n-Butanol Partitioning into Phase-Separated Heterogeneous Lipid

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n‑Butanol Partitioning into Phase-Separated Heterogeneous Lipid Monolayers Yogi Kurniawan,† Carmen Scholz,‡ and Geoffrey D. Bothun*,† †

Department of Chemical Engineering, University of Rhode Island, 16 Greenhouse Rd., Kingston, Rhode Island 02881, United States Department of Chemistry, University of Alabama in Huntsville, 301 Sparkman Dr., Huntsville, Alabama 35899, United States

‡

ABSTRACT: Cellular adaptation to elevated alcohol concentration involves altering membrane lipid composition to counteract fluidization. However, few studies have examined the biophysical response of biologically relevant heterogeneous membranes. Lipid phase behavior, molecular packing, and elasticity have been examined by surface pressure−area (π−A) analysis in mixed monolayers composed of saturated dipalmitoylphosphatidylcholine (DPPC) and unsaturated dioleoylphosphatidylcholine (DOPC) as a function of DOPC and nbutanol concentration. n-Butanol partitioning into DPPC monolayers led to lipid expansion and increased elasticity. Greater lipid expansion occurred with increasing DOPC concentration, and a maximum was observed at equimolar DPPC:DOPC consistent with n-butanol partitioning between coexisting liquid expanded (LE, DOPC) phases and liquid condensed (LC, DPPC) domains. This led to distinct changes in the size and morphology of LC domains. In DOPCrich monolayers the effect of n-butanol adsorption on π−A behavior was less pronounced due to DOPC tail kinking. These results point to the importance of lipid composition and phase coexistence on n-butanol partitioning and monolayer restructuring.

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Recent studies on alcohol-induced membrane fluidization have focused on heterogeneous membranes comprised of different lipid species and phase states (i.e., lipid domains in membranes containing saturated and unsaturated lipids), which better reflect the complex structure of cellular membranes.9,16,17 Marques et al.9 have shown that restructuring in planar supported DPPC/dioleoylphosphatidylcholine (DOPC, unsaturated, fluid phase) membranes caused by ethanol is dependent on which lipid phase is continuous. When DOPC was the continuous phase at low ethanol concentrations, ethanol preferentially partitioned into and thinned the fluid DOPC phase, increasing hydrophobic mismatch between the gel and fluid phases (a condition that would be possible if ethanol accumulated at the gel/fluid interface). Increasing ethanol concentration then thinned both the DOPC and gel DPPC domains. When gel DPPC was the continuous phase and fluid DOPC domains were present, ethanol first partitioned at the gel/fluid interface and did not lead to the expansion of the fluid domains. These results are consistent with greater partitioning of short chain alcohols into unsaturated than saturated lipid phases, and can be attributed to a greater interfacial area stemming from the poor packing properties of unsaturated lipid tails.3,10

INTRODUCTION

Primary alcohols are lipophilic and can partition into cellular membranes and cause lipid disordering, which increases membrane fluidity (reduces membrane viscosity). Cells counteract membrane fluidization through homeoviscous adaptation by altering their membrane lipid composition. Homeoviscous adaptation varies based on cell type and can involve either an increase in the ratio of saturated to unsaturated lipids, where saturated lipids restore lipid ordering, or a decrease in the ratio of saturated to unsaturated lipids, where unsaturated lipids reduce the extent of membrane expansion.1 Biophysical studies using lipid bilayers or monolayers as model cell membranes have provided a great deal of insight into how lipid structure and composition modulate membrane fluidization, and how this varies with the hydrocarbon chain length of the alcohol.2−7 With implications in physiology and biofuel production, much of what is known for alcohol-induced membrane fluidization has been gained using ethanol and homogeneous single-component lipid bilayers. Ethanol, like other short chain alcohols (up to C6), partitions to the membrane/water interface, hydrogen bonds to carbonyl groups in the lipid headgroups, leads to lipid expansion and reduced interlipid van der Waals attraction, and increases membrane elasticity.6,8−13 Above a critical concentration, short chain alcohols also induce lipid interdigitation in gel-phase membranes composted of saturated phosphatidylcholine (PC) such as dipalmitoyl-PC (DPPC).2,11,14,15 © 2013 American Chemical Society

Received: March 14, 2013 Revised: July 24, 2013 Published: July 26, 2013 10817

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Figure 1. π−A isotherms at 25 °C of DPPC/DOPC monolayers spread at air/water interfaces at DPPC:DOPC molar ratios of (A) 1:0, (B) 3:1, (C) 1:1, (D) 1:3, and (E) 0:1. n-Butanol concentrations added to the subphase, [btoh], are shown in the legend and DPPC and DOPC molecular structures are shown in (A) and (E), respectively.

