11334
J. Phys. Chem. B 2000, 104, 11334-11341
Partitioning of Small Alcohols into Dimyristoyl Phosphatidylcholine (DMPC) Membranes: Volumetric Properties Peter Westh*,†,‡ and Christa Trandum† Department of Chemistry, Technical UniVersity of Denmark, Building 206, DK-2800 Lyngby, Denmark, and Department of Chemistry and Life Sciences, Roskilde UniVersity, Building 17.2, DK-4000 Roskilde, Denmark ReceiVed: April 24, 2000; In Final Form: August 30, 2000
Volume changes associated with the partitioning of eight different alcohols and acetone into unilamellar vesicles of dimyristoyl phosphatidylcholine in the fluid state was investigated by newly designed, automated titration densitometric equipment. The change in free volume of the partitioning process, expressed by changes in the specific excess volume of the solutions, was positive for all solutes. The magnitude of the total volume change per mole alcohol partitioned, ∆Vm, was calculated by combining the volumetric data with previously published partitioning coefficients. For aliphatic 1-alcohols, ∆Vm increased gradually from 3 cm3/mol for ethanol to ∼29 cm3/mol for 1-hexanol. Values of ∆Vm for the cyclic alcohols c-hexanol and benzyl alcohol were ∼3 times smaller than for aliphatic compounds of a similar molecular weight. The composition dependence of the volumetric excess functions could be determined with sufficient accuracy to allow estimation of the partial specific excess volume of all three components (water, alcohol, and lipid). These functions elucidate the contribution of each component to the measured expansion of the system. The partial volume of vesicles and alcohols increased, whereas the volume of water was diminished as a result of the partitioning process. This result suggests that the volume increase of the membrane-alcohol complex is larger than the measured volume change of the whole system (∆Vm) because the latter is partially compensated by contraction of the solvent. The results support a molecular picture in which adsorption of alcohols promotes a loosening of the molecular packing and an increase in the water content of lipid bilayer membranes.
Introduction The modes of interactions that underlie the partitioning of small molecules into membranes are of central importance for the elucidation of a number of biological and pharmacological mechanisms. One source of such information is the thermodynamic characterization of membrane-solute interactions. This approach has a particular potential of discerning the nature and strengths of forces driving the membrane-solute association, but the combination of thermodynamic and structural information has also proven useful for more general descriptions of solute-membrane relationships. A recent development in this field has emerged from the application of highly sensitive titration calorimetric techniques, which has elucidated the thermochemistry of the process for a range of solutes and lipids.1-10 In the present work, titration densitometry is utilized to extend the thermodynamic characterization of partitioning for some of the same solutes to include volumetric properties. In addition to their general thermodynamic importance, volumetric properties may be of particular interest in discussions of membranes-solute complexes. Hence, the direct relationship between volume and the dimensions and packing arrangement of the membrane components may facilitate the merge between thermodynamic and structural information. Also, on a more specialized level, volumetric properties have been closely linked to the in vivo effect of alcohols and anesthetics.11-13 Part of this interest in volume behavior originates from the observation * Corresponding author (E-mail:
[email protected], Fax: +45 4674 3011, Telephone: +45 4674 2879). † Department of Chemistry. ‡ Department of Chemistry and Life Sciences.
