PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 6/07 p:/C|RC/DateDue.indd~p.1 .“ fi “'1 ‘1 If. n . “TV l" “"4 ABSTRACT GLYCERYL MONOOLEATE BILAYER MEMBRANES-- CHARACTERISTICS AND APPLICATIONS By Sechoing Lin The capacitance and thickness of bilayer membranes formed from dissolving glyceryl monooleate in various hydrocarbon solvents have been studied using the charge injection technique. The effects of different individual solvents, varying the mole fraction in binary solvents, and. lipid concentration on the capacitance and thickness of membranes have been-included in this study. The capacitance values obtained in single hydrocarbon solvents are in quite good agreement with values obtained by previous workers ' who used either ac or other dc techniques. The capacitance values are found to be independent of lipid concentrations for membranes in the squalene system. Both squalane and . paraffin oil have been developed into suitable solvents for the formation of membranes with little or no solvent. Binary solvents such as n-decane/n-hexadecane or n-decane/ squalene provide a way to conveniently synthesize membranes with a wide and continuous range of thicknesses. The effects of benzyl alcohol and cholesterol on the Sechoing Lin capacitances of two different membrane systems have been studied. The adsorption of benzyl alcohol into the bilayer causes a decrease in the membrane capacitance for n-hexadecane-containing membranes, while this trend is not observed for membranes with little or no solvent. The incorporation of cholesterol increases the capacitance for the n-decane-containing membranes and conversely, a decrease in the capacitance is observed for membranes with little or no solvent. An attempt to develop a bilayer membrane-based molecular sensor for polycyclic aromatic hydrocarbons is unsuccessful. The stability of bilayer membranes formed from dissolving glyceryl monooleate in n-alkanes or squalene has been found to increase drastically with the addition of a small amount of ferric chloride to the aqueous phases. In the presence of ferric chloride, the lifetime of this membrane formed on an aperture of 1.5 mm diameter is prolonged from a few minutes to more than 24 hours, and an accompanying substantial increase in the dielectric 'breakdown voltage of this membrane is observed. The effect of a recently synthesized compound, namely 1,1,2-tris-(1H-benzimidazole)ethane (TBIE) on the membrane conductance has been studied. The membrane conductance is found to increase with the addition of a small amount of TBIE to the aqueous phases. Further enhancement of the membrane conductance occurs with the addition of copper(II) chloride, but not with zinc(II), cobalt(II), and nickel(II) Sechoing Lin chlorides. A ferric(III)-stabilized bilayer membrane has been tested as a sensor. The change in resistance of this membrane caused by the selective transport of (Ph)4As+ is 'found to correlate with the concentration of (Ph)uAsCl in a linear fashion over a limited concentration range. This property is utilized in a membrane-based sensor to indicate the equivalence point in the titration of unknown mercury(II) with standard (Ph)uAsCl in 3 M NaCl solution. The results have been quite promising; an average accuracy of 5.5 % is obtained from five trials. GLYCERYL MONOOLEATE BILAYER MEMBRANES-- CHARACTERISTICS AND APPLICATIONS By Sechoing Lin A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Chemistry 1981 ACKNOWLEDGMENTS I would like to express my sincere thanks to Professor Christie Enke for his guidance and friendship during the course of my graduate study. His advice will always be highly valued. Thanks go also to Professor Weaver who served as my second reader, and to other members of my guidance committee. I wish to thank Mr. C. C. Lii for his friendship and help, and Mr. C. B. Wang for the sample donation. Finally, I wish to express my appreciation to my parents and family for their unfailing support throughout my education. TABLE OF CONTENTS CHAPTER Page LIST OF TABLES . . . . . . . . . . . . . . . . . . . . vii LIST OF FIGURES . . . . . . . . . . . . . . . . . . . ix I. INTRODUCTION . . . . . . . . . . . . . . . . . . . 1 Historical . . . . . . . . . . . . . . . . . . . . 1 Formation of Bilayer Lipid Membranes . . . . . . . 2 General . . . . . . . . . . . . . . . . . . . 2 Choice of Lipid . . . . . . . . . . . . . . . . 4 Choice of Solvent . . . . . . . . . . . . . . . 5 Mechanism for the Formation of Bilayer Lipid Membranes. . . . . . . . . . . . . . . . . . . . 6 Electrical Properties of Bilayer Lipid Membranes . 8 Unmodified Bilayer Lipid Membranes . . . . . . . 8 Modified Bilayer Lipid Membranes . . . . . . . . 10 A Preview of This Work . . . . . . . . . . . . . . 12 Measurements of Capacitance and Conductance of Bilayer Lipid Membranes . . . . . . . . . . . . . 13 General . . . . . . . . . . . . . . . . . . . . 13 Cell Assembly and Electrodes . . . . . . . . . 14 Membrane Conductance . . . . . . . . . . . . . 16 Membrane Capacitance . . . . . . . . . . . . . 17 Membrane Thickness . . . . . . . . . . . . . . . 20 Measurement of the Membrane Capacitance by A Computer-controlled Charge Injection Technique . . 21 Principles . . . . . . . . . . . . . . . . . . . 21 iii CHAPTER II. III. Instrument . . . . . . . . . . . . . . . EFFECT OF SOLVENTS AND LIPID CONCENTRATIONS ON THE CAPACITANCE AND THICKNESS OF GLYCERYL MONOOLEATE BILAYER MEMBRANES . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . Experimental . . . . . . . . . . . . . . . . . . . Materials . . . . . . . . . . . . . . . . . . Methods . . . . . . . . . . . . . . . . . . . . . Results and Discussion . . . . . . . . . . . . . . Capacitance and Thickness of Glyceryl Monooleate Bilayer Membranes in A Single Solvent System . . Effect of Lipid Concentration on the Capacitance and Thickness of Glyceryl Monooleate Bilayer Membranes . . . . . . . . . . . . . . . Development of Nearly Solvent-free Glyceryl Monooleate Bilayer Membranes by Conventional Mueller and Rudin Technique . . . . . . . . . Capacitance and Thickness of Glyceryl Monooleate Bilayer Membranes in Binary Solvent Systems. Future Work . . . . . . . . . . . . . . . . . . . APPLICATIONS OF MEMBRANE CAPACITANCE MEASUREMENT ON SOLVENT-CONTAINING (n-ALKANES) AND NEARLY SOLVENT- FREE (SQUALENE) GLYCERYL MONOOLEATE BILAYER MEMBRANE SYSTEMS . . . . . . . . . . . . . . . (A) Effect of Benzyl Alcohol on Both Solvent- containing (GMO/n-hexadecane) and Nearly Solvent- free Bilayer Lipid Membranes . iv Page 23 26 26 26 26 27 27 27 34 38 44 48 5O 5O CHAPTER IV. (B) Effect of Cholesterol on Both Solvent- containing and Nearly Solvent-free Bilayer Lipid Membranes . . . . . (C) Attempt to Use Bilayer Lipid Membranes as Molecular Sensors for the Detection of Potential Carcinogens--Polycyclic Aromatic Hydrocarbons . Introduction . . . . . Experimental Design . . Results and Discussion STABILIZATION OF GLYCERYL MEMBRANES IN THE PRESENCE Introduction . . . . . Experimental . . . . Materials . . . . . . Methods . . . . . . . Results and Discussion . THE EFFECT OF METAL(II) CONDUCTANCE IN THE PRESENCE OF 1,1,2-TRIS- (1H-BENZIMIDAZOLE)ETHANE Introduction . . . . . . Experimental . . . . . . Materials and Methods . MONOOLEATE BILAYER OF FERRIC IONS ON THE CHLORIDE MEMBRANE Preparation of 1,1,2-tris-(1H-benzimidazole)- ethane . . . . . . . Results and Discussion . Future Work . . . . . . Page 56 62 62 63 65 68 68 7O 7O 7O 71 77 77 78 .78 79 80 85 CHAPTER VI. AN ATTEMPT TO USE A BILAYER LIPID MEMBRANE AS A SENSOR AND APPLICATION TO THE DETERMINATION OF MERCURY(II) Introduction . Experimental . Reagents . . Apparatus . Procedure . Results and Discussion . Electrical Properties of the Determination of Mercury(II) Interference Problems Conclusion . Future Work . REFERENCES . . . vi Page 87 87 89 89 89 9o 91 91 93 93 97 98 mo LIST OF TABLES TABLE Page 2-1 Capacitance and Thickness of Bilayer Membranes Formed from Glyceryl Monooleate Dissolved in Various n-alkanes and Squalene . . . . . . . . . . 29 2-2 Estimates of Volume Fraction (VP) of Lipid and Various Solvents in Glyceryl Monooleate Bilayer Membranes . . . . . . . . . . . . . . . 31 2-3 Comparision of Capacitance Values for Virtually Solvent—free Glyceryl Monooleate Bilayer Membranes Formed Using Various Techniques . . . . 41 2-4 Capacitance Values of Glyceryl Monooleate (GMO) Bilayer Membranes at Various Lipid Concentrations in Paraffin Oil, Squalane, and Squalene Solvent Systems, Respectively . . . . . . 42 3-1 Effect of Cholesterol on the Capacitance and Thickness of Glyceryl Monooleate (GMO) Bilayer Membranes . . . . . . . . . . . . . . . 59 3—2 Capacitance and Thickness of Membranes which Various Polycyclic Aromatic Hydrocarbons are Incorporated . . . . . . . . . . . . . . . . . . . 66 4-1 Effect of 3.33 x 10-5 M Ferric Chloride on the Membrane Stability in 0.10 M Potassium Chloride Solution at pH = 4.28 . . . . . . . . . . . . . . 73 4-2 Effect of pH on both Membrane Resistance and Dielectric Breakdown Voltage in the Presence vii TABLE Page of 3.33 x 10-5 M Ferric Chloride in 0.10 M Potassium Chloride Solution . . . . . . . . . . . 73 Analytical Results; 30,ul of 0.1 M Mercury(II) Chloride in 30 ml of 3.0 M NaCl Solution Was Titrated With 0.05 M of Standard Tetraphenyl- arsonium Chloride Solution . . . . . . . . . . . 96 viii FIGURE 1-1 1-2 1-3 1-4 1-5 1-6 1-7(a) 1-7(b) 1-7(C) 1-7(d) 1-8 2-1 LIST OF FIGURES Page Schematic of A Physical Model of A Bilayer Lipid Membrane . . . . . . . . . . . . . . . 3 Schematic Diagram of The Thinning Process for Bilayer Lipid Membranes (Initial Thinning Process) . . . . . . . . . . . . . . . . . . . 7 Cell for the Electrochemical Study of Bilayer Lipid Membranes . . . . . . . . . . . . . . . 15 A Simplified, Equivalent Circuit of An Unmodified Bilayer Lipid Membrane . . . . . . . 18 A Simplified ac Bridge Circuit (By S. H. White) for the Measurement of Membrane Capacitance . . 18 A Schematic Circuit for the Measurement of the Measurement of the Membrane Capacitance by the dc Method . . . . . . . . . . . . . . . . ... . 19 Equivalent Circuit When An External Resistor Is Placed Across the Membrane Between Two ElectrOdes I I I I I I I I I I I I I I I I I I .22 A Short Constant Current Pulse . . . . . . . . .22 An Idealized Voltage Decay . . . . . . . . . . 