Similar to ethanol, much of what is known of the fluidizing effects of n-butanol has been gained through biophysical studies of homogeneous lipid bilayers.4,5,10,11,15,18−24 Ly and Longo4 studied the influence of short chain alcohols on the interfacial tension and mechanical properties of unsaturated stearoyloleoylphosphatidylcholine (SOPC) lipid bilayers. Their results show that n-butanol led to greater mechanical destabilization and increased bilayer expansion than methanol, ethanol, and propanol. In addition, n-butanol had the highest membrane partitioning and permeability coefficients. Kurniawan et al.17 have since shown that n-butanol partitioning in DPPC/DOPC bilayers increased with DOPC concentration, and when DOPC was the major component, n-butanol appeared to preferentially partition within the fluid phase at the interface between gel and fluid phases. These observations are consistent with increased hydrophobic mismatch caused by ethanol.9 Results from this work also suggested that DOPC reduced n-butanol interdigitation in the DPPC phase, which is in agreement with recent work by Vanegas et al.16 where DOPC prevented ethanol interdigitation in a yeast membrane model (DPPC/DOPC/ ergosterol). The emergent role that unsaturated lipids play in modulating the response of heterogeneous membranes to alcohols should provide new insight into cellular homeoviscous adaptation, where increasing the ratio of saturated to unsaturated lipid does not always correlate with improved alcohol tolerance.25 In this study, heterogeneous DPPC/DOPC monolayers have been used to determine the effects of n-butanol on lipid phase behavior, lipid packing, and monolayer elasticity. Monolayers were employed to examine n-butanol partition at DPPC/ DOPC interfaces. This was achieved by detailed analysis of surface pressure−area isotherms at 25 °C coupled with fluorescence microscopy of lipid monolayers transferred onto glass slides. Monolayers were prepared over a range of

DPPC:DOPC ratios and subjected to n-butanol subphase concentrations up to 0.27 mM (20 mg/L or 2.5 × 10−3 vol %). DPPC is a saturated lipid with dual C16 tails and a melting temperature of 41 °C, while DOPC is an unsaturated lipid with dual C18 tails (cis double bond in each tail at the 9−10position) and a melting temperature of −20 °C. DPPC/DOPC monolayers were used to yield heterogeneous monolayers with coexisting liquid expanded (LE, DOPC) and liquid condensed (LC, DPPC) phases.

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MATERIALS AND METHODS

Chemicals. 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, >99% purity), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, >99% purity), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt) (RhPE, >99% purity) were purchased from Avanti Polar Lipids (Alabaster, AL) and used without further purification. n-Butanol was purchased from Fisher Scientific, and deionized (DI) ultrafiltered water was obtained from a Millipore Direct Q-3 purification system (18.2 MΩ· cm). Surface Pressure−Area (π−A) Isotherms. The π−A isotherms were carried out in a Langmuir trough (model 102M, KSV NIMA) with a deposition area of 70 cm2 at 25 °C. Surface pressure was measured with an accuracy of ±1 μN/m using a Wilhelmy plate, which was connected to an electronic microbalance (Mini PS4, KSV NIMA). The subphase temperature was controlled by an external recirculating water bath (Haake B3). Monolayers were obtained by spreading diluted solutions of DPPC and/or DOPC in chloroform at the air/ water interface using a glass microsyringe (Hamilton). After deposition, the spreading solvent was allowed to evaporate and the films to spread for 15 min. Prior to compression by PTFE barriers, nbutanol at a desired concentration was injected from the bottom of the trough into the subphase. The system was then equilibrated for 10 min before being compressed at a speed of 10 cm2/min. All experimental isotherms were conducted in duplicate. 10818

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Figure 2. (A, B) Area per molecule, A, as a function of DOPC mole fraction, xDOPC, and [btoh] at (A) 20 mN/m and (B) 30 mN/m. (C) Change in A at [btoh] = 0.13 mM (solid lines) and [btoh] = 0.27 mM (dashed lines) at π = 20 and 30 mN/m.