that the action of anesthetics is reversed by high pressure,14,15 and it has even been suggested16 that a molecular understanding of the volume changes on solute partitioning into membranes is the key to the understanding of anesthetic mechanisms. Systematic experimental information on volume changes of membrane partitioning is sparse. In what probably remains to be the most precise and general study, Miller and co-workers17,18 reported that the partial molar volume of benzyl alcohol and two long-chain aliphatic alcohols partitioned into egg lecithin/ cholesterol bilayers was 4-5% larger than in aqueous solution. For halothane, an increase in partial volume of, respectively, 18 and 30% was observed on transfer from water to bilayers in fluid or gel states. Important information on volume properties has also been derived from the pressure dependence of membrane properties (spectroscopic or thermodynamic) in mixed solvents. Thus, based on the dependence on pressure and solute concentration of the main phase transition, Kaneshina et al.19 deduced that partitioning of four inhalation anesthetics gave rise to a volume increase of 15% of their molar volume. Auger et al.20 showed that high hydrostatic pressures lead to the expulsion of a local anesthetic from both model- and biological membranes. These studies have collectively shown that the partitioning of small solutes into membranes is characterized by a concomitant expansion of the system. Other investigations, both experimental and computational, have suggested that foreign molecules in membranes may occupy interstitial volume, which leads to zero or even negative volume changes of the partitioning process.21-24 In the present study, the volume properties of monohydroxy alcohols partitioned into fluid bilayers of dimyristoyl phos-
10.1021/jp001540l CCC: $19.00 © 2000 American Chemical Society Published on Web 10/28/2000
Volume Changes with Alcohol Partitioning Into DMPC Membranes phatidylcholine (DMPC) was investigated. It was found that aliphatic solutes induced an expansion on partitioning that scaled with the size of acyl chain of the solute and that cyclic solutes gave rise to a significantly smaller expansion. More importantly, the measurements allowed us to establish the partial volumes of all three components in membrane-solute-water suspensions. As a result, molecular origins of the observed volumetric properties can be discussed without resorting to (often rather inconsistent) literature values of partitioning coefficients. Experimental Section Dry DMPC (>99% pure) was purchased from Avanti Polar Lipids (Birmingham, AL) as a powder and used without further purification. The alcohols and acetone were of the highest purity (99.5-99.9%) from Merck (Darmstad, Germany) and were also used as delivered. Vesicles were prepared by initially hydrating weighed amounts of DMPC in a phosphate buffer (pH 7.4, 137 mM NaCl, 2 mM KCl) to yield the desired lipid concentration. The dispersions were kept 10 °C above the main transition temperature for 1 h during which time they were repeatedly shaken. Unilamellar vesicles were produced by standard extrusion techniques25 using equipment supplied by Lipex Biomembranes (Vancouver, BC, Canada). Twelve repeated extrusions through two stacked polycarbonate filters (Nucleopore, 0.1-µm pore size) were performed at a hydrostatic pressure of 25 atm at 40 °C. The principle of the experimental work is to measure the density of two-component (water + alcohol) and three component (water + alcohol + DMPC vesicles) systems as a function of the alcohol concentration (the buffer will be considered as one (pseudo) component and referred to as “water” throughout). From these density data the volume changes associated with alcohol partitioning can be evaluated (vide infra). Density measurements were conducted in a DMA602 densitometer cell (Anton Paar, Graz, Austria) build into a setup allowing automated, stepwise changes of sample composition and/or temperature. Technical details of this equipment, which is a “second generation” version of that previously described,26 will be provided in a forthcoming note. Here, we only present routines pertinent for the alcohol partitioning studies. Weighed samples of ∼7 mL (buffer or vesicle suspension) were placed in a mixing cell (a 15-mL glass tube that was closed except for 3 inlets/outlets of 1 mm i.d. Teflon tubing) and circulated by a peristaltic pump through the measuring U-tube of the densitometric cell. The cell had previously been calibrated at the experimental temperature against pure water and air. After 20 min of circulation, the peristaltic pump was stopped and the solution was allowed to equilibrate for 10 min. Subsequently, 30 “submeasurements” (simultaneous recordings of the temperature of the thermostat water close to the U-tube and the solution density) were recorded over a period of 8 min. If the slope and standard deviation of this series of submeasurements were within satisfactory limits (defined empirically from previous experiments), the temperature and density averages of the 30 recordings were saved as one data point. If not, the measurements were discarded and the routines of circulation, thermal equilibration, and recording of submeasurements were repeated. The temperature was 26.0 ( 0.004 °C in all experiments. An aliquot (10-60 µL) of alcohol was then dispensed into the mixing cell from a Metrom 665 dosimat (Herisau, Switzerland). Mixing of the added alcohol with the cell content was obtained by a magnetic stirrer bar in the mixing cell. Stirring was run for 30 min while the sample was being circulated