22 A In V versus t Straight Line. . . . . . . . . 22 Schematic of Cell Amplifier . . . . . . . . . 24 Capacitance and Thickness of Bilayer Lipid Membranes Formed from Glyceryl Monooleate Dissolved in Various n-alkanes and Squalene . .32 ix FIGURE 2-2 2-3 2-4 2-5 2-6 3-1 3-2(a) 3-2(b) 3-3 5-1 Page Volume Fraction of the Alkane Solvent and Squalene Which Are Present in the Bilayer Region . . . . . . . . . . . . . . . . . . . . 33 A Picture of the Molecule Squalene . . . . . . 35 Capacitance of Bilayer Lipid Membranes Formed from Appropriate Amounts of Glyceryl Monooleate Dispersed in n-decane, n-hexadecane, and Squalene, Respectively, As A Function of Glyceryl Monooleate Concentration.36 Capacitances of Bilayer Lipid Membranes Formed from Appropriate Amounts of Glyceryl Monooleate Dispersed in Squalene, Squalane, and Paraffin Oil, As A Function of Glyceryl Monooleate Concentration . . . . . . . . . . . . . . . . . 43 Capacitances of Bilayer Lipid Membranes Formed from Dispersing 100 mg Glyceryl Monooleate in 10 ml of Mixture Solvents . . . . . . . . . . . 46 Membrane Capacitance As A Function of Benzyl Alcohol Concentration . . . . . . . . . . . . . 52 Structure of Cholesterol . . . . . . . . . . . 57 Side View of Structure of Cholesterol . . . . . 57 Structure and Chemical Names of Polycyclic Aromatic Hydrocarbons . . . . . . . . . . ... .64 Structure of 1,1,2-Tris-(1H-benzimidazole)ethane (TBIE) . . . . . . . . . . . . . . . . . . . . 78 FIGURE ‘ Page 5-2 The Control Experiment Shows That TBIE Alone Increases the Membrane Conductance . . . . . . . 81 5-3 The Control Experiment. The Addition of Metal(II) Chloride Does Not Show Any Significant Change in the Membrane Conductance . . . . . . . . . . 82 5-4 The Effect of Various Metal(II) Ions on the Membrane Conductance in the Presence of L), 4 x 10' M of TBIE in the Aqueous Solution . . . 83 5-5 The Membrane Conductance Is Enhanced in the Presence of 1.15 x 10-5 M of Copper(II) . . . . .84 6-1 A Typical Current Versus Applied Voltage Curve . 92 6-2 A Typical Membrane Resistance Versus Time Curve . . . . . . . . . . . . . . . . . . . . . .92 6-3 A Typical Current Versus Volume of Titrant, (Ph)4AsCl, Curve . . . . . . . . . . . . . . . . 94 6-4 A Typical Titration Curve . . . . . . . . . . . .95 xi CHAPTER I INTRODUCTION Historical In 1899, Overton (1) first observed that cell membranes were very permeable to lipids and thus suggested that cell membranes contained lipids. Langmuir (2), in 1917, emphasized that lipids tended to form structured monolayers when placed in contact with water and measured the surface pressure exerted by this monolayer film on water surface. Gorter and Grendel (3), in 1925, found that lipids extracted from red cell membranes would spread on water to a thickness just about half that of the membrane itself. Assuming in accord with Langmuir that the lipid layer was one molecule thick, they concluded that the membrane was essentially a double layer of lipid molecules. Davson and Danielli (4) in 1943, on the basis of data on the low surface tensions of membranes, advanced the model to include the proteins for the outer membranes of animal cells. The membrane is depicted as a lamellar bimolecular lipid layer with the polar head groups of the lipids oriented toward the high dielectric aqueous medium which is both inside and outside the cell. This configuration allows the hydrocarbon chains of the lipids to be in their own low dielectric environment. Proteins present in the membrane are believed to be adsorbed on the surface of the lipid bilayer. This model was widely 2 accepted by biologists at the time. The first in vitro preparation of a bilayer lipid membrane was successfully performed by Mueller et al. (5) in 1962 by brushing a complex mixture of brain lipids, n-tetradecane, silicone fluid and mineral oil across a small aperture on a Teflon sheet between two aqueous compartments. Rapid progress has been made since then. Techniques of bilayer lipid membrane formation have been improved and synthetic amphiphiles have been found that provide systems more amenable to fundamental physicochemical investigation. A schematic configuration of this model membrane with a bilayer structure on the center and a relatively thick torus region surrounding it is shown in Figure 1-1. This bilayer lipid membrane is seen to be akin to the classical model of Gorter-Grendel, and is similar to the so-called smectic soap mesophase (6). In this liquid crystalline structure, the polar groups of the lipids are thought to orient in a plane which is perpendicular to the membrane. Formation of Bilayer Lipid Membranes General Bilayer lipid membranes are formed from solutions of one or more lipids in nonpolar solvents. However, the number of suitable lipids and nonpolar solvents is quite limited. The use of water-soluble solvents such as chloroform and ethanol is not precluded provided a significant portion of A Teflon support Q : I/A. oo/O/{/o [A1100 torus ._o ‘ o— 0—-—o bilayer r0 O—--—o 0——o 0—-—<) o——o o——o CF—-—o 0— Figure 1-1. Schematic of a physical model of a bilayer lipid membrane. u a water-insoluble solvent is also present. The main disadvantage of this kind of mixed solvent system is that one or more of the water-soluble solvents may dissolve out of the membrane, thus a true equilibrium structure of the membrane can not be obtained. As a consequence, the prOperties of the membrane such as composition, thickness, and tension may be time-dependent. Lipids should be strongly surface active, because the rupture of the membrane under the pressure of the London-van der waals and other thinning forces is prevented by a lipid component which must not be readily desorbed. Clearly, the more powerful the affinity of the lipid for the interface, the more effective it is likely to be as a stabilizer. Choice of Lipid The lipids known to form stable membranes include many of the naturally occurring phospholipids and some monoglycerides (7). Among those lipids suitable for bilayer lipid membrane studies, the nature and stability of the resulting membranes vary enormously. Therefore, the choice of lipid is strongly dictated by the kinds of studies being investigated. There are significant differences between the behavior of phospholipids and monoglycerides in bilayer lipid membrane systems and these differences are listed below. Characteristics of phospholipids} 1. Phospholipids do not usually dissolve molecularly in hydrocarbons but rather exist as large aggregates which 5 may nevertheless remain dispersed for long periods. The adsorption process is more complicated and takes time. Monolayers are "insoluble" and are in equilibrium only with the monolayers on the adjacent bulk phase interfaces. Phospholipids have strong affinity. Unless the lipid and hydrocarbon solvent are carefully dried, the black films obtained are liable either to be abnormally thick or to contain abnormally thick patches, and to exhibit a curious "Spider's web” appearance. Characteristics of monoglycerides: 1. 3. Monoglycerides yield molecular solutions in hydrocarbons and form micelles in the millimolar region in a manner similar to that observed for many water-soluble surfactants. At several millimoles per liter, the maximum adsorption is attained in a matter of seconds or less. Monolayers are always in equilibrium with bulk lipid solution. Careful examination of the characteristics for these two classes of lipids indicates that monoglycerides possess certain advantages over phospholipids and these advantages can become especially important in determining the composition of the membrane and in studying certain physicochemical phenomena using this lipid system. Choice of Solvent The requirement for selecting a solvent for a bilayer 6 lipid membrane is that the solvent itself should be both nonpolar and nonvolatile. Appreciable water-solubility and volatility will lead to membranes that are not at equilibrium and are therefore poorly defined. If a single solvent is to be used, the most satisfactory are the alkanes from n-octane to n-hexadecane. The most remarkable differences within this series of alkanes is the membrane thickness change attributed to a variation in the adsorption of the alkanes into the membrane which depends upon the chain length. As the solvent chain length increases, the increased exclusion of the solvent leads to a thinner membrane. In the case of glyceryl monooleate, as the solvent is varied from n-decane to n-hexadecane, the thickness of the membrane decreases by about 16 A. Mechanism for The Formationo; Bilayer Lipid Membranes A dynamic mechanism has been advanced by Snyder et al. (8) to explain the formation of bilayer lipid membranes. The mechanism is viewed as a two-step process; (a) initial thinning, and (b) surface spreading. In the first step, as shown in Figure 1—2, material flows from the center of the membrane (bilayer) to the border area (torus) near the edge of the supporting aperture. As with the flow of fluids in channels, a pressure gradient is required to maintain the thinning process. The pressure difference can be traced to the curvatures of the thinning film and the border area. The variations in surface curvature can produce bulk flow within the membrane and cause it to thin. no curvature, [AP = 0 \. Q no thinning Apio ////, % % flow along arrows Figure 1-2. Schematic diagram of the thinning process for bilayer lipid membranes (initial thinning process). 8 The rate of thinning is controlled by the bulk viscosity of the membrane solution. As thinning proceeds, the thickness of the membrane is reduced until it reaches the molecular dimensions where van der Waals forces can take over and produce local areas of bimolecular layers. Once the spots of local bimolecular layers occur, the formation mechanism changes from a simple hydrodynamic flow process to a surface Spreading phenomenon. The major driving force, at this time, is the change in bifacial tension across the torus/bilayer border. The force resulting from the pressure gradient has a negligible influence on the surface spreading process. The rate of spreading is mainly determined by "surface viscosity" if other factors are fixed. Electrical Properties of Bilayer Lipid Membranes Unmodified Bilayer Lipid Membranes A bilayer lipid membrane so formed has a very high resistance for small inorganic ions. This is attributed , to the large difference in the dielectric constants between both media-- aqueous solution (ca. e = 80) and hydrocarbon interior of the membrane (ca. e = 2.1). In order for ions to cross the relatively low dielectric hydrocarbon interior of a membrane, a great amount of energy is required (9), and this leads to the observed high resistance. If one tries purposely to lower the .