Anesthesia cutoff phenomenon: interfacial hydrogen bonding.

Anesthesia "cutoff" refers to the phenomenon of loss of anesthetic potency in a homologous series of alkanes and their derivatives when their sizes become too large. In this study, hydrogen bonding of 1-alkanol series (ethanol to eicosanol) to dipalmitoyl-L-alpha-phosphatidylcholine (DPPC) was studied by Fourier transform infrared spectroscopy (FTIR) in DPPC-D2O-in-CCl4 reversed micelles. The alkanols formed hydrogen bonds with the phosphate moiety of DPPC and released the DPPC-bound deuterated water, evidenced by increases in the bound O-H stretching signal of the alkanol-DPPC complex and also in the free O-D stretching band of unbound D2O. These effects increased according to the elongation of the carbon chain of 1-alkanols from ethanol (C2) to 1-decanol (C10), but suddenly almost disappeared at 1-tetradecanol (C14). Anesthetic potencies of these alkanols, estimated by the activity of brine shrimps, were linearly related to hydrogen bond-breaking activities below C10 and agreed with the FTIR data in the cutoff at C10.

Differential affinity of charged local anesthetics to solid-gel and liquid-crystalline states of dimyristoylphosphatidic acid vesicle membranes.

Cationic local anesthetics decreased the transition temperature of the anionic phospholipid (dimyristoylphosphatidic acid, DMPA) vesicles. The counterion concentration changes the electrical double layer effect, and affects the magnitude of temperature depression caused by anesthetics. From the counterion effect on the transition-temperature depression, the partition coefficients of cationic local anesthetics to liquid-crystalline and solid-gel DMPA membranes were separately estimated. The differences in the partition coefficients between solid-gel and liquid-crystalline membranes correlated to the nerve blocking potencies. There are at least two states in the nerve membranes: resting state at higher temperature and excited state at lower temperature. We speculate that the resting state corresponds to the liquid-crystalline state, and the excited state to the solid-gel state. The difference in the partition coefficients to the resting and excited states is the cause of local anesthesia.

Benzyl alcohol penetration into micelles, dielectric constant of the binding site, partition coefficient and high-pressure squeeze-out.

The absorbance maximum, lambda max, of a local anesthetic, benzyl alcohol, is shifted to longer wavelengths when solvent polarity is decreased. The shift was approximately a linear function of the dielectric constant of the solvent. This transition in electronic spectra according to the microenvironmental polarity is used to analyze benzyl alcohol binding to surfactant micelles. A facile method is devised to estimate the micelle/water partition coefficient from the dependence of lambda max of benzyl alcohol on surfactant concentrations. The effective dielectric constants of the sodium decyl sulfate, dodecyl sulfate and tetradecyl sulfate micelles were 29, 31 and 33, respectively. The partition coefficient of benzyl alcohol between the micelles and the aqueous phase was 417, 610 and 1089, respectively, in the mole fraction unit. The pressure dependence of the partition coefficient was estimated from the depression of the critical micelle concentration of sodium dodecyl sulfate by benzyl alcohol under high pressure up to 200 MPa. High pressure squeezed out benzyl alcohol molecules from the micelle until about 120 MPa, then started to squeeze in when the pressure was further increased. The volume change of benzyl alcohol by transfer from the aqueous to the micellar phase was calculated from the pressure dependence of the partition coefficient. The volume change, estimated from the thermodynamic argument, was 3.5 +/- 1.1 cm3.mol-1 at 298.15 K, which was in reasonable agreement with the partial molal volume change determined directly from the solution density measurements, 3.1 +/- 0.2 cm3.mol-1. Benzyl alcohol apparently solvates into the micelles close to surface without losing contact with the aqueous phase.

Interfacial adsorption of an inhalation anesthetic onto ionic surfactant micelles and its desorption by high pressure.

