Volumetric study on the protein-anesthetic binding.

Thermodynamic equations describing the volume behavior of protein-ligand mixtures in water were derived. In order to estimate the volume and binding parameters, the equations were combined with a Langmuir-type binding isotherm. Densities of aqueous solutions of mixtures of bovine serum albumin (BSA) and octanol (C8OH) were measured as a function of total BSA molality, m(M)(T), at constant total C8OH molalities, m(X)(T). The data were analyzed by the equations. The partial molar volumes at infinite dilution of BSA and C8OH, V(M)(T,0) and V(X)(T,0), respectively, were estimated. It was seen that V(M)(T,0) decreases by the addition of C8OH to the solution and that V(X)(T,0) decreases gradually with increasing m(M)(T) and approaches asymptotically to a certain value at high m(M)(T). From the concentration dependence of V(M)(T,0) and V(X)(T,0), the values of the association constant K=392 kg mol(-1), the maximum binding number b(max)=1.9, and the volume change DeltaV=-109 cm(3) mol(-1) were obtained for BSA-C8OH interaction in water. The negative value of DeltaV indicates that the hydrophobic interaction reduces the protein volume and elevation of pressure promotes BSA-C8OH binding. These results is inconsistent with the pressure reversal of anesthesia.

Does pressure antagonize anesthesia? Opposite effects on specific and nonspecific inhibitors of firefly luciferase.

Ueda and Suzuki (1998. Biochim. Biophys. Acta. 1380:313-319; 1998. Biophys. J. 75:1052-1057) reported that myristic acid inhibited firefly luciferase in microM range in competition with luciferin, whereas anesthetics inhibited it in millimeter ranges noncompetitively with luciferin. Myristate increased, whereas anesthetics decreased, the thermal denaturation temperature. The present study showed that high pressure increased the steady-state light intensity of the halothane-doped firefly luciferase but decreased that of the myristate-doped firefly luciferase. The steady-state light intensity showed a maximum at 19.1 degrees C. At 19.1 degrees C, high pressure did not affect the light intensity in the absence of the inhibitors. In the presence of 0.5 mM halothane, however, 25 MPa pressure (maximum effect) increased the light intensity to 106.0% of the control without the inhibitor. In the presence of 2.5 microM myristate, 40 MPa pressure decreased the light intensity to 90.9% of the control. When the temperature was 25 degrees C in the absence of inhibitors, 40 MPa pressure increased the light intensity 119.2% of the ambient value. At 0.5 mM halothane, 40 MPa pressure further increased the light intensity to 106.1% above the control 40 MPa value. At 2.5 microM myristate, 40 MPa pressure decreased the light intensity to 90.1% of the control 40 MPa value. From the pressure dependence of the light intensity, the volume change DeltaV of the enzyme was estimated at 25 degrees C: 0.5 mM halothane increased DeltaV = +3.93 cm3 mol-1, whereas 2.5 microM myristate decreased DeltaV = -7.66 cm3 mol-1. Present results show that there are distinct differences between the specific and nonspecific ligands in their response to high pressure. Myristate, which competes with luciferin, decreased the protein volume and stabilized the conformation against thermal perturbation. Halothane, which does not compete with the substrate, increased the protein volume and destabilized the conformation.

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.

The effects of temperature and pressure on the thermodynamic activity of anesthetics.

Anesthetic potency is often expressed by volume percent or partial pressure in the gas phase, concentrations in the aqueous phase, etc. However. these values do not represent the anesthetic activity at the action sites. Because the activity at the action sites is difficult to obtain. Ferguson (Ferguson, J., 1939. Proc. R. Soc. Lond. B 127 387-404) defined the thermodynamic activity, which is the ratio between the anesthetizing partial pressure and the vapor pressure of the pure anesthetic at the same temperature. This paper discusses the effects of temperature and pressure on the thermodynamic activity of anesthetics. It also discusses the limitations of the Meyer-Overton rule.

Do anesthetics act by competitive binding to specific receptors? Phase transition of firefly luciferase.