Figure 3. (A−E) Elastic moduli, Cs−1, corresponding to the π−A isotherms at 25 °C in Figure 1 at DPPC:DOPC ratios of (A) 1:0, (B) 3:1, (C) 1:1, (D) 1:3, and (E) 0:1. (F) Maximum elastic moduli, Cs,max−1, as a function of n-butanol concentration and DOPC concentration (xDOPC shown in graph).

DPPC:DOPC ratios from 1:0 to 0:1 at 25 °C. DPPC and DPPC/DOPC monolayers transitioned from the LE phase to coexisting LE−LC phases upon compression (plateau regions in the π−A isotherms between ca. 5 and 10 mN/m). In DPPC/ DOPC monolayers the two lipids were immiscible, and LE−LC phase coexistence reflected DPPC and DOPC phases, respectively. At high compression, above the plateau region, DPPC LC phases coexisted with DOPC LE phases. The LE− LC transition for DPPC occurred near 6 mN/m and increased with increasing DOPC concentration due to DOPC phases resisting compression.26−28 DOPC, with dual monounsaturated tails, remained in LE phases and did not form LC domains upon compression as tail “kinking” around the double bonds hindered its ability to pack tightly within the monolayer. In DPPC/DOPC monolayers, the effects of poor DOPC packing were reflected in the increasing A values for area per lipid molecule with increasing DOPC concentration at π = 20 and

Fluorescence Microscopy. A hydrophilic glass slide pretreated with piranha solution (2.54 cm × 1.27 cm) was immersed vertically into the subphase using a dipper assembly (KSV Nima). Monolayers were then formed as described in the preceding section with RhPE at 3 mol %. The labeled monolayer films were compressed at 10 cm2/min to a surface pressure of 20 or 30 mN/m, in the absence or presence of n-butanol, and equilibrated for 5 min. Monolayers were transferred to the slides during withdrawal from the subphase at a rate of 3 mm/min. Imaging was performed within 10 min of transferring at 100× magnification using a Nikon Diaphot-TMD inverted epifluorescence microscope (Nikon, Japan). The microscope was equipped with phase contrast-2 ELWD 0.52 phase-contrast condenser, a 12 V, 100 W mercury lamp (Nikon, Japan), and a digital sight DS-L2 camera (Nikon, Japan) equipped with G-2B filter cube. NIS-element software was used to analyze and capture the images.

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RESULTS Surface Pressure−Area Isotherms. Surface pressure− area (π−A) isotherms are shown in Figures 1A−E for 10819

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When DOPC was the major component (1:3 DPPC:DOPC) Cs,max−1 decreases with increasing [btoh]. Monolayer Phase Coexistence. When xDOPC was ≥0.5 (1:1 and 1:3 DPPC:DOPC, Figures 1C and 1D, respectively), the LE−LC transitions became less pronounced with increasing n-butanol concentration. To verify LE−LC coexistence and examine LC domain structure, fluorescence microscopy was conducted on supported monolayers transferred labeled with a fluorescent rhodamine lipid, Rh-DPPE, at π = 30 mN/m (Figure 4A). The images confirm, despite not being evident