‘ll.[.llll’ liir‘ l' .II: All I] {I I (,1 I 9 dielectric constant of hydrocarbon interior of a membrane by using a more polar solvent which is supposedly present in the bilayer, a decreased resistance is expected. This is indeed the case as observed by Dilger et al. (10) who employed 1-chlorodecane as solvent for a membrane-forming solution. Moreover, as mentioned previously, the composition of a bilayer lipid membrane can be varied via the changes of either lipid or solvent or both. For example, a bilayer membrane formed from an oxidized cholesterol/n-decane solution has a resistance in the order 8 2 of 10 ohm-cm , while the resistance of a bilayer membrane formed from glyceryl monooleate/n-decane solutions is in the order of 106 ohm-cmz. The two-order of magnitude greater resistance of the membrane formed from an oxidized cholesterol/n-decane solution is apparently due to the stronger hydrophobic interaction between the nonpolar portion of oxidized cholesterol in the membrane interior. 0n the one hand, the intrinsically high resistance of a bilayer lipid membrane makes it very dissimilar to biological membranes although the two systems are analogous in many other aspects (11,12). On the other hand, the high resistance of a bilayer lipid membrane facilitates the study of the effects of doping with various modifiers. The addition of certain modifiers can drastically change the resistance of a bilayer lipid membrane. This has stimulated many studies of biologically associated phenomena. 10 Modified Bilayer Lipid Membranes The intrinsic properties of a bilayer lipid membrane can be modified by introducing modifiers into the membrane system. These modifiers can be either ions or molecules. According to the mechanism by which the modifiers affect the resistance of a bilayer lipid membrane, they can be grouped into five categories; (a) those which facilitate direct transport, (b) those which act as ion carriers, (c) those which form ion-conducting channels, (d) those which vary the internal energy barriers of the membrane interior, and (e) those which interact through a receptor mechanism. Lip0philic ions (or fat-soluble ions) such as tetra- phenylborate, tetraphenylarsonium, tetraphenylphosphonium, etc. belong to category (a). The delocalization of charge among the phenyl groups makes these ions very fat-soluble. When these ions are added to the aqueous solution, they diffuse across the membrane by the direct transport mechanism. In the presence of tetraphenylborate anions, the membrane resistance decreases about three orders of magnitude even at a concentration of only 10'7 M (13) in the aqueous phase. In category (b), a representative example is valinomycin - a cyclic compound which possesses polar carbonyl groups around its center and a nonpolar portion as well. When residing at a membrane-aqueous interface, the polar groups orient toward the aqueous phase and are responsible for the selective formation of complexes with ions. Once present inside the membrane 11 interior, the portion of ion and polar groups are surrounded by the nonpolar portion of valinomycin molecule via a conformation change so that the whole complex is very hydrophobic. The ion transport across the membrane is through the ion-molecule complex acting as a recycling carrier. This process produces a drop in the membrane resistance (13,14). Molecules which fall into category (c) are polyene antibiotics such as nystatin and amphotericin B, or polypeptides such as gramicidin. Once added to the membrane, these molecules form polar channels through which small ions can travel. The decrease in the membrane resistance by way of the channel formation mechanism is generally greater than that from either of the other two mechanisms mentioned above (13,14). Molecules classified in category (d) are small, polar organic compounds such as phloretin (15) and pentachlorophenol (16,17). These molecules are adsorbed at the membrane-aqueous interface. This adsorption, in turn, varies the ion transport energy barrier of the membrane interior through the effect of the dipolar property of the adsorbed molecule. Finally, in category (e), a typical example of antigen-antibody is human serum albumin diazotized with sulfanilic acid as antigen and corresponding immune serum as antibody (18). Antigen is usually reconstituted into the membrane and the addition of antibody would make an antigen-antibody interaction take place. This mode of interaction is very specific and the resulting change in membrane resistance is 12 transient. A Preview of This Work There is no doubt that knowledge about biomembranes has been further advanced as a result of extensively using bilayer lipid membranes for studies of biologically relevant phenomena. Moreover, the accumulation of this knowledge may suggest many applications in nonbiologically related areas. To mention a few examples: (a) The bilayer lipid membrane itself is a new type of interfacial adsorption phenomena. Therefore, it can be a useful tool for the understanding of physics and chemistry of amphipatic compounds and will be relevant to the further development of interfacial chemistry and colloid science; (b) A bilayer lipid membrane can be employed as a membrane matrix and the relatively selective, sensitive interaction of compounds with or on this membrane matrix often causes the change of electrical property of the membrane. Taking advantage of this approach, bilayer lipid membranes may be developed as sensitive sensors and expected to find some applications in the bioanalytical area. This study makes of monoglycerides and emphasizes the more nonbiological applications. It begins with the characterization of the glyceryl monooleate bilayer membrane system in various solvents and lipid concentrations with the goal to better characterize this membrane system. A study of stabilizing effect of this membrane system in 13 the presence of ferric chloride follows. The conductance of this membrane system to Cu(II), Co(II), Ni(II), and Zn(II) ions is studied when a new compound, 1,1,2-tris- (1H-benzimidazole)ethane, is present in the aqueous phase. Finally, this membrane system which is stabilized by ferric chloride is applied as a sensor in the determination of Hg(II). Measurements 9f Capacitance and Conductance g; Bilayer ! Lipid Membranes General The characterization of the properties of unmodified bilayer lipid membranes is most often performed using methods of electrical measurement. Moreover, electrical measurements can provide information about the changes resulting from the interactions between the bilayer lipid membrane and its modifiers. Both dc and ac techniques have been employed for the measurement of various electrical parameters. The typical arrangement of a cell for studies of electrical properties is generally represented below: Nonpolarizable Aq. Soln. BLM on Aq. Soln. Nonpolarizable Electrode I Teflon II Electrode Support 14 Cell Assembly_and Electrodes An electrochemical cell is usually set up as shown in Figure 1-3. The cell consists of two rectangular Teflon chambers. Each chamber has a volume of approximately 45 ml and is separated by a thin Teflon sheet with a thickness of 0.25 mm. A circular aperture with a diameter between 1.0 to 2.0 mm is punched in the Teflon sheet. The Teflon sheet is made dismountable to allow for the alteration of the . aperture size by the replacement of the Teflon sheet. Both chambers have 9.5 mm holes in the center of the faces which contact the Teflon sheet. These holes are surrounded on the outside face by Viton "0" rings (2.54 cm O.D.) inset in the Teflon with 0.12 mm protrusion. When the cell is assembled, the Teflon sheet is sandwiched by the "0" rings between the two cell chambers which are in turn held together by clamps. 0n the face of each chamber opposite the side sandwiching the Teflon sheet there is a 2.54 cm hole. A circular glass window with 2.54 cm diameter is pressed fit into each chamber. This permits the membrane formation to be viewed with a micrOSCOpe through the front window when illuminated through the rear window. The electrodes used for the electrical measurements of membranes should be nonpolarizable to ensure that the voltage measured (or applied) mainly occurs across the membrane. The two types of electrodes most commonly employed are silver-silver chloride and calomel. 15 voltage current source meter 4m @4— electrodes _I/ \r/Jg‘é‘iii‘iin / L .\\\ x \_ :3 C\\\\ ‘ x" . ...— w.\\:\\ \* I I :\ glass : . window I a | Te fl on ;\ housing a? /// membrane Teflon "0" rings aperture sheet Figure 1-3. Cell for the electrochemical study of bilayer lipid membranes. 