The effects of pressure and temperature on the critical micelle concentration (CMC) of sodium dodecylsulfate (SDS) wer measured in the presence of various concentrations of an inhalation anesthetic, methoxyflurane. The change in the partial molal volume of SDS on micellization delta Vm, increased with the increase in the concentration of methoxyflurane. The CMC-decreasing power, which is defined as the slope of the linear plot between In(CMC) vs. mole fraction of anesthetic, was determined as a function of pressure and temperature. Since the CMC-decreasing power is correlated to the micelle/water partition coefficient of anesthetic, the volume change of the transfer (delta Vop) of methoxyflurane from water to the micelle can be determined from the pressure dependence of the CMC-decreasing power. The value of delta Vop amounts 6.5 +/- 1.8 cm3.mol-1, which is in reasonable agreement with the volume change determined directly from the density data, 5.5+/-0.6 cm3.mol-1. Under the convention of thermodynamics, this indicates that the application of pressure squeezes out anesthetic molecules from the micelle. The transfer enthalpy of anesthetic from water to the micelle is slightly endothermic. The partial molal volume of methoxyflurane in the micelle (112.0 cm3.mol-1) is smaller than that in decane (120.5 cm3.mol-1) and is larger than that in water (108.0 cm3. mol-1. This indicates that the anesthetic molecules are incorporated into the micellar surfaces region, i.e., the palisade layer of the micelle in contact with water molecules, rather than into the micelle core.

Partition equilibrium of inhalation anesthetics and alcohols between water and membranes of phospholipids with varying acyl chain-lengths.

From the depression of the phase-transition temperature of phospholipid membranes, the partition coefficients of inhalation anesthetics (methoxyflurane, halothane, enflurane, chloroform and diethyl ether) and alcohols (benzyl alcohol and homologous n-alcohols up to C = 7) between phospholipid vesicle membranes and water were determined. The phospholipids used were dimyristoyl-, dipalmitoyl- and distearoylphosphatidylcholines. It was found that the difference in the acyl chain length of the three phospholipids did not affect the partition coefficients of the inhalation anesthetics and benzyl alcohol. The actions of these drugs are apparently directed mainly to the interfacial region. In contrast, n-alcohols tend to bind more tightly to the phospholipid vesicles with longer acyl chains. The absolute values of the transfer free energies of n-alcohols increased with the increase of the length of the alkyl chain of the alcohols. The increment was 3.43 kJ per each carbon atom. The numerical values of the partition coefficients are not identical when different expressions for solute concentrations (mole fraction, molality and molarity) are employed. The conversion factors among these values were estimated from the molecular weights and the partial molal volumes of the phospholipids in aqueous solution determined by oscillation densimetry.

Molecular origin of biphasic response of main phase-transition temperature of phospholipid membranes to long-chain alcohols.

A statistical mechanical theory is proposed which explains the molecular mechanism of the nonlinear response of the phase-transition temperature of phospholipid vesicle membranes to added 1-alkanols. By assuming that the free energy of transfer of 1-alkanols from the aqueous phase to the membrane and the interaction energy between 1-alkanol molecules are linear functions of alkanol alkyl chain-length, the nonlinear behavior is explained in the Bragg-Williams approximation. For dipalmitoylphosphatidylcholine vesicle membranes, the theory reveals a larger free energy of transfer of 1-alkanols from the aqueous phase to the solid-gel membrane than to the liquid-crystalline membrane when the number of carbon atoms of 1-alkanol exceeds 12. When the intermolecular interaction force between 1-alkanol molecules residing in the gel phase is stronger than the interaction force between those residing in the liquid-crystalline phase, the ligand effect is to tighten the lipid matrix structure, causing the transition temperature to rise. The interaction force is a quadratic function of 1-alkanol concentration; hence, the response of the transition temperature to the 1-alkanol concentration is nonlinear. At low concentrations of the long-chain 1-alkanols that predominantly elevate the transition temperature, this intermolecular interaction force is negligible. In this case, the entropic effect of the incorporated ligand molecules, which loosens the lipid matrix, predominates, and the transition temperature decreases. The biphasic action of long-chain 1-alkanols originates from the balance of these two opposing effects: entropy and intermolecular interaction.

Stopped-flow study of anesthetic effect on water-transport kinetics through phospholipid membranes. Interfacial versus lipid core ligands.