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through the densitometric cell. Following thermal equilibration, the density of the solution at the increased alcohol concentration was determined by applying the same requirements for repeatability of submeasurements. Eight to 15 consecutive additions of alcohol were made in each trial. One of the advantages of this setup is that all quantities except Va* in eqs 1a and b (vide infra) are measured in a single titration trial using a closed system. As a result, the composition dependence of the excess volume functions can be determined without errors arising from, for example, small sample-to-sample variations of lipid concentration. Such problems have previously been claimed to hamper densitometric studies of membrane partitioning.18 Results and Data Analysis The raw data from the titration densitometry illustrates the composition dependence of the specific volume of two component (water + solute) and three component (water + solute + lipid) systems. From these results the partial specific volume of the unilamellar vesicles before addition of alcohol can be approximated as ∆V/∆mlip (≈ dV/d mlip; see eq 7), where ∆V is the volume difference of water and lipid suspension and mlip is the mass of lipid. The partial volume of the lipid was 0.975 ( 0.003 cm3/g (average of 10 experimental trials) and independent of the lipid concentration at the tested values of 50 and 100 mM. This value is in good accordance with previous data for multilamellar DMPC.27,28 The composition dependence of the measured specific volumes reflects both the density of the constituent components and effects of molecular interactions in the mixtures. To specifically illustrate the latter, we used the raw data to determine the specific excess volumes of the two- and threecomponent systems (2VE and 3VE)
V ) 2V - 2waV/a - (1 - 2wa)Vw
(1a)
V ) 3V - 3waV/a - (1 - 3wa)Vsusp
(1b)
2 E 3 E
where 2V and 3V are, respectively, the measured specific volumes of the two- and three-component systems, Va* is the specific volume of the pure alcohol (measured in a separate experiment), and Vw and Vsusp are, respectively, the specific volume of water (two-component system) and the vesicle suspension (threecomponent system) before addition of the first aliquot of solute. The concentration of the solute (alcohol or acetone) in two(2wa) or three- (3wa) component system is expressed in weight fraction units that are defined as
ma ma + mw
(2a)
ma ma + msusp
(2b)
wa )
2
3
wa )
where ma is the mass of solute added (quantified from the dispensed volume and the density of the pure organic liquid) and mw and msusp are the masses of, respectively, water and aqueous DMPC suspension transferred to the mixing cell at the beginning of an experiment (see Methods). The excess volumes defined in eq 1 signify the difference between the specific volume of a mixture and the (sum of) the volumes of its constituent components in their standard state (pure liquids for water and alcohols and a dilute suspension of unilamellar vesicles for the lipid). In other words 2VE or 3VE
11336 J. Phys. Chem. B, Vol. 104, No. 47, 2000
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Figure 2. Total volume change, ∆Vm, of the transfer of one mole of solute from aqueous solution to DMPC membranes in the fluid state at 26 °C. ∆Vm is calculated from the difference between a set of curves in Figure 1 (see eq 6) and previously published partitioning coefficients as described in the text. The abbreviations on the ordinate stands for (left to right) ethanol, 1-propanol, 1-butanol, 1-pentanol, 1-hexanol, c-hexanol, benzyl alcohol, 2-propanol, and acetone. The error bars are estimated on the basis of the experimental repeatability of the densitometric measurements reported in this study. Uncertainties of the literature partitioning coefficients used in the calculations are not included.
Figure 1. Excess volumes of, respectively, two-component systems, 2VE (water + solute), and three-component systems, 3VE (water + solute + lipid), in the form of unilamellar DMPC vesicles, plotted against the solute weight fraction, wA, of the mixtures. The symbols represents values calculated directly from the raw data (eq 1) and the lines are polynomial fits used to establish the difference between 3VE and 2VE at a given value of wA. Circles represents 2VE in all panels. Triangles and squares are 3VE at the lipid concentrations specified below. The six panels illustrate the following solutes. Panel A: Ethanol (open symbols) at the lipid concentrations 50 mM (triangles) and 100 mM (squares). Acetone (closed symbols) at a lipid concentration of 100 mM (triangles). The inset in panel A shows an enlargement of the ethanol data for wA ≈ 0.10; the curves illustrate (bottom to top) 0, 50, and 100 mM DMPC. Panel B: 1-Propanol (open symbols) and 2-propanol (closed symbols) both investigated at a lipid concentration of 100 mM (triangles). Panel C: 1-Butanol at the lipid concentrations 50 mM (triangles) and 100 mM (squares). Panel D: 1-Pentanol at the lipid concentrations 25 mM (triangles) and 50 mM (squares). Panel E: 1-Hexanol at the lipid concentration 50 mM (triangles). Panel F: c-Hexanol (open symbols) and benzyl alcohol (closed symbols) both investigated at a lipid concentration of 50 mM (triangles).
