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Langmuir Monolayer Flow across Hydrophobic Surfaces. 3. Influence of Acyl Chain Structure on Supported Bilayer Formation Rates Adam B. Steel,‡ Brady J. Cheek,*,‡ and Cary J. Miller§ Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, and MetriGenix, Inc., 708 Quince Orchard Road, Gaithersburg, Maryland 20878 Received June 18, 2001. In Final Form: September 22, 2001 The bilayer formation properties of single-component acyl chains of varying length and extent of unsaturation are compared using Langmuir monolayer flow. Four series of structural motifs in the flowing film were considered: linked oleyl chains; chains with a single cis double bond over a range of total chain lengths; chains of the same length with 1-5 cis double bonds; molecules which flow that do not have the standard cis double bond in the chain. Slower flow rates were observed for Langmuir films with increased numbers of linked oleyl chains due to the enhanced interlayer coupling between the flowing and stationary films. Slower flow rates were also observed with increased acyl chain length due to the increased intralayer coupling between the flowing molecules. More rapid flow rates were observed as the number of double bonds within the film forming material was increased, due to both the decreased inter- and intralayer coupling. While no single structural feature is common to molecules that undergo monolayer flow, all molecules that have been observed to flow exist at a liquid expanded phase state at the equilibrium spreading pressure.
Introduction Bilayer membranes are perhaps one of the most simple molecular architectures yet perform critical functions in complex cellular structures.1 Cell membranes generally contain a mixture glycerolphopholipids and proteins, and the function of the bilayer is 2-fold: a molecular barrier to hydrophilic/ionic species; a solvent for proteins. Phospholipids provide structural support for the membrane assembly and are organized on the basis of their hydrophilic properties. The composition of mixed lipids within bilayers has a pronounced influence on processes that occur at the membrane surface. Lipids generally contain two aliphatic hydrocarbon chains, one saturated and a second that can vary in length (number of carbons) and the type, number, position, and geometric configuration of unsaturated carbon-carbon bonds. Postulated models for packing in cell membranes hold that lipids pack in microscopic domains with maximally interacting saturated chains at the interior of the domain and the variable chains forming the interface between domains. The lateral packing properties of the system are determined both locally and globally by the dynamic interplay of the microscopic domain interfaces.2 By controlling the level of sterols and the extent of unsaturation in the acyl chains of lipids, mammalian cells have the capacity to produce localized regions within membranes with different elasticity. Ion permeability, channel formation, enzyme activity, and membrane fusion are all reported to coincide with phase separation and domain boundaries in nonhomo* To whom correspondence should be addressed. † University of Maryland. ‡ Current address: MetriGenix, Inc. § Current address: i-STAT Corp., Kanata, Ontario K2L 1T9, Canada. (1) Horton, R. H.; Moran, L. A.; Ochs, R. S.; Scrimgeour, K. G. Principles of Biochemistry; Prentice Hall: Englewood Cliffs, NJ, 1993. (2) Smaby, J. M.; Momsen, M. M.; Brockman, H. L.; Brown, R. E. Biophys. J. 1997, 73, 1492-1505.
geneous assemblies.2-6 In this report, the bilayer formation properties of single-component acyl chains of varying length and extent of unsaturation that make up natural lipids are compared. The tendency for lipids to accumulate in preferred orientations at aqueous phase boundaries has permitted extensive study of monolayer and bilayer structures using insoluble Langmuir films and supported bilayers.7 Monolayers of lipids are widely studied because more of the equation of state of lipid assembly can be explored by mechanical manipulation of the lipid density than is accessible in supported bilayers.8 Langmuir observed that a hydrophilic oil, such as oleic acid, has the property of spreading on water until a monomolecular layer covered the available surface area and the surplus remains gathered as a small lens. He also noted that this oil could serve as a surface piston to hold another film under constant pressure. Langmuir film forming materials of this type are called “piston oils”.9 Unsaturated acyl lipid chains, such as oleic acid (cis-9-octadecenoic acid), have a greater tendency to spread as monolayers on aqueous subphases than the corresponding saturated acid, for the present example stearic acid (octadecanoic acid).10 In addition, cis isomers of unsaturated acyl lipid chains are observed to produce more expanded films (greater surface pressure per molecular packing density) than the corresponding trans isomer.11 Expanded films can be characterized by an equilibrium spreading pressure, ΠESP, greater than 1 mN/m. Equilibrium spreading pressures (3) Koppenol, S.; Yu, H.; Zografi, G. J. Colloid Interface Sci. 1997, 189, 158-166. (4) Girshman, J.; Greathouse, D. V.; Koeppe, R. E.; Andersen, O. S. Biophys. J. 1997, 73, 1310-1319. (5) Niebylski, C. D.; Salem, N. Biophys. J. 1994, 67, 2387-2393. (6) Hoekstra, D. Biochemistry 1982, 21, 2833-2840. (7) Gaines, G. L. Insoluble Monolayers; Interscience: New York, 1966. (8) Marsh, D. Biochim. Biophys. Acta 1996, 1286, 183-223. (9) Blodgett, K. B. J. Am. Chem. Soc. 1935, 57, 1007-1022. (10) Langmuir, I. J. Am. Chem. Soc. 1917, 39, 1848. (11) Adam, N. K. Proc. R. Soc. 1922, A101, 516.