30 mN/m (A; Figure 2A,B). This demonstrates nonideal lipid mixing indicative of immiscibility. Experiments with n-butanol were conducted by adding nbutanol to the subphase of a freshly prepared monolayer before compression. To account for the possible effects of n-butanol alone, π at the air/water interface was examined at the highest subphase n-butanol concentration, [btoh] = 0.27 mM, at fixed trough areas corresponding to minimum and maximum compression. In both cases π was less than 0.8 mN/m and decreased by less than 25% over 1.5 h. This suggests that at the concentrations employed n-butanol alone had little effect on π and that minimal n-butanol evaporation occurred over the duration of the experiments. Krisch et al.29 have also shown that changes in water surface tension are minimal at the nbutanol concentrations examined. When n-butanol was added to the subphase, [btoh] = 0.07− 0.27 mM, it partitioned from water to the monolayer, binding to the lipids and occupying area at the air/water interface. Three main observations are taken from the isotherms. First, LE−LC transitions were observed when DPPC was the major component (1:0 and 3:1 DPPC:DOPC; Figures 1A and 1B, respectively). In this case increasing [btoh] progressively lowered the surface pressure associated with the LE−LC transition (πLE−LC), indicating that less pressure was needed to nucleate LC DPPC domains from LE phases. Second, area expansion in DOPC monolayers (Figure 1E) was proportional to n-butanol concentration over the entire isotherm. This behavior is consistent with previous work on alcohols and SOPC giant unilamellar vesicles examined by micropipet aspiration, where area expansion was attributed to reductions in interfacial tension.4 Third, monolayer expansion was observed in DPPC/DOPC mixtures above the LE−LC DPPC transition, and the degree of expansion exceeded that observed for DPPC or DOPC alone (Figure 2). This can be clearly seen in the change in A caused by n-butanol, ΔA, which was greatest at equimolar DPPC:DOPCa condition that coincided with the largest total interfacial perimeter between LC and LE phases. (shown in Figure 4C). Monolayer Elasticity. Elastic compressibility moduli, Cs−1, were determined based on the π−A isotherms from the following equation:28,29

(1)

Figure 4. (A) Fluorescence microscopy images of supported DPPC/ DOPC monolayers transferred at 30 mN/m. The scale bars represent 20 μm. Shown within the scale bars are the areas per molecule (Å2/ molecule) at 30 mN/m based on the isotherms in Figure 1. Images (a−c) correspond to isotherms in Figure 1B, (d−f) isotherms in Figure 1C, and (g−i) isotherms in Figure 1D. Light regions reflect RhDPPE in LE DOPC phases and dark regions reflect LC DPPC domains. (B) and (C) show the area fraction and the total perimeter associated with LC domains, respectively.

Cs−1 are shown in Figures 3A−E and correspond to the isotherms in Figures 1A−E, respectively. High Cs−1 indicates low monolayer compressibility due to tightly packed lipids and high interlipid cohesive forces (notably van der Waals attraction). DPPC formed rigid monolayers due to its saturated tails (Figure 3A), while DOPC formed less rigid monolayers due to unfavorable tail packing by its monounsaturated tails (Figure 3E).30 Mixed DPPC/DOPC monolayers contained coexisting LE and LC phases and exhibited Cs−1 between that of the pure components. The addition of n-butanol progressively shifted Cs−1 to higher A values, which can be attributed to the additional interfacial area occupied by nbutanol. The maximum elastic moduli, Cs,max−1, are shown in Figure 3F. At 1:0, 3:1, and 1:1 DPPC:DOPC a minimum was observed for Cs,max−1 between 0.07 and 0.13 mM [btoh] denoting monolayer fluidization and an increase in compressibility. At [btoh] > 0.13 mM, Cs,max−1 increased and, at 1:0 and 3:1 DPPC:DOPC, reached a maximum at 0.27 mM [btoh].

from the π−A isotherms, that the monolayers contained LC DPPC domains (dark regions) within continuous LE DOPCrich phases (light regions). The area fraction and total perimeter of LC domains were determined by analyzing at least two representative images for each condition (Figure 4B,C). It should be noted that supported monolayers were transferred at the same surface pressure; hence, A varies between 60.1 and 73.6 Å2/molecule depending on the DPPC:DOPC ratio and n-butanol concentration. The phase behavior would clearly be different if the monolayers were transferred at the same degree of compression (same A). At all DPPC:DOPC ratios the total perimeter increased from 0 to 0.13 mM [btoh] due to the presence of more abundant, smaller LC domains (Figure 4A;b,e,h). A shift from larger to smaller domains reflected a reduction in line tension at the LC domain perimeter.31 Furthermore, at all DPPC:DOPC ratios the total perimeter then decreased from