quantify the volume change generated from rearranging a certain quantity of components from standard states to a mixture. This volume change on rearrangement of a given collection of molecules thus reflects changes in packing properties arising from molecular interactions, and in the remainder of the text, changes in 2VE or 3VE will be interpreted as changes in “free volume” of the system. Excess volumes of solutions with and without vesicles (3VE and 2VE) are plotted as a function of the alcohol concentration in Figure 1. The points are the values calculated directly (eq 1), whereas the curves are polynomial fits used to determine the difference at a fixed value of wa. The uncertainty of 3VE and 2VE within a given experimental trial is 2VE for all the investigated solutes and over all investigated composition ranges. Also, in cases where two lipid concentrations have been investigated, 3VE invariably increases (becomes less negative) with increasing lipid concentration. These results collectively show that changes in free volume associated with membrane-solute contacts are positive; that is, membrane-solute interactions act to expand the system. The aim of the following is to quantify this expansion. To do so we first introduce a standard partitioning model and determine the total (integral) volume change of the system, ∆Vm, resulting from the transfer of one mole of solute from buffer to the partitioned state. Subsequently, we utilize the concentration dependence of 2VE and 3VE illustrated in Figure 1 to single out the partial specific excess volume of each of the three components (water, solute, and lipid) in the mixtures. Integral (Total) Volume Change. The molar change in volume, ∆Vm, of the partitioning process is defined by the “reaction”
A(aq) a A(lip), ∆Vm
(3)
where A is the solute dissolved in the bulk (aq) or partitioned in the lipid membrane (lip). Thus, ∆Vm is the total volume change of the system when one mole of A is transferred between these states. A straightforward access to experimental values of ∆Vm would be to directly read off the difference at a fixed value of wa between a set of curves in Figure 1. This difference, normalized with respect to the number of solute molecules partitioned as defined by the partitioning coefficient, would give a measure of ∆Vm. This approach, however, carries a systematic error. The vertical distance between a set of curves in Figure 1 reflects the volume change of a (hypothetical) experiment in which wL grams of water is removed from 1 g of water/alcohol solution and replaced by the same mass of lipid (wL is the weight fraction of lipid in the three-component system). Because water (not water/alcohol mixture) is removed, the increase in alcohol concentration in the remaining solvent will lead to a (small)
Volume Changes with Alcohol Partitioning Into DMPC Membranes volume change, which is irrelevant to the partitioning process. To account for this, the change in excess volume, ∆VE, should be defined as ∆VE ) 3VE[wa] - (1 - wL) 2VE[wa′], where square brackets indicate the variable and wa′ ) wa/(1 - wL). The volume change of eq 3 can then be expressed as
∆Vm[wa] )
3 E V [wa] - (1 - wL) 2VE[w′a] ∆VE ) nlipid nlipid a a
(4)
where the number of moles of solute partitioned, nalipid, as a function of the total number of solute molecules added, natotal, is calculated as
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with high accuracy in most cases. This information allows estimation of the partial specific excess volume of each component in the mixtures. These functions are attractive because they elucidate the contribution of each component toward the total excess volume of the system. The five partial excess functions are: 2VE(W), 2VE(A), 3VE(L), 3VE(A), and 3VE(W), where the parenthesis identify the partial volume of water (W), solute (A), or lipid (L). For the two-component system, the partial specific excess volumes 2VE(W) and 2VE(A) are readily calculated by the traditional “methods of intercepts”.31 Hence
d(2VE) dwA
V (A) ) 2VE + (1 - wA)
2 E
nlipid a
Kprm total ) n 1 + Kprm a
(5)
In eq 5, rm is the lipid-to-water mass ratio in the sample and Kp is the partitioning coefficient in molal units. The latter was taken from Katz and Diamond29 except for the solutes (1-pentanol and 1-hexanol) not reported by these workers. For 1-pentanol and 1-hexanol, values of 12 and 22, respectively, were used based on the data compiled by Janof and Miller11 and the recent work by Rowe and co-workers.3 These values are consistent with the data of Katz and Diamond in the sense that ln(Kp) depends linearly on the number of carbon atoms in the acyl chain of the aliphatic alcohols. A number of other rather deviant Kp values for, for example, hexanol have been published (see discussion in ref 30). For negative curvatures (which are dominant in Figure 1) ∆VE calculated according to eq 4 is larger than the vertical distance between 3VE and 2VE. However, for low lipid concentrations (wL )0.035 in most cases here), the difference is rather small, ranging from ∼25% in the case of ethanol at the highest concentrations (wa = 0.10) to 2 (in molal units) are considered. For solutes with lower Kp values, the difference between 2VE and 3VE is small (see, e.g., EtOH and acetone in Figure 1). As a result, ∆3VE/ ∆wL and thus 3VE(L) cannot be estimated with a satisfactory precision. The results in Figures 3 and 4 follow the same general pattern. First, the partial excess volume of the vesicles increases linearly (or with a slight negative curvature) over the investigated range of alcohol concentrations. Second, the partial excess volume of the solute is much larger in lipid suspension than in binary aqueous solution. Third, the partial excess volume of water is close to 0 in binary mixtures and lower in the vesicle suspensions.