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are determined by the surface pressure for compounds at equilibrium between the monolayer and the bulk and can be related to the interplay of cohesive intermolecular forces in the bulk and free energy of solvation of hydrophilic groups in the acyl carbon chain molecules. While monolayer studies are extremely useful to determine properties of lipids in corresponding states within bilayers, a monolayer only represents half of a bilayer and does not provide any information concerning the interlayer coupling between the layers of a membrane. Model membrane systems were developed, in part, to determine the physical and physiological properties of bilayers.5,12,13 Planar supported bilayers were introduced by McConnell and co-workers14-16 that opened the field for study tremendously. While supported bilayers and selected membrane proteins inserted therein have exhibited behavior similar to native membranes, reproducible fabrication of structurally sound supported bilayers of uniform thickness remains a difficult technological challenge.12 Methods developed around LangmuirBlodgett deposition techniques have met with mixed success because the transfer of fluid film is difficult to control and bilayers produced by this method often contain defects.17-19 Fusion of vesicles to hydrophobic surfaces has been used to enhance the reproducibility of bilayer formation and permits a dynamic measure of the bilayer formation process, providing insights into membrane dynamics.20-21 We previously introduced a method to measure the formation of supported bilayers at the air/water interface from fluid Langmuir monolayers.23 A Langmuir film “flows” into the interface between a self-assembled monolayer (SAM) modified gold substrate and an aqueous subphase producing a supported bilayer. Bilayer formation is measured as a decrease in capacitance of the modified electrode as film from the air/solution interface intrudes into the solid/solution interface. A single parameter, the flow parameter, is calculated from the capacitance-time data. The flow parameter is an empirical construct which is adequate to uniquely describe the monolayer flow rate into the solid/solution interface. Monolayer flow is characterized by a pressure driven plug flow model in that the flow rate is directly proportional to the fluid pressure and inversely proportional to the length of the flow path. According to the plug flow model, the flow parameter is given by the term (2κΠ)1/2, where κ is a term which contains intralayer (viscosity) and interlayer (friction) mechanical coupling factors and Π is the pressure of the monolayer at the air-water interface. Proportionality of the flow parameter with monolayer film pressure was demonstrated by measuring monolayer flow of a series of structurally related oleic acid derivatives where the κ term was considered reasonably conserved but the ESP was (12) Tamm, L. Biochemistry 1988, 27, 1450-1457. (13) Litman, B. J.; Lewis, E. N.; Levin, I. W. Biochemistry 1991, 30, 313-319. (14) Tamm, L. K.; McConnell, H. M. Biophys. J. 1985, 47, 105-113. (15) Seul, M.; Subramaniam, S.; McConnell, H. M. J. Phys. Chem. 1985, 89, 3592-3595. (16) McConnell, H. M. Annu. Rev. Phys. Chem. 1991, 42, 171-195. (17) Reicher, W. M.; Bruckner, C. J.; Joseph, J. Thin Solid Films 1987, 152, 345-376. (18) Osborn, T. D.; Yager, P. Biophys. J. 1995, 68, 1364-1373. (19) Stelzle, M.; Sackmann, E. Biochim. Biophys. Acta 1989, 981, 135-142. (20) Plant, A. L. Langmuir 1993, 9, 2764-2767. (21) Kalb, E.; Frey, S.; Tamm, L. Biochim. Biophys. Acta 1992, 1103, 307-316. (22) Williams, L. M.; Evans, S. D.; Flynn, T. M.; Marsh, A.; Knowles, P. F.; Bushby, R. J.; Boden, N. Langmuir 1997, 13, 751-757. (23) Steel, A. B.; Cheek, B. J.; Miller, C. J. Langmuir 1998, 14, 54795486.
