously11,21,33 for L-DPPC monolayers at the air-water interface. When (-)-carvone is added to the subphase, L-DPPC monolayers absorb twice as much heat as those with (+)-carvone or with no carvone when compressed at 30 °C (Table 1). The large negative change in entropy of compression for L-DPPC/(-)carvone monolayers (Table 1) cannot be explained by the increase of the specific molecular area. The entropy change of a two-dimensional ideal system of noninteracting particles, for a change of surface area from 200 to 47 A2 molecule-1, can be estimated as -R ln(200/ 47) ) -2.87 cal mol-1 K-1.29 This is a small value compared to the observed change in entropy of L-DPPC/(-)carvone monolayers (Table 1). Moreover, the changes in the mobility of polar groups cannot account for this large ∆S. If we assume that the interaction of (-)-carvone results in the complete loss of rotational freedom for the polar group, then ∆S ) -R ln 4π ) - 5.1 cal mol-1 K-1.30 Thus, this large negative change in entropy of compression of L-DPPC/(-)carvone monolayers can be attributed to the restriction of the conformational freedom of hydrocarbon chains because of stereospecfic interactions with (-)carvone. The results indicate that (-)-carvone interacts more strongly with L-DPPC than does its optical isomer.12 These results are consistent with our previous finding12 indicating that the average orientation of carvone molecules interacting with L-DPPC monolayers is temperature dependent and sterioselective. A strong temperature dependence of the ability to discriminate (+)- and (-)carvones was found in turtle olfactory receptors.31 These data may be related to the finding of Litereg et al.1 that (-)-carvone is a much stronger odorant than (+)-carvone on a threshold basis. The idea of stereospecific molecular recognition by phospholipid molecules does not necessarily conflict with the existence of chemospecific receptors;5 it does, however, suggest that the interaction of odorous molecules with lipid bilayers can possibly influence olfactory transduction.32 Acknowledgment. This work was supported by Grant FAA 93-G-058 (V.V.) and by the Office of Naval Research through Grant N00014-90-J-1515 (W.C.N.). We thank Mr. M. Hartell and Mr. R. O. Boddie for their technical assistance. LA970671J (29) Stishov, S. M. Sov. Phys. Usp. 1988, 31, 52-67. (30) Gershfeld, N. L.; Pagano, R. E. J. Phys. Chem. 1972, 76, 12311237. (31) Hanada, T.; Kashiwayanagi, M.; Kurihara, K. Am. J. Physiol. 1994, 266, R1816-R1823. (32) Loh, H. H.; Law, P. Y. Annu. Rev. Pharmacol. Toxicol. 1980, 20, 201-234. (33) Villalonga, F. Biochim. Biophys. Acta 1968, 163, 290-300.