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Figure 4. Excess partial specific volumes of the components in mixtures of the cyclic alcohols benzyl alcohol (BzOH) and c-hexanol (c-HeOH). The curves have the same meaning as in Figure 3 and are labeled in the benzyl alcohol data set.
Figure 3. Excess partial specific volumes of the components in mixtures of the aliphatic alcohols, 1-butanol (BuOH), 1-pentanol (PeOH), and 1-hexanol (HeOH). The partial excess volumes were calculated from the composition dependence of the (integral) excess volumes shown in Figure 1 and plotted as a function of the alcohol weight fraction, wA. The partial excess volume of the vesicles, 3VE(L), the alcohol-in-water, 2VE(A), and the alcohol-in-vesicle suspensions, 3VE(A), are shown on the left-hand ordinate and indicated by respectively dot-and-dash, solid and dotted lines. The excess partial specific volume of water in binary alcohol mixtures, 2VE(W), and vesicle suspensions 3VE(W), are shown on the right-hand ordinate and are indicated by dashed lines. In all cases, 2VE(W) is the upper and 3VE(W) the lower of these functions. Each function is indicated by labels on the butanol data set.
Discussion Integral Volume Change of Partitioning. Figure 2 shows that transfer of one mole of solute from aqueous solution to the fluid state of DMPC membranes expands the system by 3-29 cm3 depending on the chemical structure of the solute. (The main transition temperature, Tm, of DMPC is 24 °C, and all experiments in this work have been conducted at 26 °C. Small amounts of alcohols decrease Tm, whereas higher alcohol concentrations may stabilize the gel phase and thus increase Tm. This dual effect is particularly strong for small alcohols and long acyl chain lengths of the lipids. For the relatively short (C14) acyl chain of DMPC and the concentrations of alcohols used in this study, previous work suggests that the vesicles are
in the fluid state.2,32-34 This suggestion is strongly supported by the absence of inflections in the volume data in Figure 1. Freezing of the acyl chains would have induced a substantial change in 3VE; ∼1-3 µL/g, estimated from ref 27.) The observed changes in volume translate into relative expansions of 5-10% of the molecular volume of the partitioned molecules, except for 1-pentanol and 1-hexanol, for which the relative expansion is about twice as large. If instead of aqueous solution the neat organic liquid is used as the reference state, the relative volume increase of partitioning is zero for the smaller (C2C3) solutes, and ∼5% for the cyclic and 7-12% for the longer aliphatic alcohols. Partition-induced expansions of this magnitude have been reported previously,17-19 although smaller volume effects have been found for some systems (see ref 21 and references therein). Also in agreement with the present observations, the partial molar volume of aliphatic 1-alcohols in suspensions of anionic micelles was reported to be larger than the volume in dilute aqueous solution.35 The volume increase observed in the micellar systems was generally small (≈35%), but the values for the shorter, water-soluble alcohols may (as pointed out by the author) be ambiguous because the water/ micelle distribution was not accounted for in the data treatment. Because ∆Vm is the volume change of the partitioning “reaction” (eq 3), it quantifies the pressure dependence of its equilibrium constant (the partitioning coefficient, Kp). Under the assumptions that ∆Vm is the standard volume change and the system is equally compressible in the partitioned and nonpartitioned state (i.e., d∆Vm/dP ) 0), the change in partitioning coefficient on increase of the pressure from ambient, Po, to P can be expressed as
KP(Po) KP(P)
) exp
[
]
∆Vm(P - Po) RT
(9)
Volume Changes with Alcohol Partitioning Into DMPC Membranes Insertion of a typical value of ∆Vm for the present systems (10 cm3/mol) and a pressure P ) 200 atm in eq 9 shows a reduction of Kp to 92% of its value at ambient pressures. Analogously, the largest values of ∆Vm in Figure 2 will lead to a reduction of Kp to ∼80% of the ambient value following a 200 atm increase in hydrostatic pressure. This result suggests that a pressure increase of hundreds of atmospheres may expel an appreciable fraction of the alcohols molecules from a model membrane. Auger et al.20 directly observed pressure-induced expulsion of a local anesthetic from both biological and model membranes. Hence, infrared spectra of pressurized samples showed that tetracaine was “squeezed out” of