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variable at identical liquid/solid interfaces.24 Comparison of flow parameters between separate flowing monolayers requires compensation for the dependence of the flow rate on the fluid film pressure, ΠESP. The demonstrated proportionality of the flow parameter to ΠESP permits calculation of an effective flow parameters, EFP(x), that can be used to compare the monolayer flow characteristics of flowing Langmuir films. In the EFP(x) designation, the value of x indicates the monolayer film pressure at which the comparison is made. EFP(x) is calculated by multiplying measured flow parameter, FP(ΠESP) by the square root of the ratio of the effective pressure, ΠEFF, to the measured ESP, ΠESP.
EFP(x) ) FP(ΠESP)
x
ΠEFF ΠESP
(1)
For consistency throughout, the ESP of oleic acid, 32.5 mN/m, was used in the calculation of EFP(32.5) as the effective pressure for comparisons. Effective flow parameters can be compared to determine if significant differences in κ factors (monolayer viscosity or interlayer friction) exist between flowing film species. In this report, the flow parameters for systematically varied acyl lipid components have been determined and are compared using the effective flow parameter. Experimental Methods General Reagents. High-purity solvents were used in all synthesis and chromatography. Subphase electrolyte solutions were made with deionized water (Milli-Q system, Millipore). Ethanol, 95% (Pharmco) was employed as the self-assembly deposition solvent for all thiols. Potassium chloride (Sigma), trifluoroacetic anhydride (Baker), and hydrochloric acid (Baker) were used as received. Dodecanethiol (Aldrich) was purchased in the highest available purity and purified by flash chromatography (silica, cyclohexane). Piston Oils. Oleic Acid, myristoleic acid, palmitoleic acid, vaccenic acid, linoleic acid, linolenic acid, phytol (Aldrich), oleyl cyanide, 1,2-diolein, 1,3-diolein, oleyl oleate, triolein, 10-cisnonadecanoic acid, 11-cis-eicosenoic acid, 13-cis-docosenoic acid, 15-cis-tetracosenoic acid, 11,14-cis-eicosadienoic acid, 8,11,14cis-eicosatrienoic acid, 5,8,11,14-cis-eicosatetraenoic acid, 5,8,11,14,17-cis-eicosapentaenoic acid, trans-9-octadecenoic acid, ethyl myristate (NuChek Prep), and dioleyl phosphatidylcholine (Avanti) were used as received. Oleyl alcohol (Aldrich) was purified by flash chromatography (silica, chloroform). Oleyl trifluoroacetate was synthesized by refluxing oleyl alcohol with trifluoroacetic anhydride in acetonitrile for 2 h. The acetonitrile solution was extracted with heptane and the heptane fraction washed with water. The ester was collected as an oil by rotary evaporation and was purified by flash chromatography (silica, chloroform). The structure and purity of the ester were assessed by infrared spectroscopy and thin-layer chromatography. Pressure-area isotherms for all piston oils were collected on a homebuilt Langmuir trough. SAM-Modified Electrode Preparation. Gold electrodes were prepared on glass substrates with freshly cleaved smooth edges. Smooth edges were created by scoring a short segment on a plate glass face and carefully snapping the glass to give 2 freshly cleaved edges. Freshly cleaved glass edges were preferred to regular microscope slide glass edges because of the large amount of surface roughness on the latters’ edge. The plate glass was then cut into roughly 1 × 3-in. pieces and cleaned in a chromic acid bath at 50 °C. After being rinsed with copious amounts of (24) The molecular areas of these oleyl derivatives at their respective equilibrium spreading pressures are approximately the same. This allows a more close examination of the film pressure than would be afforded using oleic acid monolayers at pressures below its ESP. For oleic acid monolayers below 32 mN/m, the oleyl chain density is significantly different resulting in dramatically slower monolayer flow rates.