16 Membrane Conductance Membrane conductance is usually measured by impressing a small voltage across a bilayer lipid membrane via a pair of nonpolarizable electrodes. Under a low applied voltage (about 50 mV), Ohm's law is generally applicable. Therefore, the membrane conductance can be evaluated from the known applied voltage, V in volts, and the current, I measured in amperes. In equation form, this is expressed as Conductance = 1/R = I/V Experimentally, a simple set up, as shown in Figure 1-3, is most often used to attain an accurate, fast determination of the current at a known value of the potential across the membrane. The solution and electrode resistance (approximately 104 ohms with an aperture in place) is usually negligible with respect to the membrane resistance which is in the 8 range of 106-10 ohm-cmz. This means that essentially the entire voltage applied to the pair of nonpolarizable electrodes appears across the membrane. Because the area of a bilayer lipid membrane is often small (about 0.01 cmz), even with a membrane resistance of louohm-cm2 the error is approximately 1 % (assuming a value of 10“ ohms for the resistance of both solution and electrodes). In some cases, the membrane resistance is much greater than 108 ohm-cm2 (such as membranes formed from oxidized cholesterol/n-decane solution). The current through these membranes is extremely 17 small which requires the use of an extremely sensitive current meter. Generally Speaking, the current through any of the membranes studied under the condition when the Ohm's law is applicable is very small. Thus an electronic picoammeter designed around an electrometer amplifier with a very high input impedance is generally required for the current readout. Membrane Capacitance An unmodified bilayer lipid membrane can be regarded as a three-layered structure with a hydrocarbon portion in the center and two polar layers adjacent to it on both sides. The analysis of its capacitive behavior can therefore be carried out in terms of a model of three capacitors in series. Because of the relatively large dielectric constant and the small thickness of the polar regions (19-23), it is true in most cases that the central hydrocarbon region is the only factor which contributes significantly to the membrane capacitance (20). A simplified, equivalent circuit for an unmodified membrane as shown ianigure 1-4 is thus useful. A simplified schematic of the ac bridge designed by White et al. (24) for the measurement of the membrane capacitance is given in Figure 1-5. At balance, the membrane capacitance can be evaluated using the expression. Cm = (Rl/RZ) ck 18 l l O..— m Figure 1-4. A simplified, equivalent circuit of an unmodified bilayer lipid membrane. null detector voltage across unknown Figure 1-5. A sim lified ac bridge circuit (by S. H. White for the measurement of membrane capacitance. 19 Where R1 = 1 krl, R2= 100 kIZ, and Ck is the balance value. Also at balance, terminal 1 is at a virtual ground so that the amplifier at terminal 2 measures the ac signal appearing across the membrane. The bridge is excited via a photocoupled isolator which introduces minimal stray impedances and acts as a nearly ideal transformer. This ac bridge for the measurement of the membrane capacitance has been reported capable of obtaining a value of the membrane capacitance with a nominal accuracy of 0.05 % between the frequencies of 100 Hz and 10 KHz (24). The required voltage across the membrane is only 7 mV (rms). A dc transient technique used by Montal et al. (25) for the measurement of the membrane capacitance is shown in Figure 1-6. output —-—' membran - g V A Q? Figure 1—6. A schematic circuit for the measurement of the membrane capacitance by the dc transient method. 20 A potential step is applied across the series combination of the membrane and a resistor, and the voltage across the latter (proportional to the current) is monitored by means of an oscilloscope. Thus cm = q/V =%EI dt Where q is the charge which is stored in the membrane capacitor under the applied potential, V. The integral 8:1 dt is obtained by the graphical estimation of the area under the value of the current versus time. Although easy and relatively cheap to set up, the dc transient method tends to be less accurate than the ac method. Thus unless signal averaging techniques are employed, the integration procedure, or the evaluation of the relaxation time of the current transient, introduces uncertainties which can be greater than 1 %. Membrane Thickness The thickness of the membrane is an important piece of information in membrane studies. The measurement of the membrane capacitance permits the calculation of the membrane thickness according to the equation given below. Cm = Ct/Am = eoem/dm Where Cm is the specific membrane capacitance, Ct is the membrane capacitance measured, eO is 8.854 x 10-1)+ F/cm, em is the membrane dielectric constant, dm is the membrane thickness, and Am is the area of the bilayer portion of the membrane. A measurement of the membrane area within 21 1 % accuracy can be obtained if the circular membrane diameter is greater than 1 mm. The membrane dielectric constant can be approximated without a significant error by using that of an appropriate hydrocarbon, which is about 2.1 (26). Measurement of the Membrane Capacitance by a Computer- controlled Charge Injection Technique Principles An unmodified bilayer membrane has a resistance value in the range of 106—108 ohm-cmz. Therefore, charge injected onto the membrane capacitance will leak off very slowly if the conductance through the membrane is the only discharge pathway. This property makes it possible to measure the membrane capacitance by placing an external precision resistor between the electrodes, charging the membrane, and determining the time constant of the voltage decay through the known external resistance. When an external resistor is placed between the electrodes, an equivalent circuit can be depicted as shown in Figure 1-7(a). A charge pulse (see Figure 1-7(b)) is injected into the membrane through both the electrode and solution resistances (Rf + Rs) in a very short time, tp (usually 50-100 ns). The resulting voltage decay (see Figure 1-7(c)) is followed. The data is then linearized to acquire the slope which is equal to -1/RCm (see Figure 1-7(d)). Since the discharge pathway is through 22 Q IN Figure 1-7(a). Equivalent circuit when an external resistor is placed across the membrane between two electrodes. 100 ns 11 O t p —b time Figure 1-7(b). A short constant current pulse. v1 0 Qtotal=ltp' —9 time Figure 1-7(c). An idealized voltage decay. an a—-slope = -1/RCm 0 time Figure 1-7(d). A an versus t straight line. If (Rf+R )<< s Rext’ R = 2(Rf+R ) + R A- ~ Re XI: and the membrane capicitanEétcan be obtained from the lepe. 23 Rf + RS + R in order for the simplification to be made (i.e. R = R (Rf + Rs) must be small relative to Re ext’ xt ext if Rext§>(Rf + RS)). This can be accomplished through the use of high concentration of salt solution and large area electrodes. Instrument A commercial pulse generator (Chronetics PG-33 model) was used for this study. This pulse generator has an output voltage compliance of 12 volts, and a rise time of 6 ns maximum. The device is capable of driving 200 mA into 50 ohms, and can be triggered externally. A schematic of the cell amplifier is shown in Figure 1-8. The amplifier is placed as close to the electrodes as possible in order to minimize capacitance and stray signal pickup in the connecting wires. The input signal comes from the pulse generator via a coaxial cable. The charge pulse travels through the solution and charges the membrane which is situated between the two electrodes. The voltage transient generated is amplified by the LH0032 FET input amplifier to obtain a voltage excursion in the range of 0-4 volts at the output. The diodes (IN 914 and FD300 in series) prevent the voltage at the input of the amplifier from exceeding approximately 0.7 volts which in turn, avoids the saturation of the amplifier during the pulse application. The 10 kILresistor was included to ensure that the major current pathway for the charge pulse remains the membrane pathway. The diodes used for input protection 24 .m: CH ohm mmocmpwommmo .mmflhom CH oommm e mammcH xm .3 .meaeem comma a aHmzH xm .m .meoomq .m .mmoomq .H .umflmfiamsm Hamo wo oapmemnom .mIH mhzmflm M OH 930 :L a: M m.w M H 25 as well as reverse blocking were chosen in combination to yield fast switching characteristics as well as low leakage when in the "OFF" state. The output signal is buffered by an LH0036 cable driver (terminated with 50 ohms) to the transient recorder. A fast transient recorder (27), which employs a temporary analog storage register serves for the recording of a very fast transient signal. The analog signal is then converted into digital form and stored in the memory of a PDP 11/40 minicomputer. The transient recorder is capable of operating at a 10 MHz sampling rate. The program handling the data collection, baseline subtraction, and data storage in the floppy diskette called "MEMBRN.FTN" was developed by Last (28) from this laboratory. The corrected data was then fit into another program called ”EXPFIT.FTN" to obtain the membrane capacitance. The program, "EXPFIT.FTN", essentially linearizes the experimental data, calculates the prOper weighing coefficients, and then calls a weighted linear least squares fitting subroutines. 26 CHAPTER II EFFECT OF SOLVENTS AND LIPID CONCENTRATIONS ON THE CAPACITANCE AND THICKNESS OF GLYCERYL MONOOLEATE BILAYER MEMBRANES Introduction Glyceryl monooleate yields well defined systems which are in many ways easier to study quantitatively than phospholipids (7,29). Membrane capacitance is an important electrical property and its measurement provides us with information about the thickness, and indirectly, about the composition and structure of bilayer lipid membranes. Studies of the thickness of glyceryl monooleate bilayer membranes by means of capacitance measurement constitute part of this work. Experimental Materials Unless otherwise stated, the membrane-forming solution was made by dispersing the proper amount of glyceryl monooleate (K & K) into appropriate solvents. N-decane, neoctane and n-hexadecane (Aldrich), n-dodecane, n-tetradecane and squalene (Sigma), Squalane (Matheson, Coleman & Bell), paraffin oil (J. T. Baker), and benzyl alcohol (Fisher Scientific) were used without further purification. Cholesterol (Fisher Scientific) was recrystallized twice from absolute alcohol. The aqueous solution was 3 M 27 lithium chloride which was of analytical grade. The water used to prepare the aqueous solutions was deionized. Methods The membrane was supported by a 1.5 mm diameter hole drilled in a Teflon partition which was clamped between two symmetric Teflon chambers containing the aqueous solutions. The membrane was formed across the aperture by forming a bubble of lipid at the end of a Pasteur pipette under the surface of the aqueous solution and applying the bubble to the aperture. The membrane size was determined using a microscope reticule. The data were taken 10 minutes after the completion of thinning. The membrane capacitance was measured using a charge injection technique through a pair of nonpolarizable silver/silver chloride electrodes. This technique has been employed for study of electrochemical systems in the measurement of electrical double layer capacitance on the electrode (30) as described in the "INTRODUCTION" chapter. All measurements were performed at temperature 24.4: 1 OC. Results and Discussion Capacitance and Thickness of Glyceryl Monoolgate Bilayer Membranes in a Single Solvent System Glyceryl monooleate, when dissolved in n-alkanes, forms membranes although glyceryl monooleate itself does not form bilayer membranes in water. The extent of the adsorption 28 of the alkane in the membrane interior depends on the chain length of the alkanes. Since the area per molecule of the lipids is not appreciably affected (31,32), the adsorption of n—alkane would mainly affect the thickness of the hydrocarbon region of the bilayer. Therefore, the thickness of a bilayer membrane reflects the volume of solvent within a bilayer membrane. The thickness of a bilayer membrane can usually be estimated by the measurement of the bilayer specific capacitance using the equation, d = eoem/Cm’ where eO is 8.85 x 10-1“ F/cm, em is estimated to be 2.1 (the dielectric constant of lipid acyl chain equivalent to hydrocarbon (26)), and Cm is measured experimentally. Both the capacitance and thickness of bilayer membranes formed from glyceryl monooleate dissolved in various n-alkanes have been systematically studied by Fettiplace et al. (7) and White (33). For the purpose of comparison, their results along with ours are given in Table 2-1. As noted, our results are generally quite in good agreement with those obtained by previous workers. Furthermore, the volume fraction of lipid in a bilayer membrane can be estimated using the equation, volume fraction (VF) = ZVa/dAa’ where Va is the partial molar volume of the acyl chain in the bilayer, d is the thickness of the bilayer membrane, and Aa is the area of each lipid molecule at the interface. The density of the components in the bilayer may similarly be interpolated from those for the bulk hydrocarbon. The 29 COszHom mcmme Ho HE Hon mammHoocoE thmosz we 0H M So%% COHPSHom mcmme mo HE mom opdeoocoe HanoohHm we m.m m Emu am are use 83 2 m E GGEHO.H Thaw; WOCMMDE®E*** .00 on ad psm Homz S H. o CH COHpsHom mcmme mo HE Hog mpdeoocoE HammohHm we 0H m 809% meHom 3pm: moCMHpEmS** Hm pm cam Homz 2 H. o 2H m poshow who: memHQEmz* (1 0 HI m.mm m flown N.oI..nm.mm m I...” mum. II IIAoo 3m pmv mCmHmSUm II II m.oano.mm a HnHao II II mcmooemBOOI: To H «Hm m Hmam n.on.am a H mam m.o._lIo.mm a H can acmememxeeé So H Tam m His a.oHo.aa m H mm: adHoé: a H m6: $838372 :6 H 1:3 e H S: Tonéa a H man ..I II 3888.: To H T? 6 H mo: adHnéa m H mmm a.on.wa m H mmm mcmemeé 3.0 HI. m.m: m H no: ddHImpa m H 0mm II II ocmpoOI: as H as: a Hi: I- I. THE? m H amm 9832-: m .p mSo\mC .8 m .p Eo\m: .e m .p Eo\m: .E mHCm>Hom xhoz mH£B*** Ann .mmv eeHa3I Ammv eemHaHeeome .mCmHMSGm paw mmcmmeI: mSOHHm> CH pm>Homme opMmHoocos Hammosz 809M poEHoy mmcmnnsos pmzmHHQ Ho mmwconnv pcm mocmpHomQMU .HIN manme 30 main justification is that, in micelles of alkyl chain surfactants, the hydrocarbon interior has effectively the same density as in the bulk (34). Therefore, were 1-heptadecene assumed to be equivalent to the acyl chain of glyceryl monOoleate, the partial molar volume of lipid acyl chain could then be evaluated from the equation, Va = M/j’N, to attain a value of 475 x 10-24 cm3 (31,32); where M is the molecular weight of 1-heptadecene, f is the density of 1-heptadecene in the bulk, and N is the Avogadro's number. The area of lipid molecule at the interface, Aa, has been estimated using the adsorption isotherm technique to obtain values between ca. 37.5 and 39.5 AZ in various alkanes (31,32). More recently, White (36) obtained a value of 38.3 82 for bilayer membranes formed from glyceryl monooleate dissolved in squalene. All these values are considered to be quite close; we have chosen to use 38.5 Ag in our calculations for the volume fractions. Both the volume fraction of lipid and solvent are given in Table 2-2. The values for the volume fraction (VF) of the solvent is obtained by 1 - VF of lipid. A zero volume of mixing of solvent and lipid chains is also assumed, but this is unlikely to introduce a significant error (7). Figures 2-1 & 2-2 again indicate that both the thickness of the bilayer and the volume of solvent in the bilayer drastically decrease as the number of carbon atoms for the solvent increases. Therefore, it should be expected that a bilayer membrane with little or no solvent can be formed by the conventional Mueller-Rudin 31 msomsvm S m CH QOHPSHom mcmme mo HE pom we 0H m Song . 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32
Thickness, A
50 —
45 "m
40 _
35 _.
30 — \
§
25 —
23 l I l l l I l l l J I I‘
6 8 10 12 14 16 18 20 22 24 26 28 30
Number of Carbon Atoms
Figure 2-1. Capacitance and thickness of bilayer lipid
membranes formed from glyceryl monooleate
dissolved in various n-alkanes and squalene.
Volume Fraction of Solvent
33
1.0 I-
0.8 -
0.4 -
0.2 _
0.0 l l l l J I l 1
6 8 10 12 14 16 18 20 22 24 26 28 30
Number of Carbon Atoms
Figure 2-2. Volume fraction of the alkane solvent and
squalene which is present in the bilayer
region. Note that the volume fraction
decreases drastically to very low value
at high carbon numbers of alkane.
34
technique if a hydrocarbon solvent with a high enough carbon
number can be found which is still a liquid under normal
experimental conditions.
Effect of Lipid Concentration on the Capacitance and
Thickness of Glyceryl Monoqleate Bilayer Membranes
Because of relatively short thinning time in the
formation of bilayer membranes, earlier workers made very
dilute membrane-forming lipid solutions near the critical
micelle concentration (for example, 1.75 mM for n-decane)
above which the lipid content of the bilayer membrane is
expected to remain constant (31). Recently, Waldbillig
et al. (36) measured the capacitances of membranes formed
from glyceryl monooleate dissolved in n-hexane and n-decane,
respectively, at various lipid concentrations. They
observed a linear dependence of membrane capacitance on the
lipid concentration and further took advantage of this
property to form solvent-depleted bilayer membranes by
preparing a very highly concentrated membrane-forming
solution.
It is well-established that the volume of solvent
within the bilayer can be estimated by the measurement of
the membrane capacitance (32,37-39) and this is also
demonstrated in the previous section. This approach is
employed here to evaluate the distribution of solvent
between the bilayer and torus at various lipid concentrations.
The solvent systems chosen in the work by Waldbillig et al.
(36) are both n-hexane and n-decane. As one may notice, the
35
membranes formed from glyceryl monooleate dissolved in both
solvents contain relatively large amounts of solvent. This
study extends the observation of the solvent distribution
to include membrane systems that include less solvent. The
three solvent systems studied are n-decane, n—hexadecane
and squalene (see Figure 2-3 for the structure). With a
concentration of 10 mg/ml of glyceryl monooleate for each
solvent, the volume fractions of solvent that remained in
the bilayer are 0.46 for n-decane, 0.21 for n-hexadecane,
and about 0.00 for squalene. As seen in Figure 2-4, in all
three solvent systems a linear relationship of membrane
capacitance versus lipid concentration is observed, while
the rate of change of membrane capacitance versus lipid
concentration (i.e. the sensitivity) appears to be different.
The concentration effect is greater in the case of n-decane
— CCCCC
(%§‘;;\‘§%§‘§;Y:};§CY;AY:/§H:/‘\cyl§%:/‘\cy,§%:-_'
Figure 2-3. A picture of the molecule squalene. Squalene
(C 0H5 ) is a liquid at room3temperature. It
ha a 8ensity of 0.3584 g/cm and an extended
length of about 29 . Unlike cholesterol,
squalene lacks a hydroxyl group, which may
anchor it to the interface. Note that the
rings are not closed.
36
800 -
35. §———§ §
squalene
700 ..
N
E
O
\ -
ELI
:
- 1"
3 600 )— }._._—————————- n-hexadecane
if!
«4
O
m
9" -
m
L)
A
g: 500 —
,0
E
(D
E - X
// n-decane
400 _ .
350 I l I l I l I
0 02 0.04 0.06 0.08
Glyceryl Monooleate Conc., M
Figure 2-4. Capacitances of bilayer lipid membranes formed
from appropriate amounts of glyceryl
monooleate dispersed in n-decane, n-hexadecane
and squalene, respectively, as a function of
glyceryl monooleate concentration. The
aqueous solution was unbufgered 3 M LiCl and
the temperature was 24:|:1 0 Note that in
squalene system the membrane capacitance is
independent of glyceryl monooleate
concentration.
37
than in n-hexadecane. In the case of squalene, the membrane
capacitance remains constant and does not increase with
increasing lipid concentration, i.e. the sensitivity equals
zero. As in n-decane and n-hexadecane systems, the increase
of membrane capacitance with lipid molarity implies that
membranes formed from concentrated lipid solutions are
thinner and thus contain less solvent. Apparently, the
solvent content of the bilayer is primarily determined by
the solvent content of the membrane-forming solution. The
linearity of membrane capacitance on lipid concentration
allows us to synthesize different thickness of membranes
conveniently by preparing the appropriate concentrations of
lipid solutions. The different sensitivities indicate that
the effect of lipid concentration on the membrane capacitance
depends on the volume of solvent in the bilayer. For the
n-hexadecane system, the volume of solvent is less than that
for the n-decane system and therefore, a lower sensitivity
in n-hexadecane system is observed. For the squalene system,
the membrane capacitance is essentially solvent-free (35)
and thus the membrane capacitance is independent of lipid
concentration.
In summary, it appears to us that the linear dependence
of membrane capacitance on the lipid concentration
apparently is a characteristics for those membrane systems
with appreciable amounts of solvent remaining in the bilayer.
Moreover, the linear dependence of membrane capacitance on
the lipid concentration such as n-decane and n-hexadecane
38
systems provides a very convenient way to form membranes
with different thicknesses by preparing lipid solutions of
the appropriate concentrations. For a membrane system with
little or no solvent such as membranes formed from lipid
dissolved in squalene, the membrane capacitance is
independent of lipid concentration.