1. Firefly luciferase (FFL) emits a flash of light when mixed with ATP and luciferin in the presence of molecular oxygen. 2. Halothane inhibited FFL at 0.22 mM, while myristic acid inhibited it at 0.68 microM. Under steady-state conditions, myristic acid competed with luciferin. The Lineweaver-Burk plots of anesthetics were nonlinear. 3. The Hill numbers for anesthetics were above 1, while that of myristic acid was below 1 (0.89). Anesthetics interact with FFL cooperatively at multiple sites. The binding site of myristic acid is limited. 4. FFL undergoes a phase transition at 37-40 degrees C. Anesthetics decreased, while myristic acid increased the transition temperature. 5. The contrasting effects of anesthetics and myristic acid are discussed according to Koshland's transition-state model for myristic acid, which involves specific receptors, and Eyring's unfolded-state model for anesthetics, which involves nonspecific conformational change. Fatty acids are receptor-binders and structure-maker of FFL. Anesthetics are non-specific binders and structure-breaker of FFL.

Temperature-dependent effects of high pressure on the bioluminescence of firefly luciferase.

This study measured the effect of high pressure on the enzyme kinetics of firefly luciferase. When firefly luciferase is mixed with luciferin and ATP, a transient flash of light is produced, followed by a weak light, lasting hours. The first stage reaction produces an enzyme-luciferin-AMP complex and pyrophosphate. Addition of pyrophosphate to the reaction mixture decelerated the reaction rate, and the initial flash was prolonged to a plateau, showing a quasi-equilibrium state. The effects of temperature and pressure were analyzed at the plateau. The temperature scan showed that the maximum light intensity was observed at about 22.5 degrees C. When pressurized below the temperature optimum, pressure decreased the light intensity, while increasing it above the temperature optimum. According to the theory of absolute reaction rate, the following values were obtained for the bioluminescent reaction: delta V++ = 823.7 - 2.8 T cm3/mol and delta V = -280.47 + 0.94T cm3/mol, where T is the absolute temperature, delta V++ and delta V are, respectively, activation volume and the volume change due to thermal unfolding. The optimal temperature for the maximum light output occurs because the reaction rate increases with the temperature elevation at low temperature range, but the thermal unfolding of the enzyme decelerates the reaction velocity when the temperature exceeds a critical value. The intensity of luminescence is modified by the influence of pressure on both delta V++ and delta V. So long as the volume of the activated complex (V++) exceeds the average volume of the nonactivated complex (VN), pressure will slow down the reaction. At the point where the volumes become equal, there is no change in the rate under pressure. When the volume of the activated complex is less than that of the reactants, pressure will speed up the rate. This study showed that firefly luciferase is not exceptional to other enzymes in responding to high pressure.

Malignant hyperthermia and calcium-induced heat production.

The abnormal increase in intracellular Ca++ in malignant hyperthermia (MH) is well documented, but the link between the increased Ca++ concentration and high temperature remains speculative. We investigated the possibility that the Ca(++)-induced change in the state of cell membranes may contribute to the temperature elevation. Calcium ion transforms phospholipid membranes from the fluid to solid state. This is analogous to the freezing of water, and liberates latent heat. Differential titration calorimetry (DTC) measures heat production or absorption during ligand binding to macromolecules. When CaCl2 solution was added to anionic dimyristoylphosphatidic acid (DMPA) and dimyristoylphosphatidylglycerol (DMPG) vesicle membranes in incremental doses, DTC showed that the heat production suddenly increased when the Ca++ concentration exceeded about 120 microM. At this Ca++ concentration range, these lipid membranes underwent phase transition. The latent heat of transition was measured by differential scanning calorimetry (DSC). The values were 7.1 +/- 0.7 (SD, n = 4) kcal.mol-1 of DMPA and 6.8 +/- 0.7 (SD, n = 4) kcal.mol-1 of DMPG. The study shows that Ca++ produces heat when bound to lipid membranes. We are not proposing, however, that this is the sole source of heat. We contend that the lipid phase transition is one of the heat sources and it may trigger a hypermetabolic state by elevating the temperature of cell membranes. Because Ca++ is implicated as the second messenger in signal transduction, multiple systems may be involved. More studies are needed to clarify how Ca++ increases body temperature.

Structure-selective anesthetic action of steroids: anesthetic potency and effects on lipid and protein.