Langmuir Monolayer Flow across Hydrophobic Surfaces water, the substrates were placed in a radio frequency sputtering chamber with the cleaved edges normal to the metal source. Before being coated with the metal layers, the substrates were cleaned with a 50 W argon plasma for 30 s. To promote gold adhesion, a chromium underlayer of ca. 50 nm was coated on the substrates prior to the deposition of ca. 200 nm of gold. The goldcoated substrates were placed in SAM deposition solutions (∼20 mM dodecanethiol in ethanol) immediately upon removal from the sputtering chamber, and the SAM was allowed to form for at least 12 h before use. Just prior to monolayer flow measurements, a section of SAM-coated substrate was cut to give a rectangular glass block with the cleaved edge and the 2 opposing plate faces coated with gold and SAM and 2 uncoated edges of hydrophilic glass. Cut pieces were reimmersed in the appropriate deposition solution for at least 10 min prior to flow measurements. Monolayer flow substrates were positioned at the air/solution interface in a Teflon trough (36 cm2 surface area) with the smooth SAM-coated edge in contact but slightly raised above the solution surface. The substrate is pulled up from the solution to ensure that only the bottom face is wetted. The SAM-coated electrode serves as the working electrode in a three-electrode cell. A saturated calomel electrode (SCE) and platinum wire, located in a surface-isolated chamber of the trough, served as the reference and counter electrodes, respectively. The subphase electrolyte was 10 mM potassium chloride, pH 2 (HCl), to ensure the carboxylic headgroup of oleic acid was protonated. Subphase conditions were kept constant for piston oil comparisons. The temperature of the subphase was in the range 20-23 °C for all experiments reported here. The piston oils used herein exist in the liquid expanded phase at temperatures between 20-23 °C. The capacitance of the SAM-coated electrode was calculated from the ac admittance. The admittance magnitude and phase angle were measured using a lock-in amplifier (Stanford Research Systems, model SR530) under computer control at a frequency of 10 Hz with a 10 mV alternating current excitation. At the 10 Hz frequency, the admittance phase angle was between 86 and 90 deg, which is indicative of a predominantly capacitor-like circuit. The lock-in amplifier was connected to the cell using a potentiostat (EG&G PAR, model 360) that holds the system at 0.0 V versus the SCE reference. Piston oils were applied in excess of monolayer coverage by placing a drop of the neat piston oil on the surface a few centimeters away from the bilayer formation electrode using a small glass rod. The flow process begins a few seconds after deposition and occurs over several minutes until a complete bilayer is formed. Molecules that did not give a constant pressure within 10 s were not included in this study. The glass rod with reservoir of piston oil was left at the solution interface for the duration of the experiment. The surface pressure was measured by differential weight measurements using a filter paper (Whatman, No. 1) Wilhelmy plate suspended from an analytical balance (Denver Instruments, model 100A).
Results and Discussion Previous work demonstrated that monolayer flow is driven predominantly by the ESP of the Langmuir film and that compounds with similar structure flow at a rate proportional to the square root of the ESP.23 The previous work has also shown that SAM composition can have a substantial effect on the monolayer flow rate. In an attempt to minimize SAM effects, the SAM portion of the flow system was conserved for all experiments. For structurally similar compounds, the effective flow parameters at a common reference pressure are identical. However, when the flow rates of varied chemical species are compared, the effective flow parameter provides a measure of the differences in viscosity and interlayer coupling. Monolayer viscosity is highly dependent on the structure and molecular packing of the film.25 In general, the viscosity of liquid films, like the piston oils at the ESP used in Langmuir monolayer flow, increases with the length of acyl chain and decreases as the free volume in the film (25) Harkins, W. D. The Physical Chemistry of Surface Films; Reinhold: New York, 1952; pp 141-143.
Langmuir, Vol. 17, No. 25, 2001 7853 Table 1. Influence of Linking Oleyl Chains Together on Monolayer Flow Ratesa flowing film
chains
ESP (mN/m)
FP EFP(32.5) (µm s-1/2) (µm s-1/2)
oleyl trifluoracetate oleyl cyanide oleic acid oleyl alcohol oleyl oleate 1,2- diolein 1,3-diolein dioleyl phosphatidylcholine triolein
1 1 1 1 2 2 2 2
6.3 ( 0.9 16.8 ( 0.3 32.5 ( 0.5 36.4 ( 0.5 4.6 ( 0.5 29.4 ( 0.6 30.9 ( 0.3 47.5 ( 0.5
3
13.1 ( 0.3 28.6 ( 1.7 45.0 ( 2.7
28.5 ( 2.6 48.7 ( 2.0 63.1 ( 2.7 72.7 ( 1.8 20.4 ( 0.7 45.8 ( 2.2 55.1 ( 3.2 61.2 ( 1.6
64.7 ( 5.9 67.7 ( 2.8 63.1 ( 2.7 68.6 ( 1.7 54.2 ( 1.9 48.2 ( 2.3 56.5 ( 3.3 50.2 ( 1.3
a
The number of oleyl groups that are linked together in each flowing film is denoted in the table under the heading of Chains. The headgroup for each molecule can be found in the Experimental Section. The equilibrium spreading pressure (ESP), measured flow parameter (FP), and effective flow parameter at 32.5 mN/m (EFP(32.5)) are reported as the average of at least 3 independent determinations along with the corresponding standard deviation.