DMPC membranes at pressures of ∼2700 atm. A similar conclusion could be reached about alcohols based on the present data because pressures of several thousand atmospheres is predicted (eq 9) to reduce the partitioning coefficients to 5-25% of their ambient values. Partial Specific Excess Volumes. The partial excess volumes presented in Figures 3 and 4 furnish a more direct and detailed representation of the experimental data. Thus, they (i) provide a measure of the contribution of each components toward the measured (total) volume change and (ii) do not depend on literature values of partitioning coefficients. (The error bars in Figure 2 indicate the estimated uncertainty, based on the experimental repeatability, of the densitometric measurements. It is possible however, that the uncertainty of the partitioning coefficients underlying the values of ∆Vm (eq 6) gives rise to a similar or larger uncertainties). The dependence of the partial specific excess volume on the solute concentration in systems containing aliphatic alcohols is illustrated in Figure 3. All three systems (1-butanol, 1-pentanol, and 1-hexanol) follow the same pattern. For 1-pentanol (panel B), for example, at an alcohol concentration of 0.5% (w/w; wPeOH ) 0.005) the partial volume of the lipid molecules,3VE(L), is larger by ∼13 µL/g than the volume of lipid in pure water. This volume increase does not reflect the volume occupied by the pentanol molecules incorporated in the membrane. Rather, it is the increase in the contribution of the lipid molecules toward the total volume of the system. Thus, the positive slope of 3VE(L) shows that addition of pentanol acts to loosen the packing of DMPC molecules. At a total alcohol concentration of 0.5%, this increase in free volume (13 µL/g lipid) exceeds 1% of the lipid volume (975 µL/g). Using the same example (wPeOH ) 0.005), we find that the excess partial volumes of the alcohol in water (2VE(A)) and 50 mM DMPC (3VE(A)) are, respectively, -110 and -44 µL/g. The negative signs show that the volume of pentanol is decreased on transfer from the pure state to either of these solvents. The higher (less negative) value in the vesicle suspension illustrates that its interaction with DMPC acts to increase the volume of pentanol. Because the volume of pure pentanol at 26 °C, Va*, was measured to 1234.3 µL/g, the effective volume of pentanol is ∼5% larger in a 50-mM suspension of unilamellar DMPC vesicles than in water. Finally, the partial excess volume of water at wPeOH ) 0.005 is -0.12 µL/g in binary mixtures and -0.40 µL/g in vesicle suspensions. Thus, in contrast to the alcohol and the lipid, the volume of water decreases as a result of the partitioning process. Because this picture is qualitatively the same for both aliphatic (Figure 3) and cyclic (Figure 4) alcohols, it is concluded that the increase in free volume associated with alcohol-membrane interactions (Figure 1) can be discerned into expanding contributions from lipid and solute, which are partly compensated by contraction of the water. Judging from the numbers in the example just given, the contraction of water may appear to be negligibly
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TABLE 1
alcohol 1-butanol 1-pentanol 1-hexanol benzyl alcohol c-hexanol
concentration contribution to VE in µL/g (weight fraction) no. of VE ) Σ wi VE(i) alcohol lipid comp alcohol lipid water 0.010 0.010 0.0040 0.0040 0.0030 0.0030 0.0050 0.0050 0.0060 0.0060
0 0.067 0 0.034 0 0.034 0 0.034 0 0.034
2 3 2 3 2 3 2 3 2 3
-0.79 -0.52 -0.46 -0.16 -0.35 -0.07 -0.12 -0.01 -0.16 -0.07
0.27 0.34 0.37 0.13 0.10
0.04 -0.07 -0.12 -0.29 -0.08 -0.29 -0.02 -0.06 0.00 -0.07
small. However, to evaluate the contribution of a component toward the volume of the entire system, its partial function should be weighted according to its concentration (cf. eq 8; VE ) Σ wi VE(i)). Thus, the contribution to the excess volume in two- and three-component systems, wiVE(i), where i ) A, L, or W, was calculated and listed in Table 1. For conformity with the previous discussion, the composition of the systems listed in the table was chosen to yield an alcohol concentration in the vesicles of 300 mmol/kg lipid. It should be emphasized that although the alcohol concentrations chosen in Table 1 to exemplify the volume changes are based on the partitioning coefficient, the volumetric results in columns 5-7 are (extensive) thermodynamic data characteristic for the investigated systems and independent of Kp and indeed any model for membrane-alcohol