Dipole potential of lipid membranes

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Chemistry and Physics

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Chemistry and Physics of Lipids 73 (1994) 57-79

Dipole potential of lipid membranes Howard Brockman The Hormel Institute, Universityof Minnesota, Austin, MN 55912, USA

Abstract

Of the individual potentials which comprise the potential profile of a membrane, the least well understood is the dipole potential. In general, the dipole potential is manifested between the hydrocarbon interior of the membrane and the first few water layers adjacent to the lipid head groups. Changes in dipole potential caused by spreading a lipid at an air- or oil-water interface can be measured directly and the dipole potential of bilayers can be estimated from the conductances of hydrophobic ions. For a typical phospbolipid, like phosphatidylcholine, its measured value is ~ 400 mV in monomolecular films and ~ 280 mV in bilayer membranes, with the hydrocarbon region being positive relative to the aqueous phase. The difference between dipole potentials measured in monolayers and bilayer membranes appears to arise from the use of the lipid-free air- or oil-water interface as the reference point for monolayer measurements and can be corrected for. The species-specific correction term is a lipid concentration-independent potential, the existence of which suggests the ability of lipid headgroups to globally reorganize water structure at the interface. The dipole potential arises from the functional group dipoles of the terminal methyl groups of aliphatic chains, the glycerol-ester region of the lipids and the hydrated polar head groups. Classical methods for obtaining partial dipole moments for each of the three contributing regions are all based on questionable assumptions and give conflicting results. More sophisticated mean-field models of dipole potential origin recognize the important role of interfacial water in determining its value but still cannot adequately describe the microscopic nature of the interactions from which it arises. In part this is because the dipole potential develops in a region over which the dielectric constant of the medium is changing from 2 to 80. Despite of our limited understanding of the dipole potential, it is an important regulator of membrane structure and function. Membrane-membrane and membrane-ligand interactions are regulated by the hydration force, the value of which can be related to the dipole potential of the membrane. For thermotropically phase-separated or multicomponent membranes the size and shape of lipid domains is controlled by the balance between the line tension at the domain borders and the difference in dipole density between the domains. Line tension tends to make the domains compact and circular whereas dipole repulsion promotes transitions to complex domain shapes with larger perimeters. The role in enzyme regulation of mean interracial dipole potential and differences in its lateral distribution is a promising area for future investigation and will contribute to our understanding of lipid-mediated cellular signaling in cells. An important tool in these investigations and in better understanding the origins of the dipole potential on a microscopic level will be the recentiy-developed Maxwell stress microscope.

Keywords: Dipole potential; Surface potential; Bilayer membranes; Monolayers; Lipid domains; Hydration force

1. Introduction Biomembranes impose a thin layer of low dielectric medium between two aqueous compartments of high dielectric constant. From the bulk aqueous phase to the interior of the membrane the dielectric constant changes from a value of 80

to about 2 over a distance of about 1 nm. This occurs in the vicinity of the lipid head groups and glycerol ester moieties. In biological systems the aqueous medium bathing the membrane contains ions and the membrane itself is composed of lipid and protein molecules which carry formal charges and possess functional groups with substantial

0009-3084/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved.

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H. Broclonan / Chem, Phys. Lipids 73 (1994) 57-79

dipole moments. As a consequence of the amphipathic nature of the membrane constituents, charged and dipolar groups are relatively fixed with respect to their orientation and their distance from the center of the membrane. Because of these restrictions the dipoles and charges can be compensated only partially by water dipoles and solution electrolytes. This gives rise to a complex array of electric fields about and within the membrane which are of importance in regulating membrane structure and function. It is conceptually useful to model the total potential distribution in and adjacent to the membrane as the sum of three components considered to be uniformly distributed in the plane of the membrane and each at a particular distance from its center [1]. These are the potential generated by ion gradients across the membrane [the diffusion or transmembrane potential], the potential arising from fixed charges located in the aqueous phase at the membrane surface [the surface or double-layer potential], and the potential due to dipoles [the dipole potential] in the lipid-water interface. Although such a model of additive potentials is conceptually useful, it is experimentally limited in two respects. First, electrical potential difference is, strictly speaking, defined as the work necessary to move a charge between two points in a vacuum. In a biological system, the potential difference between two points in a single phase must be defined in terms of chemical as well as electrostatic work, because ions, not point charges, are the mobile species [2]. Secondly, moving a charged species across a membrane involves the transfer of that charge from the aqueous phase into the oil-like phase of low dielectric which constitutes the membrane interior. This gives rise to Born-image charge effects and renders the potential extremely dependent on the chemical properties, like size and hydrophobicity, of the mobile species. More importantly, the measurement of a potential between the aqueous phase and the apolar interior of the membrane is thought to be impossible [3,4] or, at the least, extremely difficult [5]. However, with appropriate simplifying assumptions, it is possible to model the work necessary to move a specific charged