increases. Interlayer coupling, or friction, results from van der Waals interactions between the terminal groups of the two layers of a bilayer.8 In monolayer flow the two layers of the bilayer are the pseudocrystalline SAM monolayer on the solid electrode and the lipid chains of the Langmuir film. For fluid bilayers, interlayer coupling is considered small relative to the intralayer coupling; however, the interlayer coupling can be significant for supported bilayers as is the case in monolayer flow.15 The data in this report were generated using the same SAM, dodecanethiol, and comparisons are reserved for compounds within a series that should conserve the molecular interfaces between the layers. Four series of structural motifs in the flowing film are considered: linked oleyl chains; chains with a single cis double bond over a range of total chain lengths; chains of the same length with 1-5 cis double bonds; molecules which flow that do not have the standard cis double bond in the chain. Molecules with more than one acyl chain linked to a single molecular headgroup is a common structure in biology, for example, phospholipids. In the context of Langmuir monolayer flow, linking chains together should increase the interlayer coupling between the flowing monolayer and the SAM since each flowing molecule will have as many contact points with the SAM as there are linked chains. An increase in coupling should result in a slower flow rate. Table 1 includes the results from experiments with molecules that include 1, 2, or 3 linked oleyl chains. Four single oleyl chain molecules with differing headgroups are included as are four molecules with 2 oleyl groups, attached to 3 distinct headgroups, and one molecule with three oleyl groups. The dual oleyl headgroups include two doubly substituted triglyceride, phosphatidylcholine, and an ester. Triolein is a triply substituted triglyceride. The effective flow parameter for all molecules was calculated at 32.5mN/m, the ESP for oleic acid. The effective flow parameters for the single oleyl molecules range from 6.31 to 6.86 µm s-1/2 with a mean value of 6.60 µm s-1/2. The effective flow parameters for the double and triple oleyl species are less than the single chain. Molecules with two oleyl groups have a mean effective flow parameter of 5.23 µm s-1/2, a roughly 20% decrease. Triolein has an effective flow parameter of 4.5 µm s-1/2. A requirement to ascribe the decrease in effective flow parameters for these disparate chemical structures to interlayer coupling is that the molecules exist at similar chain densities at the ESP. Comparison of the pressurearea isotherms indicates that all of the molecules have
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Table 2. Influence of Single Cis-Unsaturated Fatty Acid Chain Length on Monolayer Flow Ratesa flowing film
carbons
ESP (mN/m)
FP (µm s-1/2)
EFP(32.5) (µm s-1/2)
myristoleic acid palmitoleic acid oleic acid vaccenic acid nonadecenoic acid eicosenoic acid docosenoic acid tetracosenoic acid
14(8/4) 16(8/6) 18(8/8) 18(10/6) 19(9/8) 20(10/8) 22(12/8) 24(14/8)
34.7 ( 0.5 33.5 ( 0.4 32.5 ( 0.5 33.4 ( 0.5 31.0 ( 0.3 29.7 ( 0.7 21.5 ( 0.4 18.3 ( 0.5
85.8 ( 7.0 77.1 ( 5.4 63.1 ( 2.7 73.4 ( 1.4 59.2 ( 3.3 71.5 ( 5.2 39.6 ( 1.9 15.5 ( 0.9
83.0 ( 6.7 75.8 ( 5.3 63.1 ( 2.7 72.4 ( 1.4 60.6 ( 3.4 74.8 ( 5.4 48.7 ( 2.4 20.7 ( 1.2
a The number of carbons in the fatty acid is denoted in the table under the heading of Carbons. The values in parentheses following the number of carbons denotes the number of carbons below and above the double bond. The equilibrium spreading pressure (ESP), measured flow parameter (FP), and effective flow parameter at 32.5 mN/m (EFP(32.5)) are reported as the average of at least 3 independent determinations along with the corresponding standard deviation.