We have compared ligand effects between polar and apolar anesthetic molecules upon water transport across phospholipid membranes by kinetic analysis of the osmotic swelling rate, using a stopped-flow technique. Chloroform and 1-hexanol were used as interfacial ligands, and carbon tetrachloride and n-hexane were used as their counterparts, representing lipid core action. Because anesthetics transform the solid-gel membrane into a liquid-crystalline state, and because phospholipid membranes display an anomaly in permeability at the phase transition, dimyristoylphosphatidylcholine vesicles were studied at temperatures above the main phase transition to avoid this anomaly. All these molecules increased the osmotic swelling rate. However, a significant difference was observed in the activation energy, delta Ep, between polar and apolar molecules; delta Ep was almost unaltered by the addition of polar molecules (chloroform and 1-hexanol), whereas it was decreased by apolar molecules (carbon tetrachloride and n-hexane). The obtained results were analyzed in terms of the dissolution-diffusion mechanism for water permeation across the lipid membrane. It is suggested that polar molecules affect water permeability by altering the partition of water between the membrane interior and water phase, and apolar molecules affect it by altering both the partition and the diffusion of water within the membrane interior.

Alcohol effects on rapid kinetics of water transport through lipid membranes and location of the main barrier.

The effect of 1-alkanols (from 1-butanol up to 1-dodecanol) on the water permeability of dimyristoylphosphatidylcholine vesicle membranes was studied by measuring the osmotic swelling rate as functions of 1-alkanol concentrations and temperatures above the gel-to-liquid-crystalline phase transition. For 1-butanol and 1-hexanol, the activation energy for water permeation was invariant with the addition of alkanols, whereas for 1-octanol, 1-decanol and 1-dodecanol, the activation energy decreased depending on the alkanol concentration, and the extent of the decrease was larger for alkanol with a longer hydrocarbon chain. These results suggests that hydrocarbon moiety beyond seven or eight carbon atoms from the head group in phospholipid molecules constitutes the main barrier for water permeation through the dimyristoylphosphatidylcholine vesicle membrane. The relative volume change of the vesicle due to osmotic swelling increased with the addition of 1-alkanols. Presumably, the membrane structural strength is weakened by the presence of 1-alkanols in the membrane. Contrary to the dependence of the swelling rate upon the alkanol carbon-chain length, no significant difference in the effect on the relative volume changes was seen among the 1-alkanols. This result suggests that weakening of the membrane structure is caused by perturbation of the membrane/water interface induced by incorporation of 1-alkanols into the membrane.

High pressure antagonism of alcohol effects on the main phase-transition temperature of phospholipid membranes: biphasic response.

The combined effects of high pressure (up to 300 bar) and a homologous series of 1-alkanols (ethanol C2 to 1-tridecanol C13) were studied on the main phase-transition temperature of dipalmitoylphosphatidylcholine (DPPC) vesicle membranes. It is known that short-chain alkanols depress and long-chain alkanols elevate the main transition temperature. The crossover from depression to elevation occurs at the carbon-chain length about C10-C12 in DPPC vesicle membranes coinciding with the cutoff chain-length where anesthetic potency suddenly disappears. Alkanols shorter than C8 linearly decreased the transition temperature and high pressure antagonized the temperature depression. Alkanols longer than C10 showed biphasic dose-response curves. High pressure enhanced the biphasic response. In addition, alkanols longer than the cutoff length depressed the transition temperature under high pressure at the low concentration range. These non-anesthetic alkanols may manifest anesthetic potency under high pressure. At higher concentrations, the temperature elevatory effect was accentuated by pressure. This biphasic effect of long-chain alkanols is not related to the 'interdigitation' associated with short-chain alkanols. The increment of the transition temperature by pressure was 0.0242 K bar-1 in the absence of alkanols. The volume change of the transition was estimated to be 27.7 cm3 mol-1. This value stayed constant to the limit of the present study of 300 bar.

Depression of phase-transition temperature by anesthetics: nonzero solid membrane binding.