species across a membrane in a manner consistent with the measurable rate of transport of that ion (e.g., [1] and references therein). The field of membrane electrostatics and its implications for biology is too large to be adequately described in the present format. Therefore, this review will only very briefly describe the first two components of the potential distribution so that the dipole potential can be more fully described. Of the three potential contributors it is the least well understood, but certainly not least important. Simplest and best understood with respect to its origin and magnitude is the transmembrane potential. It exists as a consequence of charge separation across the bilayer and, hence, is the potential difference between the aqueous phases partitioned by the membrane. Under steady-state conditions the value of the transmembrane potential depends on both the concentrations and membrane permeabilities of the diffusing ions. In biological systems this potential ranges from 10-100 mV with the inside aqueous compartment negative relative to the outside (e.g., [1]). It is important for many transmembrane transport and bioenergetic processes [6], can cause the accumulation of proteins on the membrane surface [7] and may regulate enzyme activities [8]. Because a transmembrane potential of 100 mV generates internal electric fields of up to 105 V/cm, it affects the conformation of proteins involved in ion transport, energy transduction and protein transport [1]. In addition, such applied fields will alter the orientation of dipolar molecules and proteins within the membrane. The presence of charged species affixed to the surface of a membrane is a consequence of the amphipathic nature of charged lipids such as phosphatidylserine, phosphatidylinositol, phosphatidic acid and glycolipids bearing charged carbohydrate moieties. The cationic lipid, sphingosine, is generated in biological systems, but only in small quantities necessary for its role in signal transduction [9,10]. However, because it is generated locally by the hydrolysis of the sphingomyelin, it may produce significant positive charge density in a localized area of the membrane on a transient basis. Charges anchored at

H. Brockman/ Chert Phys.Lipids 73 (1994)57-79 the lipid-water interface but still exposed to the aqueous medium attract counterions from the bulk aqueous phase to the interface. This concentrating effect creates a diffusional flux of counterions away from the interface. When these fluxes are equal in magnitude the excess concentration of counterions and, hence, the electrical potential, decay to the bulk value over a distance of several nanometers. This potential is commonly referred to as the surface potential of membranes. The ionic distribution near the interface is approximately described by the Gouy-Chapman theory of the diffuse double layer (for reviews see [6,11,12]). It is most seriously flawed in describing the electrostatic properties of the membrane within a few nanometers of the lipid-water interface where hydration effects predominate [6] but is reasonably accurate at greater distances. The biological importance of surface potentials is well documented. The presence of charged groups on the membrane surface attracts not only small ions but proteins. Positively charged proteins, like myelin basic protein, are readily adsorbed at surfaces composed of negatively charged lipids (e.g., [13]). As shown by Bazzi and Nelsestuen and reviewed by McLaughlin [12], the phosphorylation of substrates by protein kinase C requires that the substrates interact with negatively charged phospholipids in the membrane. In the simplest sense, the surface charge serves to concentrate the substrate in the vicinity of the enzyme. A sort of inverse charge effect has been demonstrated with the adsorption of melittin to vesicles of a zwitterionic phospholipid [14]. In this case the primary adsorption of the basic peptide to the charge-neutral interface is driven by its amphipathic nature. As more protein becomes adsorbed, the accumulated positive charges generate a surface potential which tends to limit further adsorption in a manner described by Gouy-Chapman theory. Although it was mentioned above that the transmembrane potential affects many membrane processes, the effects of transmembrane and surface potentials are additive with respect to those processes which are regulated within the membrane. For example, the surface potential may serve to gate or otherwise regulate a transport event driven by the trans-