very nearly the same area/oleyl chain, 38 A2/molecule, at their respective ESP.26 Therefore, it is reasonable to explain the observed slower flow rates for the Langmuir films with more contacts per molecule as the number of chains increase by an increase in coupling between the layers that inhibits the movement of the Langmuir film across the hydrophobic surface. Natural lipids predominately incorporate fatty acid chains from 14 to 24 carbons in length.1 The chain length determines the free volume within the hydrophobic portion of the lipid bilayer as well as determining the transition temperature, or melting point, when the headgroup is conserved. As the molecule is lengthened, an increase in the van der Waals interactions within the flowing film is expected along with a commensurate rise in the film viscosity. Investigations of the surface viscosity of long chain fatty acids by Harkins and Boyd revealed that the viscosity of the liquid monolayers rise rapidly with the length of the hydrocarbon tail.27 On the basis of the inverse relationship between monolayer viscosity and the flow parameter, higher melting temperature is anticipated to correlate with slower effective flow parameters. The ESP, flow parameter, and effective flow parameter for cisunsaturated fatty acid chains between 14 and 24 carbons are listed in Table 2. For each flowing film the total number of carbons is given along with the number of carbons below and above the cis double bond in parentheses. For example, oleic acid or cis-9-octadecenoic acid is represented as 18(8/8). Two trends are apparent as the chain length increases; both the ESP and the EFP(32.5) decrease. The tendency for the ESP to decrease with increasing chain length was described by Langmuir.10 Similarly, the decrease in EFP(32.5) can be attributed to the increase in van der Waals interactions between molecules as the chain length increases. There are two additional points of interest from the data in Table 2. First, oleic and vaccenic acids are of equal length and both contain a single cis double bond; however, the position of the point of unsaturation differs. The double bond in oleic acid is at the 9-position, and the double bond is 2 carbons closer to the tail to the 11-position in vaccenic acid. In general, introducing a cis double bond at the center of a hydrocarbon chain, as is the case for oleic acid, induces greater fluidity than positioning the double bond near the (26) Mingotaud, A.; Mingotaud, C.; Patterson, L. K. Handbook of Monolayers; Academic Press: San Diego, CA, 1993. (27) Harkins, W. D.; Boyd, G. E. J. Chem. Phys. 1939, 7, 203.
head or tail.28 Increased fluidity is expected to result in an increased monolayer flow rate. The observed EFP values for oleic (6.31) and vaccenic (7.24) acids are contrary to the anticipated results and suggest perhaps that an interlayer component for the shorter segment above the double bond in vaccenic acid gives rise to the faster flow rate. Langmuir film balance studies reveal no measurable difference between oleic and vaccenic acids, lending ancillary support to an interlayer coupling difference for the two molecules.29 Second, nonadecenoic acid has an odd number of carbons, 19, and is not found in biological systems.1 Melting studies of fatty acids show a pronounce odd/even effect where chains with an odd number of carbons tend to have a lower melting temperature then the proximal even numbered carbon chains.28 According to the monolayer flow model proposed herein and the correlation between lower melting temperature and lower viscosity, odd length chains would be expected to have higher monolayer flow rates. Again, in contrast the anticipated result, the EFP(32.5) for nonadecenoic acid is lower than that for the two 18mers and 11-eicosenoic acid. When the intralayer coupling arguments fail to correlate with the measured data, we postulate that a difference in interlayer coupling is determining the observed behavior. A more comprehensive sample of odd length fatty acids is needed prior to drawing any conclusions to an oddeven effect in monolayer flow. The extent of unsaturation in acyl chains of natural phospholipids varies according to the function of the membrane in which the lipids are embedded.4 For example, the most highly unsaturated lipids occur in the membranes of cells involved in information processing such as neurons and rod cells.5 The properties of lipids with varying extent of unsaturation have been studied intently to discern why particular membranes require these structures.2,28,30 Just as the number of cis double bonds in a lipid acyl chain controls bilayer properties, the extent of unsaturation is expected to result in changed monolayer flow behavior. The presence of cis double bonds in the molecule produce pronounced bends, or kinks, within the hydrocarbon tail. Kinks prevent the formation of closely packed, wellordered monolayers; hence, van der Waals interactions decrease between molecules and the viscosity of the Langmuir film decreases. According to the Langmuir monolayer flow model, both decreased relative viscosity and weaker van der Waals interaction between layers translate into increased effective flow parameter. Table 3 depicts the results of flow measurements using oleic acid and eicosenoic acid series with an increasing number of double bonds. Increasing the number of double bonds resulted in a significant increase in the flow rate for both series of molecules. In Figure 2, the EFP(32.5) for the 18 and 20 carbon fatty acids is plotted versus the number of cis double bonds. The EFP(32.5) increases for both series with each additional double bond producing an incrementally smaller increase (e.g. the difference between the EFP(32.5) values for the first and second molecules in the series is less than the difference the second and third, and so on). Similar incremental behavior has been observed in differential scanning calorimetry studies of phosphatidylcholines and phosphatidylethanoloamines.28 The addition of the fifth double bond to the 20 carbon fatty acid results in a dramatic decrease in the flow rate. (28) Huang, C.; Lin, H.; Li, S.; Wang, G. J. Biol. Chem. 1997, 272, 21917-21926. (29) Welles, H. L.; Zografi, G.; Scrimgeour, C. M.; Gunstone, F. D. In Monolayers; Goddard, E. D., Ed.; American Chemical Society: Washington, DC, 1975; pp 135-152. (30) Wang, G.; Li, S.; Lin, H.; Huang, C. Biophys. J. 1997, 73, 283292.