The anesthetic-induced depression of the main phase-transition temperature of phospholipid membranes is often analyzed according to the van't Hoff model on the freezing point depression. In this procedure, zero interaction between anesthetics and solid-gel membranes is assumed. Nevertheless, anesthetics bind to solid-gel membranes to a significant degree. It is necessary to analyze the difference in the anesthetic binding between the liquid-crystal and solid-gel membranes to probe the anesthetic action on the lipid membranes. This article describes a theory to estimate the anesthetic binding to each state at the phase-transition temperature. The equations derived here reveal the relation between the partition coefficients of anesthetics and the anesthetic effects on the transition characters: the change in the transition temperature, and the broadening of transition. The theory revealed that the width of transition temperature is determined primarily by the membrane/buffer partition coefficients of anesthetics. Our previous data on the local anesthetic action on the transition temperature of the dipalmitoylphosphatidylcholine vesicle membrane (Ueda, I., Tashiro, C. and Arakawa, K. (1977) Anesthesiology 46, 327-332) are analyzed by this method. The numerical values for the partition of local anesthetics into the liquid-crystal and solid-gel dipalmitoyl-phosphatidylcholine vesicle membranes at the phase-transition temperature are: procaine 8.0 x 10(3) and 4.7 x 10(3), lidocaine, 3.7 x 10(3) and 2.3 x 10(3), bupivacaine 4.1 x 10(4), and 2.6 x 10(4), and tetracaine 7.3 x 10(4) and 4.7 x 10(4), respectively.

Alcohol interaction with high entropy states of macromolecules: critical temperature hypothesis for anesthesia cutoff.

Nerve excitation generates heat and decreases the entropy (review by Ritchie and Keynes (1985) Q. Rev. Biophys. 18, 451-476). The data suggest the existence of at least two thermodynamically identifiable states: resting and excited, with a thermotropic transition between the two. We envision that nerve excitation is a transition between the two states of the excitation machinery consisting of proteins and lipids, rather than the sodium channel protein alone. Presumably, both proteins and lipids change their conformation at excitation. We proposed (Kaminoh et al. (1991) Ann. N.Y. Acad. Sci. 625, 315-317) that anesthesia occurs when compounds have a higher affinity to the resting state than to the excited state of excitable membranes, and that there is a critical temperature above which the affinity to the excited state becomes greater than to the resting state. When the temperature exceeds this critical level, compounds lose their anesthetic potency. We used thermotropic phase-transition of macromolecules as a model for the excitation process. Anesthetic alcohols decreased the main transition temperature of dipalmitoylphosphatidylcholine (DPPC) membranes and also the temperature of the alpha-helix to beta-sheet transition of poly(L-lysine). The affinity of alcohols to the high- and low-temperature states of the DPPC membranes were separately estimated. The difference in the affinity of n-alcohols to the liquid (high-temperature) and solid (low-temperature) states correlated with their anesthetic potency. It is not the total number of bound anesthetic molecules that determines the anesthesia, rather, the difference in the affinity between the higher and lower entropy states determines the effects. The critical temperatures of the long-chain alcohols were found to be lower than those of the short-chain alcohols. Cutoff occurs when the critical temperature of long-chain alcohols is below the physiological temperature, such that the anesthetic potency is not manifested in the experimental temperature range.

Anesthetic-protein interaction. Random versus helix polylysine monolayers and interaction with 1-alkanols.

Penetration of 1-alkanols into monolayers of hydrophobic polypeptides, poly(epsilon-benzyloxycarbonyl-L-lysine) and poly(epsilon-benzyloxycarbonyl-DL-lysine), was compared with their adsorption on the air/water interface in the absence of monolayers. The polypeptide prepared from L-lysine is generally considered to be in the alpha-helical form whereas DL-copolymer polypeptide contains random-coiled portions due to the structural incompatibility between the two isomers. The free energy of adsorption of 1-alkanols on the air/water interface at dilute concentrations was -0.68 kcal X mol-1 per methylene group and 0.15 kcal X mol-1 for the hydroxyl group at 25 degrees C. In the close-packed state, the surface area occupied by each molecule of 1-alkanols of varying carbon chain-lengths showed nearly a constant value of about 27.2 A2, indicating perpendicular orientation of the alkanol molecules at the interface. About 75% of the water surface was covered by 1-butanol in this close-packed state. The mode of adsorption of 1-alkanols on the vacant air/water interface followed the Gibbs surface excess while the mode on the polypeptide membranes followed the Langmuir adsorption isotherm, indicating that the latter is characterized by the presence of a finite number of binding sites. The free energies of adsorption of 1-alkanols on the L-polymer monolayers were more negative than those on the vacant air/water interface and less negative than those on the DL-copolymer monolayers. Thus, the affinity of 1-alkanols to the interface was in the order of vacant air/water interface less than L-polymer less than DL-copolymer. The difference between the air/water interface and L-polymer was about 0.54 kcal X mol-1 and that between L-polymer and DL-copolymer was 0.17 kcal X mol-1 at 25 degrees C: the adsorption of 1-alkanols to the DL-copolymer was favored compared to the L-polymer. The polar moieties of the backbone of the DL-copolymer may be exposed to the aqueous phase at the disordered portion. Dipole interaction between this portion and 1-alkanol molecules may account for the enhanced adsorption of the alkanols to the DL-copolymer.