Development of Nearly Solvent-free Glyceryl Monooleate
Bilayer Membranesgby Conventional Mueller and Rudin
~ Technique (5)
The solvents commonly used in the formation of bilayer
membranes are normally not present in biological membranes
(40). The presence of the solvent does not prevent the use
of such membrane systems for studies of physicochemical
activities in many cases. However, some problems arising
from the presence of solvents in the bilayer have been
reported (41). In an effort to remove the difficulty
caused by the presence of solvents in the bilayer. Montal
et al. (42) modified the technique by Takagi et al. (43) to
form bilayer membranes from lipid monolayers. This method
suffers the disadvantages of (a) a complicated formation
procedure; (b) the use of a smaller hole (ca. 0.1-0.2 mm in
diameter) (7); and (c) the requirement of a nonpolar
solvent such as petroleum jelly to form the torus in order
to stabilize the membrane (44,45). Although this method
has disadvantages, it should still be credited for being
able to form asymmetric membranes which are assembled by
two different monolayers from different kinds of lipids.
39
Further, White (46) made solvent-free membranes using a
solvent "freeze-out" technique by lowering the temperature
to below the melting point of the solvent so that the
solvent is frozen out into the torus while the bilayer still
remains at liquid-crystalline state. Waldbillig et al. (36)
made solvent-depleted membranes using very highly
concentrated lipid solutions. More recently, the property
of large molecule insolubility in the bilayer (47) has been
employed to form solvent-free glyceryl monooleate bilayer
membranes (35). Apparently, this latest technique offers
the easiest way to form bilayer membranes with little or no
solvent.
The amount of the solvent in the bilayer decreases
drastically with increasing number of carbon atoms of the
alkanes used as solvents in the preparation of membrane-
forming solutions as demonstrated in the previous section.
This approach can be advantageous in forming nearly solvent-
free bilayer membranes by selecting a large, long-chain,
and nonpolar hydrocarbon as the solvent for a membrane-
forming solution. Hydrocarbons which are large, long-chain,
and simultaneously exist as liquid states at normal
conditions are rare. After a careful examination, we
decided to investigate squalane (C30H62), a saturated
hydrocarbon with six methyl groups at side chains, and
paraffin oil, a long chain hydrocarbon mixture, as solvents
for glyceryl monooleate bilayer membranes.
At a concentration of 10 mg glyceryl monooleate per ml
40
of the solvent, the capacitance values of the membranes are
750 nF/cm2 when squalane is the solvent, and 790 nF/cm2 in
the case of paraffin oil. As noted in Table 2-3, these
capacitance values are comparable with those nearly solvent-
free membranes formed from other techniques. The high value
of the capacitance suggests that both squalane and paraffin
oil can be used as solvents in yielding membranes which are
virtually‘solvent-free.
Moreover, the capacitances of bilayer membranes formed
by dissolving lipid in either squalane or paraffin oil at
various lipid concentrations have been studied. The results,
as seen in Table 2-4 and Figure 2-5, do not show a lipid
concentration dependence. This independence of membrane
capacitances versus lipid concentration is consistent with
the observation made from studies earlier —- a bilayer lipid
membrane with the presence of solvent in the bilayer region,
such as in the case of lipid/n-decane, gives a linear,
increasing dependence of membrane capacitances versus a
increase of lipid concentrations, while a bilayer membrane
with little or no solvent such as in the case of lipid/
squalene does not. One may also note in Figure 2-5, there
are differences in the membrane capacitances among three
solvent systems. One possible, but not unlikely explanation
is that the membrane formed from these solvent system may
not actually be entirely solvent-free. The difference in
capacitance values may be due to the undetectable trace
amount of solvent remaining in the bilayer and this amount
41
Table 2-3. Comparison of capacitance values for virtually
solvent-free glyceryl monooleate bilayer
membranes formed using various techniques.
methods capacitance, uF/cm2
monolayer 0.75-0.81
solvent freeze-out 0.790
dispersed in squalene 0.777
dispersed in paraffin 0.790
oil
dispersed in squalane 0.750
dispersed in squalene 0.780
reference
Montal et al.
(42)
White (46)
White (35)
this work
this work
this work
42
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2
Membrane Capacitance, nF/cm
9OO
800
43
paraffin oil
1} @—
squalene
5‘ I
squalane
700 l I, I I I l I
.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09
Glyceryl Monooleate Concentration, M
Figure 2-5. The capacitances of bilayer lipid membranes
formed from appropriate amounts of glyceryl
monooleate dispersed in squalene, squalane,
and paraffin oil, as a function of glyceryl
monooleate concentration. The aqueous
solution was unbuffered 3 M LiCl and the
temperatures were 24:1:1 C. Note that in
all three solvent systems, membrane
capacitance is independent of glyceryl
monooleate concentration because of the
absence of solvent in the bilayer.
44
of solvent may depend upon the solvent chosen, thus
affecting the capacitance values to a different degree.
However, these differences in membrane capacitances are
within 6 % at maximum.
In summary, a large, long-chain, and nonpolar
hydrocarbon can be employed in the membrane-forming
solution. A bilayer membrane with little or no solvent is
then able to be formed using this lipid solution by the
conventional Mueller and Rudin technique. We have extended
these solvents to include squalane and paraffin oil. Again,
as in squalene (35), it always takes a much longer time to
begin the thinning process with these solvents than with
the analogous short alkanes. This is attributed to the
increased bulk viscosities that these solvents possess and
the resulting slower development of the pressure gradient
which is responsible for the initiation of the thinning
process.
Capacitance and Thickness of Glyceryl MonooleategBilayer
Llp d Membranes in Binary Solyent Systems
The hydrocarbon solvents present in synthetic bilayer
lipid membranes are not present in biological membranes (40).
Since the use of a single solvent in the formation of
bilayer lipid membranes is already troublesome, what merit
would the study of bilayer lipid membrane in binary solvents
have ? The interesting behavior observed in the
following studies gives the best answer to this
45
question.
Two binary solvent systems employed in the preparation
of membrane-forming solutions are n-decane/n-hexadecane and
n-decane/squalene. When membrane capacitances versus
bulk solvent volume fractions are plotted, two very
different kinds of results are observed as seen in Figure 2-6.
For n-decane/n-hexadecane system, a linear relationship is
obtained, while the membrane capacitance versus bulk solvent
volume fraction for n-decane/squalene system is non-linear.
This difference may be explained in terms of closeness of
molecular size, structure and the underlying solubility in
the bilayer for both n-decane and n-hexadecane relative to
those for both n-decane and squalene.
The use of n-decane/n-hexadecane offers a way to make
any desired thickness of membranes ranging from 3.1 nm to
4.6 nm. An empirical equation can be derived for the.
convenience of membrane preparation using these binary
solvents.
Cm = 031.0% + 0:1,th
= 01%,dvd 03mm vd)
= Com + (Grid Grim) Vd
where Vd + Vh = 1
apply C = eOe/d
eoe/dm = eoem,h/dm,h + (eoem,d/dm,d ' eoem,h/dm,h) Vd
since e = e = e
m
2
Membrane Capacitance, nF/cm
Figure 2-6.
46
800..
opure
squalene
I.
700-
600L. Pure
d////p'n-hexadecane
500L.
400*- pure
n-decane
EQIILIILIILIJ
IO I2 I“ I6 I8 100
VF of bulk solvent
Capacitances of bilayer lipid membranes formed
from dispersing 100 mg glyceryl monooleate
in 10 ml mixture solvents. As noted, a linear
dependence of membrane capacitance upon the
volume fraction is observed for the n-decane/
n-hexadecane system while a nonlinear
relationship is seen for the n-decane/Squalene
system.
47
1/d = 1/dm,h + (1/dm’d - 1/dm,h) Vd
m
where Vd I bulk volume fraction of n-decane.
Vh I bulk volume fraction of n-hexadecane.
Cm I membrane capacitance in n-decane/n-hexadecane.
cg’d I membrane capacitance in pure n-decane.
C;,h I membrane capacitance in pure n-hexadecane.
eO I 8.854 x 10‘1“ F/cm.
em I membrane dielectric constant, ca. 2.1.
em d I dielectric constant of pure n-decane.
!
em h I dielectric constant of pure n-hexadecane.
!
dm I membrane thickness in binary solvents.
dIn d I membrane thickness in pure n-decane.
9
dm h I membrane thickness in pure n-hexadecane.
9
By varying the bulk volume fraction of n-decane, Vd’
different thicknesses of membranes can be made. On the
other hand, although no simple relationship between membrane
capacitance and bulk solvent volume fraction can be derived
for n-decane/Squalene system, the preparation of any desired
thickness of membranes between 2.4 nm to 4.6 nm is
demonstrated.
In summary, the use of binary solvents permits the
synthesis of membranes of any desired thickness, instead of
the fixed, intermittent values of thickness obtained by
varying the hydrocarbon chain length from n-octane to
n-hexadecane in single solvent systems.
48
Future Work
The ion transport rate across the bilayer lipid
membrane is an interesting topic that has been extensively
studied (13). The effect of membrane thickness on the
electrical conductance has been reported in studies such as
(a) ion transport using the pore mechanism such as
o-pyromellithyl-gramicidin channels (48); (b) ion tranSport
using the carrier mechanism such as valinomycin carriers
(49-52); and (c) direct ion transport such as dipicrylamine
(53,54), tetraphenylborate,tetraphenylarsonium, and
tetraphenylphosphonium ions (55). In all these experimental
results, it is observed that the conductance has decreased
as membrane thickness increased, although discrepancies
exist between theoretical and experimental results.