59

membrane potential. An additional factor for transport processes is that their rate is dependent on the surface, rather than bulk, concentration of the species being transported [11]. For charged species this depends on the surface potential. The focus of this review is the component of the total membrane potential which arises from -dipoles located, for the most part, in the transition region between the aqueous phase and the hydrocarbon-like interior of the membrane. These dipoles include those of the lipid molecules themselves and of water molecules in the transition region between the phases. They can be measured or estimated for both monomolecular films at the oil- or gas-water interface and lipid bilayer membranes. With respect to this dipole potential, some confusion exists in the literature. This is the result of the terms used by researchers in the monolayer and bilayer fields to refer to different components or groups of components of the membrane potential profile. These, in turn, reflect the different techniques used to measure the potential components. As noted above, the term surface potential is used to refer to that component of the potential arising from fixed charges adjacent to the interface in the aqueous phase. The same term is also used in the monolayer field to denote changes in total potential across an interface that can be measured when a lipid monolayer is interposed between the aqueous and air or oil phases (e.g., [2]). These changes are due to the imposition of charges and dipoles in the interface. In this review the term dipole potential, abbreviated AV, will be used throughout to refer to the potential arising from dipoles and their effect on water polarization in both monolayers and bilayers. If the measured potential contains a contribution from charges, i.e., a surface potential, that contribution will be noted. 2. Measurement of the dipole potential

2.1 Measurement of A V using monomolecular films Consider an aqueous phase adjacent to an air or oil phase. The potential jump, Xw, in moving from within the water phase, where the potential is 4~,~,to just outside it in the air or oil phase, q'w,

H. Brockman/ Chem.Phys.Lipids73 0994) 57-79

60

cannot be directly determined, but its value has been estimated at 100-500 mV negative toward air [5,15,16]. If the air or oil phase contains a metal electrode there is a second potential jump, Xm, between the bulk metal interior potential, ~m, and the air or oil phase value, ~bm. It can be shown that when the phases are connected the difference, ~w-~bm = V can be measured [2]. Because ~w - ~bm = V is the difference in potentials within a single phase, it can be measured• Three simplified circuits showing how this is done in principle are shown in Fig. 1 and the advantages and limitations of each will be described in more detail below. Below each equivalent circuit the relation of @m to ~bw at the time of measurement is indicated• If the potential difference is measured first at an air- or oil-water interface in the absence of lipid, ~bw -~bm, and then in the presence of a lipid monolayer at the interface, ~ w + l -- ~/m, the change in surface potential, ~w+ 1 -~b w resulting from imposition of the lipid monolayer equals the difference in the measured potentials in the presence and absence of the monolayer. This is defined as the Volta potential, AV, or contact potential. For membranes, its value represents the sum of the dipole and, if uncom-

A Vibrating Plate

E

P

pensated charges are present, the surface potential. As noted above, the symbol AV will be used throughout this review to refer to the dipole potential and any material contribution to AV from the surface potential will be specified.

2.1.1. The tibrating plate (Kelvin) method This widely used method of measuring contact potentials between dissimilar materials was first reported by Lord Kelvin and impro~,ed upon by Zisman [17,18]. Modem instruments for making such measurements follow the Zismfin approach but use more contemporary components like piezoelectric vibrators and phase sensitive detection (e.g., [19,20]). This method is based on the capacitance which is developed across the nonconducting air or oil gap between aqueous phase and the metal electrode placed just above it (Fig. la). When the metal electrode is oscillated, typically at a frequency of 150-200 Hz, an AC current is set up in the circuit as a result of the difference in potential, ~ w - Ore, between the metal electrode and the aqueous interface. Application of a DC voltage, V, opposite to that between the electrodes compensates the interfacial potential and when the compensating voltage

B Ioniz/Null

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Fig. 1. Simplified circuits for the measurement of air-water or oil-water dipole potentials. X denotes the difference between the potential, ¢P, of the bulk phase metal electrode (m) or water (w) and the potential of the bulk air or oil phase, ~ , adjacent to it.