Pressure-anesthetic antagonism on the phase separation of non-ionic surfactant micelles.

An aqueous solution of non-ionic surfactants becomes suddenly turbid when heated to a critical temperature, known as the cloud point, and concomitantly expands the volume. The volume expansion is caused by release of structured water molecules from the hydrophilic polyoxyethyelene moieties. Inhalation anesthetics decreased the cloud-point temperature of hexaoxyethylene dodecyl ether micelles. The concentrations of methoxyflurane, halothane and enflurane causing a 1 degree C depression of the cloud-point temperature were 0.51, 0.71 and 0.78 mmolal, respectively. Hydrostatic pressure increased the cloud-point temperature in the absence and presence of the anesthetics. The change of the apparent molal volume at the cloud point was estimated to be 2.2 cm3/mol in the absence of anesthetics. This value decreased in the presence of the anesthetics, dose dependently. The results indicate that the anesthetics favor dehydration of the hydrophilic surface of the non-ionic surfactant micelles.

Specific and non-specific binding of long-chain fatty acids to firefly luciferase: cutoff at octanoate.

Firefly luciferase emits a burst of light when the substrates luciferin and ATP are mixed in the presence of oxygen. We (I. Ueda, A. Suzuki, Biophys. J. 75 (1998) 1052-1057) reported that long-chain fatty acids are specific inhibitors of firefly luciferase in competition with luciferin in microM ranges. They increased the thermal transition temperature. In contrast, 1-alkanols of the same carbon chain length inhibited the enzyme non-competitively in mM ranges and decreased the transition temperature. The present study showed that the action of fatty acids switched from specific to non-specific when the carbon chain length was reduced below C8 (octanoate). The fatty acids longer than C10 inhibited the enzyme in microM ranges whereas those shorter than C8 required mM ranges to inhibit it. The longer fatty acids increased whereas shorter fatty acids decreased the transition temperature. The Hill coefficients of longer chain bindings were less than one whereas those of shorter chain were more than one. The shorter fatty acids interacted with the enzyme cooperatively at multiple sites. Binding of the longer fatty acids is limited. Fatty acids longer than C10 are high-affinity specific binders and followed Koshland's induced-fit model. Those shorter than C8 are low-affinity non-specific denaturants and followed Eyring's rate process model. These results contradict the general consensus that the size of the receptor cavity discriminates specific binders.

Saturable and unsaturable binding of a volatile anesthetic enflurane with model lipid vesicle membranes.

Presence of specific receptors for volatile anesthetics has recently been proposed (Evers, A.S. et al. (1987) Nature 328, 157-160) by a finding that halothane uptake by the rat brain was characterized, in part, by saturable binding. We report here that volatile anesthetics bind model lipid membranes also with saturable and unsaturable kinetics. Binding of enflurane to dipalmitoylphosphatidylcholine vesicle membranes was measured by gas chromatography. At low anesthetic concentrations, comparable to the clinical level, the interaction was saturable. After reaching a temporary saturation, a sudden increase in the anesthetic binding to the membrane occurred, when the anesthetic concentration in the aqueous phase exceeded 2.7 mM, or 6.3 x 10(-2) atm partial pressure in the gas phase in equilibrium with the aqueous phase. The secondary binding was linear to the aqueous anesthetic concentrations and was unsaturable to the limit of this study. We also found that enflurane self-aggregated in water above 4 mM. When the aqueous concentration exceeded 6 mM, the aggregation number was about 8. We conclude that the saturable binding indicates adsorption onto the vesicle surface, and the unsaturable binding indicates multilayer stacking of the enflurane molecules, where the initially adsorbed molecules provide the binding sites to the succeeding molecules according to the multilayer condensation kinetics. The tendency of enflurane to self-aggregate in water promotes the multilayer stacking at the surface of the membrane.