Alphaxalone was a clinically used steroid anesthetic. Its analog delta 16-alphaxalone is nonanesthetic. The only difference between the two is the presence of a double bond at the hydrophobic end of the delta 16-alphaxalone molecule. This study determined the anesthetic potency of alphaxalone and delta 16-alphaxalone in goldfish and compared it with their effects on dipalmitoylphosphatidylcholine (DPPC) membranes and an alpha-helix polypeptide, poly(L-lysine). The goldfish EC50 values were: alphaxalone 5 mumol/L and delta 16-alphaxalone 80 mumol/L. Because these steroids are insoluble to water, the bulk of the steroid in water is absorbed by the fish. Larger containers hold more steroids than smaller containers at the same steroid concentrations. Then, EC50 values vary according to the size of the container. By assuming that the total amount of steroids in the container is distributed into the fish, the EC50 values expressed by the concentration in the fish body become 1.9 mmol/L for alphaxalone, and 30.5 mmol/L for delta 16-alphaxalone. A monoamino acid peptide, poly(L-lysine), can be formed into random-coil, alpha-helix, or beta-sheet. Addition of 0.07 mmol/L alphaxalone to the alpha-helix poly(L-lysine) partially transformed it to a beta-sheet structure. An equivalent change was observed with 3.0 mmol/L delta 16-alphaxalone. These values translate into 3.5 mmol/L for alphaxalone and 0.15 mol/L for delta 16-alphaxalone, when expressed by the concentration in the peptide. The change from alpha-helix to beta-sheet is accompanied by dehydration of the surface of poly(L-lysine). The steroids decreased the phase-transition temperature of DPPC membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Local anesthetics destabilize lipid membranes by breaking hydration shell: infrared and calorimetry studies.

Differential scanning calorimetry (DSC) showed that local anesthetics decreased the pretransition (L beta'-->P beta') temperature of dipalmitoylphosphatidylcholine (DPPC) vesicle membranes four- to five-fold more than the main transition (P beta'-->L alpha) temperature. Because pretransition is mainly a change in the hydrophilic head property (tilted-rippled), the stronger effect on the pretransition suggests that the primary action site of local anesthetics is the lipid-water interface. The interfacial effect was analyzed by Fourier-transform infrared spectroscopy (FTIR) in water-in-oil (CCl4) reversed micelles. FTIR showed that the local anesthetics released hydrogen-bonded water molecules from the phosphate (P = O bands) and glycerol (sn-2 C = O) moieties. The N-H stretching band of the local anesthetics was deconvoluted into two bands: hydrogen bonded to the phosphate moiety of the lipid and free (unbound to lipid). The formation constants between lipid P = O and anesthetic N-H were estimated in CCl4 from the spectral changes: 110 M-1 for lidocaine and 250 M-1 for dibucaine. This small difference in the formation constants cannot explain the ten-fold stronger effect on the phase-transition temperature of dibucaine over lidocaine. By comparing the local anesthetic adsorption to the air/water interface in the presence and absence of lipid monolayers, we have previously shown (Lin et al. (1980) Biochim. Biophys. Acta 598, 51-65) that lipid-anesthetics interaction involves three forces: lipophilic effect, hydrophobic effect, and anesthetic-anesthetic interaction. The anesthetic potency depends mainly on the hydrophobic effect (the difference in the standard molar free energies of local anesthetics in water and at the interface) and anesthetic-anesthetic interaction energy. The anesthetic-anesthetic interaction means cooperativity of local anesthetics for the interfacial density: local anesthetics condense at the membrane surface when there are enough anesthetic molecules present at the interface to attract more anesthetics. The present data suggest that anesthetic action is directed to the interface between water and macromolecule, whether it is lipid membranes or proteins.

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.

Biphasic effects of alcohols on the phase transition of poly(L-lysine) between alpha-helix and beta-sheet conformations.

Poly(L-lysine) exists as a random-coil at neutral pH, an alpha-helix at alkaline pH, and a beta-sheet when the alpha-helix poly(L-lysine) is heated. The present Fourier-transform infrared (FTIR) study showed that short-chain alcohols (methanol, ethanol, and 2-propanol) partially transformed alpha-helix poly(L-lysine) to beta-sheet when their concentrations were low. At higher concentrations, however, these alcohols reversed the reaction, and the alcohol-induced beta-sheet was transformed back to alpha-helix structure. The reversal occurred at 1.40 M methanol, 0.96 M ethanol, and 0.55 M 2-propanol. The alcohol effects on the secondary structure were further investigated by circular dichroism (CD) on the thermally induced beta-sheet poly(L-lysine). Methanol, ethanol, and 1-propanol, but not 1-butanol, shifted the negative mean-residue ellipticity at 217 nm of the beta-sheet poly(L-lysine) to the positive side at low concentrations of the alcohols and to the negative side at high concentrations. With 1-butanol, only the positive-side shift was observed. The positive-side shift at low concentrations of alcohols indicates enhancement of the hydrophobic interactions among the side chains of the polypeptide in the beta-sheet conformation. The negative-side shift indicates a partial transformation to alpha-helix. The shift from the positive to negative side occurred at 7.1 M methanol, 4.6 M ethanol, and 3.1 M 1-propanol. The alcohol concentrations for the beta-to-alpha transition were higher in the CD study than in the IR study.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

The alpha-helix to beta-sheet transition in poly(L-lysine): effects of anesthetics and high pressure.