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Table 3. Influence of the Number of Cis Double Bonds within a Fatty Acid Chain on Monolayer Flow Ratesa flowing film
CIS
ESP (mN/m)
FP (µm s-1/2)
EFP(32.5) (µm s-1/2)
cis-9-octadecenoic acid (oleic acid) cis,cis-9,12-octadecadienoic acid all-cis-9,12,15-octadecatrienoic acid cis-11-eicosenoic acid cis,cis-11,14-eicosadienoic acid all-cis-8,11,14-eicosatrienoic acid all-cis-5,8,11,14-eicosatetraenoic acid all-cis-5,8,11,14,17-eicosapentaenoic acid
1 2 3 1 2 3 4 5
32.5 ( 0.5 30.6 ( 0.3 29.4 ( 0.3 29.7 ( 0.7 29.1 ( 0.4 28.0 ( 0.2 24.4 ( 0.2 31.2 ( 0.4
63.1 ( 2.7 77.1 ( 3.7 80.1 ( 1.9 71.5 ( 5.2 80.9 ( 5.7 86.3 ( 1.2 85.7 ( 2.2 60.5 ( 1.0
63.1 ( 2.7 79.5 ( 3.8 84.2 ( 2.0 74.8 ( 5.4 85.5 ( 6.0 93.0 ( 1.3 98.9 ( 2.5 61.7 ( 1.1
a The number of double bonds in the fatty acid is denoted in the table under the heading of CIS. The position of the double bonds is given under the name of the flowing film. The equilibrium spreading pressure (ESP), measured flow parameter (FP), and effective flow parameter at 32.5 mN/m (EFP(32.5)) are reported as the average of at least 3 independent determinations along with the corresponding standard deviation.
Figure 1. Electrode geometry for monitoring Langmuir monolayer flow. A SAM-modified gold electrode is positioned at the air-solution interface. A piston oil is introduced to the subphase and quickly spreads across the entire subphase surface. The Langmuir film then moves into the electrodesolution interface at a much slower rate. The electrode capacitance is monitored as a function of time (t) during the experiment, as depicted by the inverted view of the electrodesolution interface. Langmuir monolayer flow is complete when a bilayer is formed at the interface.
Interestingly, highly unsaturated lipids have very specialized roles in cellular membranes.1 Eicosapentaenoic acid is found in the photoreceptor membrane of the eye in greater proportion than elsewhere in the body. It is assumed that this highly unsaturated acyl chain provides a highly mobile environment for Rhodopsin, a visual pigment, in the retina.31 The 5 cis double bonds provide mobility by imparting free volume within the bilayer without sacrificing structural rigidity of the membrane.5 We propose that eicosapentaenoic acid displays similar rigidity as a monolayer at the air-water interface and during monolayer flow, possibly resulting in the observed smaller effective flow parameter. The result for the 5 cis double bonds could also be due to oxidation, resulting in a change of monolayer properties. Oxidation is a fundamental reaction in lipid chemistry, and the rate of oxidation is known to increase with degree of unsaturation.32 (31) Kunau, W. H. Angew. Chem., Int. Ed. Engl. 1976, 15, 61-74. (32) Gunstone, F. D. Topics in Lipid Chemistry; Logos: London, 1970.