Atypical Langmuir adsorption of inhalation anesthetics on phospholipid monolayer at various compressional states: difference between alkane-type and ether-type anesthetics.

Adsorption of chloroform, halothane, enflurane and diethyl ether on the air/water interface was compared with adsorption on the dipalmitoylphosphatidylcholine monolayer, spread on the air/water interface, at four compressional states; 88.5, 77.0, 66.5 and 50.5 A2 surface area per phosphatidylcholine molecule. Anesthetics were administered from the gas phase. The affinities of these agents to the phosphatidylcholine monolayer varied according to the state of the monolayer. Chloroform and halothane showed a stronger affinity to the highly compressed phosphatidylcholine monolayer (50.5 A2) than to the expanded monolayer (88.5 A2) or to the air/water interface without the monolayer. Diethyl ether behaved in reverse; a stronger affinity to the expanded monolayer was exhibited than to the compressed monolayer. Enflurane showed the highest affinity to the intermediately compressed monolayer (77.0 A2). The adsorption isotherm of anesthetics to the monolayer was characterized by atypical Langmuir-type, in which available number of binding sites changed when anesthetics were adsorbed. The mode of adsorption onto the monolayer was dissimilar to adsorption onto air/water interface, where adsorption followed the Gibbs surface excess. A theory is presented to explain the above differences. The adsorbed anesthetic molecules do not stick to phosphatidylcholine molecules but penetrate into the monolayer lattice and occupy the phosphatidylcholine sites at the interface. Quantitative agreement between the theory and the experimental data was excellent. For the monolayer at 50.5 A2 compression, the changes in the transfer free energy accompanying the anesthetic adsorption from the gas phase to the monolayer were in the order of chloroform greater than halothane greater than enflurane greater than diethyl ether, in agreement with the clinical potencies.

Stopped-flow rapid kinetics of anesthetic-induced phase transition in phospholipid vesicle membranes: nonlocalized fluctuation.

Kinetics of the gel to liquid-crystalline phase transition of dipalmitoylphosphatidylcholine vesicle membrane was studied by the stopped-flow technique with turbidity detection. The observed change in turbidity was well characterized by a single-exponential decay curve with relaxation time in the millisecond range, although the existence of a faster process than the dead-time of the stopped-flow apparatus was inferred from the amplitude analysis. Relaxation times were determined as functions of 1-hexanol concentration and temperature just below phase transition. From the analysis based on the theories of nonequilibrium relaxation, it is concluded that the phase transition induced by 1-hexanol is governed by a nonlocalized fluctuation mechanism. The anesthetic-induced nonequilibrium state is unstable rather than metastable.

A statistical mechanical analysis of the effect of long-chain alcohols and high pressure upon the phase transition temperature of lipid bilayer membranes.

Long-chain n-alcohols decrease the main phase-transition temperature of lipid vesicle membranes at low concentrations but increase it at high concentrations. The nonlinear phenomenon is unrelated to the interdigitation and is analyzed by assuming that alcohols form solid solutions with solid as well as liquid phases. The biphasic response originates from the balance of the free energy difference of alcohols in the liquid and solid membranes (delta gA) and the alcohol-lipid interaction free energy difference (delta u) between the two phases. When delta gA less than 0 and delta u greater than 0, or delta gA less than delta u less than 0, the transition temperature decreases monotonously according to the increase in the alcohol concentration. When delta gA greater than 0 and delta u less than 0, or delta gA greater than delta u greater than 0, it increases monotonously. Biphasic response occurs with a minimum temperature when delta u greater than delta gA greater than 0, and with a maximum temperature when delta u less than delta gA less than 0. When the alcohol carbon-chain length becomes closer to the lipid carbon-chain length, delta u is equalized by delta gA, and the temperature minimum of the main transition is shifted to extremely low alcohol concentrations. Hence, long-chain alcohols predominantly elevate the main transition temperature and lose their anesthetic potency. High pressure decreased both delta gA and delta u. Presumably, high pressure improves the packing efficiency of liquid membranes and decreases the difference between the solid and liquid membrane properties.