interactions. The data in Table 1 can be rationalized by comparing the differences between two- and three-component systems for each alcohol. If again pentanol is used as an example, we find that the contribution of the alcohol at wPeOH ) 0.004 to the volume of 1 g of mixture is increased by 0.30 µL in 50 mM DMPC (wL ) 0.034). Analogously, the volume of water is decreased by 0.17 µL. For the lipid, the effect of membrane-lipid interactions is reflected directly in column 6. The free volume of the lipid molecules in 1 g of mixture with wL ) 0.034 increases by 0.34 µL on addition of pentanol to wPeOH ) 0.004. Following the same line of arguments, the changes in free volume of each component in 1 g of mixtures have been estimated and illustrated for all five solute systems in the histogram in Figure 5. This presentation emphasizes that interactions of the alcohols with unilamellar vesicles of fluid DMPC generate an increase in the free volume of the lipid and alcohol molecules and a decrease in the free volume of water. Moreover, it appears that the two positive contributions are similar in size whereas the contraction of water is somewhat smaller ranging from 25 to 80% of the two former terms. Figure 5 may be particularly useful in attempts to interpret the present results on a molecular level. For example, the negative volume changes of water oppose previous suggestions that the overall expansion characteristic of solute partitioning reflects the release of hydration water from the membrane surface on adsorption of the solute.16 This suggestion is primarily based on spectroscopic evidence36-38 (supported by calorimetry37,39) indicating that the binding of solutes displaces water from the phosphate moiety of the lipid headgroup. Because the volume of surface-associated water is very low due to the ability of water molecules to occupy interstitial spaces in the “rugged” membrane surface region40 as well as the electrostrinction induced by the charges of the lipid headgroup,16 displacement to the bulk will expand the system. The data in Figure 5, however, clearly show that in alcohol-DMPC systems, membrane-solute interactions act to decrease the partial volume of
11340 J. Phys. Chem. B, Vol. 104, No. 47, 2000
Figure 5. Changes in contributions to the total excess volume determined from the data in Table 1. The columns illustrate the change in free volume of respective lipid (no fill pattern), alcohol (diagonal fill), and water (crosshatch fill) generated from membrane-solute interactions at an alcohol concentration in the membrane of 300 mmol/ kg lipid. The lipid concentration is 50 mM; see text for details.
water. This result is compatible with a recent study by Ho and Stubbs41 in which the penetration of water into lipid membranes was monitored by fluorescence spectroscopy. It was found that the addition of aliphatic alcohols increased the water content in the headgroup region. The present data may reflect the same mechanism, because transfer of water from the bulk to the headgroup region is likely to be accompanied by a negative volume change. Namely, in analogy to membrane hydration in the absence of alcohols,40 the penetrating water will have a small partial volume because of the availability of voids in an environment of much larger molecules. The difference between this picture and the dehydration hypothesis may arise from the comparison of average and specific structural properties. The spectroscopic investigations36-38 suggest that water is displaced from specific interactions with the phosphate group whereas the present and other works41-43 provide evidence that the adsorption of a variety of solutes to membranes increases their average water content. The different conclusions about water might also simply reflect specifics of the investigated systems because evidence on dehydration primarily comes from work with partially hydrated membranes or inversed micellar systems.36-38 Membrane partitioned alcohols, such as those in Figure 5, are thought to be located with the hydroxyl group at the membrane-water interface and the nonpolar group inserted in the acyl chain region.44,45 Thus, Figure 5 indicates that intercalation of a flexible aliphatic alcohol generates a pronounced loosening in the packing of the lipids. The strongest effect is observed for hexanol, which spans approximately half a membrane leaflet. Intuitively, it seems probable that this arrangement will lead to inefficient packing, and further studies of alcohols with chain lengths comparable to the those of the