The thickness of the hydrocarbon core of the membrane
has been changed in two different ways; (a) varying the
lipid chain length; and (b) varying the solvent chain
length. The membrane thickness obtained from both methods
would be expected to be fixed and intermittent, instead of
continuous. Furthermore, the discrepancies of experimental
results mentioned above led Hladky (56) to conclude that
membrane thickness is not the only variable of importance
in electrical conductance. Apparently, both ways of
changing membrane thickness would drastically change the
orderliness of the membrane core due to different
structures of either lipids or solvents as observed by
McIntosh et al. (57), and this would reasonably be expected
49
to lead to changes in electrical conductance by changing
the mobility or the solubility of the ions in the membrane
core in both carrier and direct ion transport cases.
The results from membrane thickness values in binary
solvent systems (for example, squalene/n-decane) in this
study suggest that membrane thickness could be changed to
obtain any desired thickness of membranes between 2.4 nm to
4.6 nm. It should be very interesting to test the‘
electrical conductance and membrane thickness correlation
using these membrane systems.
A new series of phosphonium salts are found to be
effective as anti-trypanosoma cruzi (58,59) and anti-
schistosoma mansoni (60) drugs. These phosphonium cations
are lipophilic and selectively transport across the
membrane. How these drugs act is still not known. Studies
of membrane conductances caused by these drugs may permit
us to gain information such as the dependence of drug
activity on the magnitude of membrane conductances.
Furthermore, the effects of membrane thickness on the
conductance of these phosphonium salts would also be
interesting.
50
CHAPTER III
APPLICATIONS OF MEMBRANE CAPACITANCE MEASUREMENT ON SOLVENT-
CONTAINING (N-ALKANES) AND NEARLY SOLVENT-FREE (SQUALENE)
GLYCERYL MONOOLEATE BILAYER MEMBRANE SYSTEMS
(A) Effect of Benzyl Alcohol on Both Solvent-containing
(GMO/n-hexadecane) and Nearly Solvent-free Bilayer Lipid
Membranes
The anesthetic potency of n-alkanes is closely related
to an increase in absorption of the alkane into bilayer
membranes (61,62). The expansion of membrane thickness is
pr0posed to explain the blockage of the nerve impulse by
n-alkanes in axons. By incorporating a local anesthetic
molecule, benzyl alcohol, into a n-tetradecane-containing
bilayer membrane, a similar result is observed and this
simultaneously led Ashcroft et al. (63,64) to arrive at the
same hypothesis for the action of benzyl alcohol. Since the
membrane-forming solvent may modify the benzyl alcohol
adsorption parameters or conversely, benzyl alcohol may
modify the amount of solvent in the membrane, we decided to
make membrane capacitance measurement while incorporating
benzyl alcohol into both solvent-containing and nearly
solvent-free glyceryl monooleate bilayer membranes. These
measurements may be a suitable test of the hypothesis for
the mechanism proposed for the anesthetic action of benzyl
alcohol.
51
Equal amounts of benzyl alcohol were added into each
aqueous phase. The result in Figure 3-1 shows that benzyl
alcohol actually increases the capacitance of the bilayer
membrane which is essentially solvent-free. The membrane
capacitance in the absence of benzyl alcohol is 0.78 uF/cmz,
which corresponds to a membrane thickness of 23.8 A. The
capacitance increase is about 6 % at 75 mM of benzyl alcohol.
Conversely, the membrane capacitance decreases substantially
in the presence of benzyl alcohol when n-hexadecane is
present in the membrane. This behavior is qualitatively
similar to the observation obtained by Ashcroft et al. (63,
64) and apparently is not unique to a particular lipid-
solvent combination when the solvent is present in the bilayer.
The remarkable differences of the membrane capacitances
in the absence and presence of solvents infer an interesting
and contrasting action of benzyl alcohol on these two kinds
of glyceryl monooleate bilayer membranes. The specific
capacitance of a bilayer membrane is given by CIn = eoem/d,
where Cm is the specific capacitance of the membrane, eO is
8.85 x 10-1“ F/cm, em is the dielectric constant of the
membrane interior, and d is the membrane thickness. Because
the dielectric constant of benzyl alcohol (e = 13.1 at 20 0C)
is much greater than that of hydrocarbon (e = 2.1), the
incorporation of benzyl alcohol into the bilayer would
make one expect that the change in dielectric constant of
the membrane interior, e , may be greatly affected and this
In
change would, in turn, affect the membrane capacitance.
800
N
8
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52
I I
(a) squalene
I
(b) n-hexadecane
I
300-4630! I I I I I
Figure 3-1.
0.0075 0.75 75
Benzyl Alcohol Conc., mM
Membrane capacitance as a function of benzyl
alcohol concentration. In (a), the membrane-
forming solution is glyceryl monooleate
dispersed in squalene (10 mg/ml). The membrane
formed from this solution is essentially
solvent-free. In (b), the membrane-forming
solution is glyceryl monooleate dispersed in
n-hexadecane (10 mg/ml). The membrane formed
from this solution contains significant
amount of solvent. The aqueous solution was
unbuffered 3 M LiCl and the temperatures were
2401 0c.
53
However, this dielectric constant effect appears to be
relatively small. Since the membrane capacitance changes
were primarily due to increases in dielectric constant of
the membrane interior via the incorporation of benzyl
alcohol, the membrane capacitances should increase for both
solvent-containing and nearly solvent-free bilayer membranes.
Clearly, this is not the case. There are two most probable
reasons to account for the small benzyl alcohol-induced
change in dielectric constant. First, the effective
dielectric constant of benzyl alcohol in the membrane interior
is probably much lower than 13.1. The presence of hydroxyl
group on benzyl alcohol is the key factor in the high bulk
dielectric constant of benzyl alcohol relative to toluene
which has a dielectric constant of 2.38 at 21 OC. However,
if the hydroxyl group is oriented to the interface, it will
be unlikely to contribute appreciably to the membrane's
dielectric constant. Second, owing to the relatively low
concentration of benzyl alcohol employed in the aqueous
phase, the partition of benzyl alcohol into the membrane
interior would be relatively small. Thus, the observed
changes in membrane capacitance can't be due primarily to
changes in dielectric constant. Therefore, they must be
due, in large part, to changes in membrane thickness, d.
If the effect of benzyl alcohol on the change in
dielectric constant of the membrane interior is small, then
what causes the increase in membrane thickness when
n-hexadecane is present in the bilayer as we observed ?
54
As previously noted, both solvent-containing and nearly
solvent-free planar bilayer membranes are in equilibrium
with a torus. It is likely that the manner in which solvent
partitions between the torus and the bilayer region of the
membrane will be a factor of considerable importance. In
view of the observation made by Hui et al. (65), that
alcohol may associate in nonpolar liquids at these
concentration ranges, it seems plausible to consider the
activity effect of benzyl alcohol in the membrane. The
effect of benzyl alcohol on the n-hexadecane-containing
bilayer membrane may therefore be rationized as follows:
the incorporation of benzyl alcohol into the bilayer region
may be responsible for the decrease in chemical potential
of n-hexadecane (and lipid) in this region. The decrease
of this chemical potential would cause the redistribution
of solvent molecules to reach a new equilibrium. As a
consequence, an increased partition of solvent molecules is
in favor of the membrane interior. Since n-hexadecane has
been observed to lie parallel to the acyl chain of the lipid
in the bilayer region (57), this amount of increased
n-hexadecane may further straighten out the acyl chains of
glyceryl monooleate and thus increase the membrane thickness.
In contrast, for nearly solvent-free membrane system, the
slight increase in the membrane capacitance is likely due
to the increase in the dielectric constant of the membrane
interior owing to the partition of benzyl alcohol into the
bilayer region. Therefore, the membrane thickness is
55
unlikely to be much changed. This conclusion is
qualitatively consistent with the result obtained by
Turner et al. (66) who used high-field deuterium nuclear
magnetic resonance spectroscopy and calculated a thickness
reduction of 2 A for liquid crystalline state bilayers of
dimyristoyllecithin in the presence of large amount of
benzyl alcohol (3 to 1 benzyl alcohol/lipid mole ratio).
In summary, the incorporation of a third component such
as benzyl alcohol in these studies acts to vary the chemical
potential of n-hexadecane, thus resulting in an increase in
membrane thickness. However, this behavior does not occur
in nearly solvent-free membranes.
56
(B) Effect of Cholesterol on Both Solvent-containing and
Nearly Solyent-free Bilayer Lipid Membranes
Cholesterol (see Figures 3-2(a) & 3-2(b) for the
structure) is widely distributed in animal membranes and is
found in larger amounts in plasma membranes of cells-rather
than in the intracellular membranes. The incorporation of
cholesterol into bilayer membranes leads to the
rigidification of the membrane (67-72) and simultaneously to
an increase in the stability of the membrane (12).
The capacitance of bilayer membranes has been observed
yto increase with the incorporation of cholesterol into the
membrane (32,52,54,73) when the membrane contains n-decane.
The contribution due to the dielectric constant of
cholesterol alone does not account for this increase (74).
Therefore, a reduction in the average extension of the chain
length is invoked to explain the observed result (32,74).
The difference in the capacitance and thickness of the
bilayer membrane formed from dispersing glyceryl monooleate
in n-decane and in squalene is significant. The critical
comparison of the capacitances of these two kinds of
membranes with cholesterol incorporated in them would
facilitate us to further examine the solvent effect on the
membrane structure.
When incorporated into the bilayer membrane, cholesterol
molecules orient to an interface with hydroxyl groups
oriented toward the aqueous solution while both the ring
and the branching chain at the 17 position is directed
57
I 17
HO
Figure 3-2 (a). Structure of cholesterol with polar
hydroxyl group at 3 position and a branched
hydrocarbon side chain at 17 position.
Figure 3-2 (b). Side view of structure of cholesterol.
The steroid nucleus is a fused, bulky,
reduced tetracyclic ring system that is
hydrophobic and stereochemically rigid. The
length of the steroid nucleus along the long
molecular axis is 9 .