⎛ ∂π ⎞ Cs−1 = −A⎜ ⎟ ⎝ ∂A ⎠T

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DOPC bilayers is 35% greater than in DPPC bilayers. Furthermore, Kurniawan et al.17 have shown that n-butanol partitioning into DPPC/DOPC vesicles increases significantly with DOPC concentration. Greater alcohol partitioning into DOPC is attributed to the acyl tail structure. DOPC occupies more space than DPPC, yielding additional “void area” at the lipid/water interface where the hydrocarbon tails are exposed to water. Short chain alcohols, which are known to partition at the lipid/water interface, exhibit greater partitioning into unsaturated lipids than saturated lipids due to the presence of these voids. This ability is related to the extent in which the monolayer must expand or restructure to accommodate the alcohol. It has been shown for ethanol that monolayer expansion (increasing A) for DPPC was greater than that for palmitoyloleyolphosphatidylcholine (POPC).5 Results for DPPC and DOPC with n-butanol support this concept (Figures 1A,E and 2A). Based on previous work, increasing DOPC concentration within the DPPC/DOPC monolayers increased the net nbutanol partitioning. However, a cooperative effect of DPPC + DOPC was observed that increased monolayer expansion beyond what was expected based on pure DPPC or DOPC, which reflected the additional area expansion due to nonideal miscibility and interlipid repulsion. n-Butanol exacerbated this nonideal mixing behavior, and a likely explanation for the large area expansion is that n-butanol accumulated at LE−LC phase boundaries. n-Butanol accumulation at the LE−LC interface would reduce the effects of hydrophobic mismatch between LE and LC phases and could further explain why πLE−LC decreased within [btoh] in DPPC-rich monolayers. This concept is supported by the work of Marques et al.9 where ethanol effects on supported DPPC/DOPC bilayers were examined by AFM. At 1:1 DPPC:DOPC they showed that ethanol partitioning increased hydrophobic mismatch between gel and fluid domains, a condition that could be favorable if ethanol accumulated at the domain interface and shielded the exposed acyl tails from water. When DPPC was the continuous phase (92:8 DPPC:DOPC), ethanol preferentially thinned the fluid DOPC domains at the gel/fluid interface. Interestingly, the concept of n-butanol accumulation at LE−LC interfaces is also supported by the work of Jørgensen et al.38 where anesthetics were shown to exhibit high affinity for gel-fluid phase boundaries, relative to the gel or fluid phases, in lipid bilayers that were “dominated by kink-like lipid-chain conformations”. A remaining question is to what extent DOPC concentration and n-butanol partitioning into LE or LC phases may have influenced monolayer phase behavior. From 0 to 0.27 mM [btoh] the LE area fraction increased significantly relative to the LC domains at constant surface pressure (30 mN/m). However, the LC domain perimeter was largely preserved due to the formation of large irregularly shaped LC domains with “rough” edges. This behavior may be attributed to preferential n-butanol partitioning into LE phases, relative to the LC phases, which would cause the LE phase to expand at the expense of the LC area fraction at constant surface pressure. Additional fluorescence microscopy images are shown for supported monolayers at 1:1 DPPC:DOPC transferred at 20 mN/m, which further confirm this behavior (Figure 5). The fact that the LC domain perimeter was preserved at 30 mN/m despite the loss in LC fraction area with increasing [btoh] suggests that additional LE−LC interface was created to accommodate the n-butanol, consistent with previous observations for anesthetics.38 It should be noted that further

0.13 to 0.27 mM [btoh], and larger, irregularly shaped LC domains were observed (Figure 4A;c,f,i). This was accompanied by a decrease in LC area fraction. At 1:1 and 1:3 DPPC:DOPC the irregularly shaped LC domains were large and resembled “snowflakes” with high perimeter per domain. Domains such as these (ice-like) have been attributed to diffusion-limited aggregation (DLA) of smaller LC domains.32,33 At this point it is unclear whether DLA played a role in LC domain structure and additional kinetic experiments would need to be conducted to address this question.