lipid molecule seems promising in attempts to elucidate this property. The expansion induced by the cyclic alcohols (at the same concentration in the membrane) is 2-4 times smaller than that of the aliphatics. This difference may reflect a shallower penetration into the acyl chain region and/or lower flexibility of the solute molecule. A rigid molecule, such as cholesterol, has been shown to occupy interstitial (free-) volume in the membrane46 and hence enhance packing density of bilayer membranes. However, no simple relationship between flexibility and volume properties of membrane-solute complexes can be established at this time. Hexane, for example, which is highly flexible and, judging from investigations of multilamellar
Westh and Trandum membranes under osmotic stress, accumulates in the core of lipid membranes near the methyl groups of the lipid molecules,47 enhances the packing density of the complex.48,49 This complexity of location and physical properties of partitioned solutes is currently limiting the detailed interpretation of volumetric data. On the other hand, it suggests that volumes properties are highly sensitive to the molecular arrangement of membrane-solute complexes, and therefore that, if systematically investigated, this type of work holds an appreciable potential for the elucidation of partitioning processes. In summary we have used titration densitometry to quantify the (total) volume change, ∆Vm, for the transfer of small monoalcohols and acetone from aqueous solution to fluid bilayers of DMPC. It was found that ∆Vm was positive and amounted to 5-20% of the volume of the solute in aqueous solution. More importantly, the partial excess volumes of alcohol, water, and lipid was derived from the composition dependence of the measured specific volumes and utilized to discuss changes in free volume of each component resulting from membrane-solute interactions. It was shown that the lipid molecules become more loosely packed when the solute adsorbs to the membrane. This effect is particularly strong for aliphatic alcohols. Water, on the other hand, rearranges to occupy a lower volume. These observations support a molecular picture in which partitioning of solutes into membranes is accompanied by a disordering of the phospholipids and an increase in the water content of the membrane. Acknowledgment. This work was supported by the Hasselblad and Lundbeck Foundations. The excellent technical assistance by H.C. Jehg on setting up the densitometer and the comments and suggestions by Dr. Martin Zuckermann are highly appreciated. References and Notes (1) Selig, J.; Ganz, P. Biochemistry 1991, 30, 9354. (2) Zhang, F.; Rowe, E. S. Biochemistry 1992, 31, 2005. (3) Rowe, E. S.; Zhang, F.; Leung, T. W.; Parr J. S.; Guy, P. T. Biochemistry 1998, 27, 2430. (4) Heerklotz, H. H.; Binder, H.; Epand, R. M. Biophys. J. 1999, 76, 206. (5) Heerklotz H. H.; Seelig, J Biophys. J. 2000, 78, 2435. (6) Trandum, C.; Westh, P.; Jørgensen, K.; Mouritsen, O. G. Thermochim. Acta 1999, 328, 129. (7) Trandum, C.; Westh, P.; Jørgensen, K.; Mouritsen, O. G. J. Phys. Chem. 103B, 1999, 4751. (8) Trandum, C.; Westh, P.; Jørgensen, K.; Mouritsen, O. G. Biochim. Biophys. Acta 1999, 1420, 179. (9) Westh, P.; Trandum, C. Biochim. Biophys. Acta 1999, 1421, 261. (10) Trandum, C.; Westh, P.; Jørgensen, K.; Mouritsen, O. G. Biophys. J. 2000, 78, 2486. (11) Janoff, A. S.; Miller, K. W. In Biological Membranes, Vol. 4; Chapman, D., Ed.; Academic Press: London 1982. (12) Seeman, P. Pharmacol. ReV. 1972, 24, 583. (13) Kaminoh, Y.; Kamaya, H.; Tashiro, C.; Ueda, I. Toxicol. Lett. 1998, 100, 353. (14) Miller, K. W.; Wilson, M. W. Anesthesiology 1978, 48, 104. (15) Trudell, J. R.; Hubbell, W. L.; Cohen, E. N.; Kending, J. J. Anesthesiology 1973, 38, 207. (16) Ueda, I. Colloids Surf. 1989, 38, 37. (17) Kita, Y.; Bennett, L. J.; Miller, K. W. Biochim. Biophys. Acta 1981, 647, 130. (18) Kita, Y.; Miller K. W. Biochemistry 1982, 21, 2840. (19) Kaneshina, S.; Kamaya, H.; Ueda, I. J. Colloid Interface Sci. 1983, 93, 215-224. (20) Auger, M.; Jarrell, H. C.; Smith, I. C. P.; Wong, P. T. T.; Siminovitch, D. J.; Mantsch, H. H. Biochemistry 1987, 26, 8513. (21) Bano, M.; Pajdalova, O. Biophys. Chem. 1999, 80, 53. (22) Ipsen, J. H.; Mouritsen, O. G.; Bloom, M. Biophys. J. 1990, 57, 405. (23) Mouritsen, O. G.; Dammann, B.; Fogedby, H. C. Ipsen J. H.; Jeppesen. C.; Jørgensen, K.; Risbo, J.; Sabra, M. C.; Sperotto M. M.; Zuckermann, M. J. Biophys. Chem. 1995, 55, 55.
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