58
toward the membrane core. The actual composition of
glyceryl monooleate and cholesterol in the membrane remains
unclear.
As seen in Table 3-1, for n-decane-containing membranes,
the capacitance increases and the thickness decreases with
the amount of cholesterol in membranes. This result is
consistent with that obtained by the earlier workers (32,52,
54,73). At a 1 to 1 molar ratio of glyceryl monooleate and
cholesterol in the membrane-forming solution, a 29 %
decrease of membrane thickness is observed: 'The largest
estimate for the dielectric constant of cholesterol is 2.27
(32). Such a dielectric contribution for cholesterol would
have less than 3 % effect on the overall dielectric constant
of the membrane considered. This would not explain the
large difference observed above. The n-decane-containing
membrane has a thickness of 48 A which is greater than twice
of fully extended length of glyceryl monooleate (about 44.6
8). Experimental evidence (33,57) supports that n-decane
molecules reside in the center zone of the bilayer. The
decrease of the membrane thickness in the presence of
cholesterol would then be rationalized as (a) the exclusion
of more n-decane molecules from the bilayer into the torus
region. This may be caused by the stronger interactions
between cholesterol and the acyl chains of glyceryl
monooleate, which then provides the driving force for the
flow; (b) subsequent kinking and bending of the acyl chain
toward the branching alkyl group of cholesterol to maximize
59
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60
the interaction (75) so that there will not be any empty
space in the center of the bilayer. This kinking would
decrease the membrane thickness. The significantly shorter
extended length of cholesterol (about 17.5 8) relative to
that of glyceryl monooleate (about 22.3 A) along with the
greater fluidity in the center of the bilayer (74,75) makes
this kinking possible.
On the other hand, the nearly solvent-free membrane as
formed from diSpersing glyceryl monooleate in squalene has
a thickness of 23.8 A. This value is much smaller than
twice of extended length of glyceryl monooleate (about 44.6
A). The large discrepancy infers that the acyl chains of
lipid molecules must be highly deformed. As cholesterol is
incorporated into the membrane, both the stronger interaction
between the ring portion of cholesterol and the acyl chains
of glyceryl monooleate, and the intrinsically rigid ring
structure of cholesterol would prevent the bending of acyl
chains of glyceryl monooleate. In order to accommodate the
bulky branching group of cholesterol at C-17 position, the
acyl chains of glyceryl monooleate have to be straightened
to a significant extent. This explains the increase of
the membrane thickness as seen in Table 3-1 for membranes
formed from squalene solvent. The extension of the acyl
chain here is the dominant factor for the observed
capacitance decrease with the presence of cholesterol in the
membrane. Were the dielectric contribution of cholesterol
significant, the capacitance would increase instead of
61
decrease. One additional interesting point to note from
Table 3-1 is that even at 1 to 0.25 molar ratio of glyceryl
monooleate and cholesterol, the decrease of the thickness
for the n-decane-containing membrane is less than 1 % while
the increase is about 6 % for the nearly solvent-free
membrane.
In summary, the effect of cholesterol on both n-decane-
containing and nearly solvent-free membranes is very
different. The incorporation of cholesterol causes the
thickness to decrease for the n-decane-containing membrane
through the exclusion of the n-decane into the torus mostly,
and conversely, the thickness to increase for the nearly
solvent-free membrane through the stretch of the acyl chains
of lipids in order to accommodate the bulky branching
group of cholesterol at C-17 position.
62
(C) Attempt to use Bilaygr Lipid Membranes as a Molecular
Sensor for the Detection of Potential Carcinogens--
Polycyclic Aromatipflydrocarbons
Introduction
The interaction of small molecules with bilayer lipid
membranes has attracted recent interest since many well-
characterized small molecules are toxicants or drugs (76).
The small molecules of interest could be either polar ones
such as pentachlorophenol (17,77), 2,3,5,6-tetrachloro-
phenoxyacetic acid (78), and 2,4-dichlorophenoxyacetic acid
(79-81), or nonpolar ones such as n-alkanes, benzene,
adamantane, and so on. Most of polar molecules, due to
their high hydrophilicity in nature, are believed to
interact with polar head groups of lipid molecules at the
membrane-aqueous interface to cause a lowering of the
dipolar field in the membrane interior and a change of
underlying ion transport rate. In contrast, the nonpolar
molecules often go into the membrane interior and interact
with the acyl chains to lead to the variation of either
membrane thickness, the motion of the acyl chains, or both.
Benzene, adamantane and their derivatives have been
found to increase the motion of the acyl chains in the
membrane interior (82), while n-alkanes (61-63,83,84) and
p-di-t-butylbenzene (85) are responsible for the decreased
capacitance and the increased thickness of the membrane.
Bilayer membranes possess considerable internal order by
virtue of the packing arrangement of their fatty acyl
63
chains. The inherent anisotropy of the bilayer structure
places certain constraints on the interaction of small
molecules with these membranes (86). The perturbation of
this peculiar structure via molecule-membrane interactions,
if they take place, may find some applications. Based on
the results of earlier work and an awareness of the
imporatnce of carcinogens in living tissues, it appeared
that the development of a molecular sensor for the detection
of carcinigens would be possible and worthwhile. In this
part of work, the measurement of the capacitance of the
bilayer membrane with a group of potential carcinogens
incorporated, namely polycyclic aromatic hydrocarbons, are
examined to test the feasibility of using a bilayer membrane
as a molecular sensor.
ExperimentalDesign
The compounds employed in this study are listed in
Figure 3—3. Pyrene, anthracene (both from Eastman),
phenanthrene (Mallinckrodt), fluoranthene and fluorene
(both from Aldrich) were recrystallized twice from ethanol.
The individual compound was then saturated in the membrane-
forming solution which was prepared by dispersing 300 mg of
glyceryl monooleate in 10 ml of squalene. Saturation was
intentional to ensure the maximum incorporation of.
polycyclic aromatic hydrocarbon into the membrane interior.
In other words, we were seeking the maximum possible change
of capacitance. The membrane formed from this squalene-
based membrane-forming solution is essentially solvent-free
64
Figure 3-3. Structure and chemical names of polycyclic
aromatic hydrocarbons.
chemical names structure
anthrac ene
phenanthrene 0"
naphthal ene
fluoranthene 0.0
\\.
fluorene I
//
biphenyl
65
and the acyl chains in the membrane interior are believed
to be highly deformed (35). This membrane system has the
advantage of avoiding the interference of solvent molecules,
while facilitating the maximum interaction between polycyclic
aromatic hydrocarbon molecules and acyl chains of the
membrane. The membranes were formed in 3 M NaCl aqueous
solution and all the measurements were performed at
O
238:1 C.
Results and Discussion
As seen in Table 3-2, essentially no change of membrane
capacitance or thickness was observed within experimental
error. Two possible explanations may account for this
observation: First, in order for these potential
carcinogens to cause the appreciable difference in membrane
thickness, the potential carcinogens present in the membrane
interior must reach a certain level of concentration. A
low concentration of potential carcinogens in the membrane
interior may not alter the membrane thickness at all.
Second, the concentration of potential carcinogens in the
membrane interior may be appreciable, but these molecules
may perfectly fit into the void volume between acyl chains
without causing the extension of highly deformed length of
acyl chains.
The lipophilicity of these potential carcinogens is
intrinsically high and this property makes these compounds
very soluble in the membrane core (87). The partition of
these molecules into the membrane interior by the method
66
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67
employed in this work would be expected to be greater than
the physiologically active concentration. Since no chamge
in the membrane capacitance or thickness is observed, we
would conclude that the utilization of a bilayer membrane
as a molecular sensor for polycyclic aromatic hydrocarbons
via the change of the membrane capacitance or thickness is
not feasible.
68
GMFERIV
STABILIZATION OF GLYCERYL MONOOLEATE BILAYER MEMBRANES
IN THE PRESENCE OF FERRIC CHLORIDE
Introduction
Many applications using a glyceryl monooleate bilayer
membrane as a model membrane in physicochemical
investigations have appeared in the literature (29,48,88-96)
although the membrane made from this lipid has limited
stability (97-99). This requires workers who use this model
membrane to either form the membrane on a very small
aperture (97) or dope the membrane with a stabilizer such as
cholesterol. The former method suffers the disadvantage of
decreased accuracy and sensitivity toward the phenomena of
interest, while the latter rigidifies the membrane and
drastically changes the resistance.
Calcium ion is known to stabilize phospholipid bilayer
membranes (100,101). Uranyl ion has been reported to
stabilize both phOSpholipid (98,102) and glyceryl monooleate
bilayer membranes (98). Lanthanum ion has been shown to
stabilize oxidized cholesterol bilayer membranes (103).
These ions are believed to interact with the polar head
groups of the lipid molecules in some way that increases
the stability of the membrane. Furthermore, Snyder et al.
(8) examined the relative stability of cholesterol-
oxidized cholesterol bilayer membranes in lithium
69
chloride, sodium chloride and potassium chloride solutions,
and found that the membrane formed in lithium chloride
solution has greater stability than in the other two. They
related that the lack of the stability of the membrane in
both potassium and sodium chloride solutions is due to the
disruption of the electric double layer as well as to
interference with the internal van der Waals interaction
by these two cations because of their lesser ability to
arrange water molecules around the interface region. All
these studies demonstrate that the composition of the ionic
solution affects the stability of the membrane.
Ferric(III) ion has been observed to produce a large
change in bilayer resistance (104,105) and it has been
shown to be adsorbed on the polar head groups of lipid
molecules (105). However, no stabilizing effect from
ferric(III) ion has been reported. This highly charged ion,
according to Frank et al. (106), should be categorized as
having properties similar to lithium and thus is expected
to play some kind of role in the region at the membrane-
aqueous interface. Qualitative observation indicated that
glyceryl monooleate bilayer membrane exhibits a decreasing
stability in the alkali ionic solutions in the order LiCl<:
NaCl