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DISCUSSION The objective of this study was to examine the effects of nbutanol on heterogeneous monolayers that exhibit LE−LC phase coexistence. Few studies have been reported on nbutanol partitioning and restructuring in lipid bilayers, and there have been no studies, to the authors’ knowledge, reporting the effects of short chain alcohols on heterogeneous monolayers. Comparisons to existing literature will be based on methanol or ethanol with lipid monolayers or bilayers. However, these comparisons are complicated by the fact that DPPC/DOPC is an immiscible two-phase system. Brezesinski et al.34 have proposed that ethanol binding to dimyristoyl-PC (DMPC) monolayers dehydrates the PC headgroups and leads to lipid condensation, consistent with reductions in A and the degree of chain tilt relative to the air/ water interface. Simulations by Patra et al.5 have further shown that dehydration is due to ethanol hydrogen bonding with the ester groups in the glycerol backbone of PC lipids. While FTIR studies have also demonstrated DPPC dehydration by nbutanol,35 lipid condensation was not observed in this work. Rather, monolayer expansion was observed at all DPPC:DOPC ratios. It is assumed that this was due to the longer acyl tail of nbutanol, which allowed it to penetrate deeper into the lipid hydrocarbon region within the monolayer. This concept is supported by simulation work comparing density profiles of ethanol and n-butanol in lipid bilayers.36 Increases in A with nbutanol suggest that the lipid tilt angle increased to minimize voids within the hydrocarbon region and to maximize van der Waals interactions between acyl tails. This phenomenon has been proposed for ethanol and PC bilayers, but increasing tilt is unstable and leads to the formation of an interdigitated lipid phase (not possible in monolayers).2 The observed behavior in C s,max −1 at 1:0 and 3:1 DPPC:DOPC was unexpected based on the reported effects of methanol or ethanol on DPPC monolayers, which reduce the elastic modulus (increase elasticity).37 This is reflected in eq 1 where reductions in A due to lipid dehydration lead to lower Cs−1. However, unlike methanol or ethanol, A increased with nbutanol concentration due to monolayer expansion. It was this increase in A, due to n-butanol penetration within the monolayer and increased DPPC tilt, that led to high Cs,max−1 values when DPPC was in abundance. At equimolar DPPC/ DOPC, or when DOPC was the major component, Cs,max−1 with n-butanol did not exceed that of the single component monolayers. This reflects the fact that, unlike DPPC, the monounsaturated tails of DOPC prevented it from tilting or packing tightly, which caused DOPC to occupy high interfacial areas and yielded lower interlipid attraction relative to DPPC. In addition to LE−LC phase coexistence, a difference between this and previous work is that preferential alcohol (n-butanol) partitioning into LE or LC phases should influence the monolayers. Cantor3 has shown that ethanol partitioning in 10821

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due to dissimilarities between lipid tail structure and phase coexistence. This work suggests that n-butanol partitioning into monolayers increases with unsaturated lipid concentration and significantly affects phase behavior. Consistent with theories for homeoviscous adaptation, the unsaturated lipid (DOPC) accommodated high n-butanol concentrations without significant expansion. However, lipid packing based on saturated to unsaturated lipid ratios represents a new variable for understanding alcohol fluidization, particularly in heterogeneous membrane with coexisting phases.

Figure 5. Fluorescence microscopy images of supported 1:1 DPPC:DOPC monolayers transferred at 20 mN/m at (a) 0, (b) 0.13, and (c) 0.27 mM [btoh]. The scale bars represent 20 μm. Shown within the scale bars are the areas per molecule (Å2/molecule) at 20 mN/m based on the isotherms in Figure 1C.

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compressing the monolayers to constant A would certainly increase the LC area fraction and domain perimeter. The effects of n-butanol on DPPC, DOPC, and DPPC/ DOPC monolayer are summarized schematically in Figure 6 for

AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]; tel +1-401-874-9518; fax +1-401874-4689 (G.D.B.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Science Foundation Energy for Sustainability program (CBET-0966818).