Poly(L-lysine) exists in a random-coil formation at a low pH, alpha-helix at a pH above 10.6, and transforms into beta-sheet when the alpha-helix polylysine is heated. Each conformation is clearly distinguishable in the amide-I band of the infrared spectrum. The thermotropic alpha-to-beta transition was studied by using differential scanning calorimetry. At pH 10.6, the transition temperature was 43.5 degrees C and the transition enthalpy was 170 cal/mol residue. At pH 11.85, the measurements were 36.7 degrees C and 910 cal/mol residue, respectively. Volatile anesthetics (chloroform, halothane, isoflurane and enflurane) partially transformed alpha-helix polylysine into beta-sheet. The transformation was reversed by the application of hydrostatic pressure in the range of 100-350 atm. Apparently, the alpha-to-beta transition was induced by anesthetics through partial dehydration of the peptide side-chains (beta-sheet surface is less hydrated than alpha-helix). High pressure reversed this process by re-hydrating the peptide. Because the membrane spanning domains of channel and receptor proteins are predominantly in the alpha-helix conformation, anesthetics may suppress the activity of excitable cells by transforming them into a less than optimal structure for electrogenic ion transport and neurotransmission. Proteins and lipid membranes maintain their structural integrity by interaction with water. That which attenuates the interaction will destabilize the structure. These data suggest that anesthetics alter macromolecular conformations essentially by a solvent effect, thereby destroying the solvation water shell surrounding macromolecules.

Anesthetic-protein interaction: effects of volatile anesthetics on the secondary structure of poly(L-lysine).

Effects of volatile anesthetics (chloroform, halothane, and enflurane) on the secondary structure of poly(L-lysine) were analyzed by circular dichroism (CD). The relative proportions among alpha-helix, beta-sheet, and random-coil conformations were calculated by the curve-fitting method on the CD data. Volatile anesthetics partially transformed alpha-helix to beta-sheet but not to random-coil under the present experimental condition. When expressed by the anesthetic partial pressures in the gas phase in equilibrium with the solution, the values that partially transformed alpha to beta conformation by 10% were 1.1 x 10(-2), 4.7 x 10(-2), and 7.9 x 10(-2) atm for chloroform, halothane, and enflurane, respectively. The order of potency is in reasonable agreement with the order of the anesthetic potencies of the agents. The alpha-to-beta transition was completely reversible when anesthetics were purged by nitrogen gas. Volatile anesthetics disrupted the hydrogen bonds of alpha-helix backbones and rearranged them to form the beta-sheet conformation. The beta-sheet conformation is stabilized mainly by the hydrophobic interaction among methylene side groups of poly(L-lysine). Volatile anesthetics promoted the transition by enhancing the hydrophobic interaction among side-chains and by rearranging the hydrogen bonds in the peptide backbone.

Interfacial dehydration by anesthetics: an electrocapillary study of surface charge density of adsorbed monolayer.

We have proposed that anesthetics destruct the hydration shell of macromolecules irrespective of lipid membranes or proteins. These macromolecular structures are supported by the hydrogen-bonded matrix of water molecules. A loss of this support destabilizes the membranes and proteins. The disordered structures are suboptimal for the assigned biological functions, and anesthesia may ensue. We postulated that the dehydration is prompted mainly by the decrease in the interactions of the surface charges with the water dipole. To prove or disprove the above hypothesis, this study measured the effect of volatile anesthetics (chloroform, halothane, and enflurane) on the surface charge density in adsorbed monolayers by an electrocapillary method. The oil phase was methylisobutylketone (MIBK) with cetyltrimethylammonium chloride (CTAC). The aqueous phase was 0.1 M NaCl. The anesthetics decreased the surface charge density, and the effect paralleled the clinical anesthetic potency. At concentrations that induce surgical stage anesthesia in 50% of the population, these anesthetics reduced the surface charge density by 5%.

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.