Figure 2. Effective flow parameter at 32.5 mN/m for 18 and 20 carbon fatty acids plotted versus the number of cis double bonds in the molecule. The solid points are for the 20 carbon series, and the open points are for the 18 carbon series. The error bars represent the standard deviation of at least three determinations
An original motivation to study monolayer flow was the development of a monolayer chromatography where the flowing Langmuir film would act as a mobile phase.23 Sorption-mediated chromatographic separations are effected by a difference in partition coefficients between the mobile and stationary phases, where the partition coefficients are determined, in part, by the solubility of solutes in the solvent. Whereas conventional chromatographic methods benefit from the availability of a range of solvents, Langmuir monolayer chromatography would similarly benefit from a range of monolayer solvents with varying structural features to effect solute solubility. All of the examples of flowing Langmuir monolayers described above contained at least one cis-unsaturated double bond. Some extent of unsaturation generally indicates that a molecule will undergo monolayer flow as described herein; however, this structural feature is not a requirement. While no single structural feature is common to molecules that undergo monolayer flow, all molecules that have been
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Table 4. Molecules That Flow as Monolayers without Containing a Cis Double Bonda flowing film
ESP (mN/m)
FP (µm s-1/2)
EFP(32.5) (µm s-1/2)
oleic acid (9-cis) elaidic acid (9-trans) myristoleic acid (9-cis) ethyl myristate phytol
32.5 ( 0.5 22.5 ( 0.2 34.7 ( 0.5 18.8 ( 0.3 32.2 ( 0.6
63.1 ( 2.7 44.2 ( 1.2 85.8 ( 7.0 56.3 ( 3.7 59.1 ( 1.7
63.1 ( 2.7 53.1 ( 1.4 83.0 ( 6.7 74.0 ( 4.9 59.4 ( 1.7
a Oleic and myristoleic acid are included as reference values. The equilibrium spreading pressure (ESP), measured flow parameter (FP), and effective flow parameter at 32.5 mN/m (EFP(32.5)) are reported as the average of at least 3 independent determinations along with the corresponding standard deviation.
structural and empirical data, pressure-area isotherms and Langmuir flow, provide additional support for the assertion that increased relative intralayer coupling is a mechanism to decrease EFP(32.5). A second comparison from Table 4 is made for ethyl myristate and myristoleic acid. Ethyl myristate is the ethyl ester of the 14 carbon saturated fatty acid myristic acid and contains to point of unsaturation, whereas myristoleic acid has a cis double bond at the 9 position of the same length chain. The ester is a liquid and has an appreciable ESP at room temperature. Calculation of the compressibility from the pressure-area isotherm for ethyl myristate shows that the film exists in the liquid expanded phase state up to the collapse point at the ESP. The area/molecule at the ESP is roughly 35 A2/molecule, which is a similar chain density to that for myristoleic acid at its ESP (data not shown). Ethyl myristate yielded a significant EFP(32.5), albeit somewhat smaller than for myristoleic acid, even without containing a cis double bond. A final entry in Table 4 is made for phytol, a side chain component in chlorophyll and bacteriochlorophyll pigments that is found in bacterial membranes.1 Phytol is a 16 carbon hydrocarbon chain with a hydroxyl headgroup with a trans double bond at the 2 position and pendant methyl groups present every 4 carbons beginning with the 3 position. The pressure-area isotherm of phytol is consistent with a liquid expanded film at pressures near the ESP26 with a limiting area of roughly 35 A2/molecule. The ESP of phytol is quite close to oleic acid as is the EFP(32.5). Conclusion
Figure 3. Structural diversity of piston oils in Table 4.
observed to flow exist at a liquid expanded phase state at the ESP. Table 4 contains the ESP, flow parameter, and EFP(32.5) for select molecules that flow without containing a cis double bond. Line drawings of the chemical structures of the molecules in Table 4 are shown in Figure 3. The first comparison from Table 4 is made for the cis and trans isomers of 9-octadecenoic acid, oleic, and elaidic acids, respectively. The ESP of the trans isomer is significantly smaller than for the cis, and the EFP(32.5) is also smaller for elaidic acid. From a structural consideration, the trans double bond in elaidic acid permits greater van der Waals interactions between chains. Empirically, higher density chain packing at pressures up to and including the ESP was observed in the pressurearea isotherm of elaidic acid relative to oleic acid.29 The
A technique was described wherein bilayers were formed by the flow of a Langmuir film into the interface between a SAM-modified gold substrate and an aqueous subphase. Slower flow rates were observed for Langmuir films with increased numbers of linked oleyl chains due to the enhanced interlayer coupling between the flowing and stationary films. Slower flow rates were also observed with increased acyl chain length due to the increased intralayer coupling between the flowing molecules. More rapid flow rates were observed as the number of double bonds within the film-forming material was increased, due to both the decreased inter- and intralayer coupling. The dynamic formation of the bilayer provides useful insights into the interplay of forces within the structure. By control of molecular architecture and use of a pressure compensating factor, the relative predominance of intralayer and interlayer mechanical coupling factors was inferred for structurally similar bilayer assemblies. The mobility of molecules is hindered by increasing the contact between the layers and by increasing the contact between molecules within flowing monolayer. The observations made herein are consistent with bilayer formation properties within cellular structures. Acknowledgment. The State of Maryland and the National Science Foundation (Grant CHE-9417357) supported this research. LA0109056