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REFERENCES

(1) Weber, F. J.; de Bont, J. A. Adaptation mechanisms of microorganisms to the toxic effects of organic solvents on membranes. Biochim. Biophys. Acta 1996, 1286, 225−245. (2) Adachi, T.; Takahashi, H.; Ohki, K.; Hatta, I. Interdigitated structure of phospholipid-alcohol systems studied by X-ray-diffraction. Biophys. J. 1995, 68, 1850−1855. (3) Cantor, R. S. Bilayer partition coefficients of alkanols: predicted effects of varying lipid composition. J. Phys. Chem. B 2001, 105, 7550− 7553. (4) Ly, H. V.; Longo, M. L. The influence of short-chain alcohols on interfacial tension, mechanical properties, area/molecule, and permeability of fluid lipid bilayers. Biophys. J. 2004, 87, 1013−1033. (5) Patra, M.; Salonen, E.; Emma, T.; llpo, V. Under the influence of alcohol: the effect of ethanol and methanol on lipid bilayers. Biophys. J. 2006, 90, 1121−1135. (6) Barry, J. A.; Gawrisch, K. Effects of ethanol on lipid bilayers containing cholesterol, gangliosides, and sphingomyelin. Biochemistry 1995, 34, 8852−8860. (7) Dickey, A. N.; Faller, R. How alcohol chain-length and concentration modulate hydrogen bond formation in a lipid bilayer. Biophys. J. 2007, 92, 2366−2376. (8) Holte, L. L.; Gawrisch, K. Determining ethanol distribution in phospholipid multilayers with MAS-NOESY spectra. Biochemistry 1997, 36, 4669−4674. (9) Marquês, J. T.; Viana, A. S.; De Almeida, R. F. M. Ethanol effects on binary and ternary supported lipid bilayers with gel/fluid domains and lipid rafts. Biochim. Biophys. Acta 2011, 1808, 405−414. (10) Terama, E.; Ollila, O. H. S.; Salonen, E.; Rowat, A. C.; Trandum, C.; Westh, P.; Patra, M.; Karttunen, M.; Vattulainen, I. Influence of ethanol on lipid membranes: from lateral pressure profiles to dynamics and partitioning. J. Phys. Chem. B 2008, 112, 4131−4139. (11) Vierl, U.; Löbbecke, L.; Nagel, N.; Cevc, G. Solute effects on the colloidal and phase behavior of lipid bilayer membranes: ethanoldipalmitoylphosphatidylcholine mixtures. Biophys. J. 1994, 67, 1067− 1079. (12) Nizza, D. T.; Gawrisch, K. A layer model of ethanol partitioning into lipid membranes. Gen. Physiol. Biophys. 2009, 28, 140−145. (13) Toppozini, L.; Armstrong, C. L.; Barrett, M. A.; Zheng, S.; Luo, L.; Nanda, H.; Garcia Sakai, V.; Rheinstader, M. C. Partitioning of ethanol into lipid membranes and its effect on fluidity and permeability as seen by X-ray and neutron scattering. Soft Matter 2012, 8, 11839−11849.

Figure 6. Schematic of monolayer structure and partitioning behavior of n-butanol at 25 °C into (A) LC DPPC phases, (B) LE DOPC phases, and (C) mixed LE−LC DPPC/DOPC phases.

LC, LE, or LC/LE monolayers, respectively. In the case of DPPC without n-butanol at 25 °C, the hydrocarbon chains of DPPC were packed in an all-trans conformation and tilted at the interface. When n-butanol was added, it adsorbed to PC headgroups, penetrated into the monolayer, and increased the area per lipid, which increased lipid tilt. In the case of DOPC, nbutanol adsorption and penetration presumably occurred in a similar fashion, but the effect on lipid orientation and expansion were less pronounced than DPPC due to the interfacial voids caused by DOPC tail kinking. In the case of mixed DPPC/ DOPC monolayers, n-butanol preferentially partitioned into the LE phase and accumulated at the interface between LE and LC phases to reduce tail mismatch between DPPC and DOPC.

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CONCLUSIONS This is the first study depicting the effects of n-butanol partitioning on heterogeneous monolayers with coexisting LE and LC phases arising from a mixture of unsaturated and saturated lipids, respectively. Approaches such as this provide new insight into how complex, multicomponent cellular membranes are adapted to elevated alcohol concentrations. The effects of n-butanol on monolayer structure are not simply the cumulative effects on saturated + unsaturated lipids. Rather, the presence of both lipids exacerbates the effects of n-butanol 10822

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dx.doi.org/10.1021/la400977h | Langmuir 2013, 29, 10817−10823