Alcohols dehydrate lipid membranes: an infrared study on hydrogen bonding.

The effects of alcohols (methanol, ethanol, and n-butanol) on the hydrogen bonding of dipalmitoylphosphatidylcholine (DPPC) were studied by Fourier-transform infrared spectroscopy (FTIR) in water-in-oil (carbon tetrachloride) reversed micelles. The bound O-H stretching mode of water, bonded to DPPC, appeared as a broad band at around 3400 cm-1. The O-H bending mode of this complex appeared as a weak broad band at 1644 cm-1. No free O-H signal was observed. When alcohols were added, a part of DPPC-bound water was replaced by the alcohols. The released 'free' water appeared at 3680 cm-1. This free O-H stretching band represents water-alcohol complex. A new broad band of O-H stretching appeared at 3235 cm-1, which represents the alcohol molecules bound to the phosphate moiety of DPPC. When the alcohol concentration was increased, the intensities of the free O-H stretching and bending bands increased. The P = O- antisymmetric stretching band at 1238 cm-1 became broader and shifted to lower frequencies. This means that alcohols interacted with the phosphate moiety and replaced the bound water. In the deconvoluted spectra of the C = O stretching mode, the ratio between the free sn-2 and the hydrogen-bonded sn-2 bands increased; a part of the bound water at the sn-2 carbon in the glycerol skeleton is also released and the free sn-2 signal increased. From the change in the intensity of the P = O- stretching band, the partition coefficients of alcohols between the phosphate region of DPPC and water were estimated: methanol 7.8, ethanol 16.7 at 22.0 degrees C in mole fraction bases. In molality, these values translates into methanol 0.21 and ethanol 0.45. These results indicate that short-chain alcohols interact with lipid membranes at the phosphate moiety at the hydrophilic head, weaken the membrane-water interaction, and destabilize membranes.

Lateral conductance parallel to membrane surfaces: effects of anesthetics and electrolytes at pre-transition.

The effects of dilute salts and anesthetics were studied on the impedance dispersion in the dipalmitoylphosphatidylcholine (DPPC) liposomes. Below the pre-transition temperature, the apparent activation energy for conductance in DPPC-H2O without salts was equivalent to pure water, 18.2 kJ mol-1. This suggests that the mobile ions (H3O+ and OH-) interact negligibly with the lipid surface below the pre-transition temperature. At pre-transition temperature, the apparent activation energy of the conductance decreased by the increase in the DPPC concentrations. The effects of various salts (LiCl, NaCl, KCl, KBr, and KI) on the apparent activation energy of the conductance were studied. Changes in anions, but not in cations, affected the activation energy. The order of the effect was Cl- less than Br- less than I-. Cations appear to be highly immobilized by hydrogen bonding to the phosphate moiety of DPPC. The smaller the ionic radius, the more ions are fixed on the surface at the expense of the free-moving species. The apparent activation energy of the transfer of ions at the vesicle surface was estimated from the temperature-dependence of the dielectric constant, and was 61.0 kJ mol-1 in the absence of electrolytes. In the presence of electrolytes, the order of the activation energy was F- greater than Cl- greater than Br- greater than I-. When the ionic radius is smaller, these anions interact with the hydration layer at the vesicle surface and the ionic transfer may become sluggish. In the absence of electrolytes, the apparent activation energy of the dielectric constant decreased by the increase in halothane concentrations. In the presence of electrolytes, however, the addition of halothane increased the apparent activation energy. We propose that the adsorption of halothane on the vesicle surface produces two effects: (1) destruction of the hydration shell, and (2) increase in the binding of electrolytes to the vesicle surface. In the absence of electrolytes, the first effect predominates and the apparent activation energy is decreased. In the presence of electrolytes, the latter effect predominates and the apparent activation energy is increased.