H. Brodeman/ Ch¢~ Phys.Lip~ 73 (I994)57-79 equals that of the interfacial potential current is reduced to the negligible value required by the null-measuring device. At this point, V = ~ w ~bm. In theory, this method is superior to those described below because there is essentially no net current flow in the AC circuit. However, as reviewed by Rossi [21], the value of V is dependent upon the spacing between the metal electrode and the surface. With one recent variation of the Kelvin apparatus [22], AV values have been obtained which are equal in magnitude but opposite in sign of those measured by all other investigators. This result has, however, been challenged on instrumental grounds [23] and, as described below, directly contradicts dipole potential measurements in monolayer and bilayer membranes. As normally configured, instruments for measuring V by the Kelvin and other methods are generally macroscopic, sensing the average potential over a relative large area of the surface. However, replacing the metal electrode with a raster-scanned, electrochemically-etched tungsten wire of the type used in scanning tunneling microscopy allows V to be measured with a lateral resolution of 5 p m [24,25]. Z1.2. The ionizing electrode method A second approach to measuring the potential difference between two materials at the gas-liquid or gas-solid interface is to reduce the resistance of the gap between a stationary metal electrode and the aqueous phase sufficiently that a finite current can flow in the circuit, driven by the potential difference between the phases. To render the air layer conducting, the metal electrode is coated with an ionizing source such a s 241An'lor 21°po. Traditionally, this technique was applied with a compensating potential as with the Kelvin method. When current flow in the circuit is reduced to the detection limit of the null device, V = ~bw - ~m (Fig. lb). With the present availability of high impedance ( > 1013 1)) operational amplifiers, a compensating voltage and the circuitry to provide it have become unnecessary unless changes in V of less than 1.0 mV must be measured (Fig. lc). A simple circuit, based on an Analog Devices FET Input Electrometer operational amplifier having 10 TM l'l impedance, is

Ol

shown in Fig. lc. With the ionizing electrode method a voltage follower should be used on the shield covering the metal electrode cable if it extends to the vicinity of the electrode. If a grounded shield is used, the ionized gas around the electrode can provide a conductive path to ground, leading to erroneous potential measurements [26]. As with the Kelvin method, AV is the difference in V values measured in the presence and absence of a lipid monolayer. The measurements are typically sequential, but may be alternating [27] or simultaneous [28] if a clean reference interface is provided. These variations are well suited to experiments in which adsorbates in the aqueous phase might interfere with the reference electrode, e.g., calomel, normally in the aqueous phase. However, they suffer the disadvantage that the reference surface must remain uncontaminated or be periodically cleaned throughout the course of the experiment.

2.1.3. Specialized methods The direct measurement of the capacitance which develops across a gas- or oil-water interface has also be used to determine surface potentials [29,30]. A related technique involves the grounding of both the aqueous phase and metal electrodes at the gas-liquid interface and measuring the current which flows to ground as a lipid monolayer on the aqueous phase is compressed [31]. A novel instrument for measuring AV across supported monolayers, the light-addressable potentiometric sensor, has been described by Haleman et al. [32]. It is sensitive to the potential across a semiconductor interface, can be referenced to an adjacent control surface and offers advantages to earlier instruments based on the chemically sensitive field-effect transistor. Field gradient electrophoresis of lipid domains yields differences in AV between lipid domains in twophase monolayers which correlate with known AVs of the phases [33]. However, none of these methods can be presently recommended for routine measurement of AV. An exciting development in the electrostatic characterization of thin films is the development of the scanning Maxwell stress microscope. As opposed to the scanning Kelvin technique microscope, which relies on the

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H. Brockman / Chem. Phys. Lipids 73 (1994) 57-79

detection of null current [24,25], the Maxwell stress microscope uses harmonic analysis of the electric field-induced oscillations of a cantilever of the type used in atomic force microscopy [34,35]. From this analysis surface potential, surface dielectric constant and surface topography differences can be detected with submicron resolution [36]. Although it has only been demonstrated to operate in air, improvements in cantilever size should allow it to be used with biological samples under water [36].

monolayers in general (e.g., [39]) and for AV measurements at both the gas- [40] and oil-water [41] interfaces in particular. For example, Taylor et al. [40] showed that on aged purified water the surface pressure-area isotherm of stearic acid was unaltered relative to that obtained with freshly prepared water but that the AV-area behavior in the gaseous to liquid-expanded transition region was markedly perturbed. The choice of spreading solvent for lipid monolayers has also been shown to affect AV measurements more than those of surface pressure [42].

2.1.4. General experimental considerations At the oil-water interface, only the Kelvin method is available to measure AV. For typical monolayer compression experiments at the gasliquid interface, there is no obvious choice for whether to use the Kelvin or ionizing electrode method. When the two methods were compared with spread monolayers of octadecyl sulfate on both neutral and acidic substrates [37] or for the adsorption of serum albumin [38], comparable results were obtained. From the instrumentation standpoint the uncompensated, ionizing electrode method is dearly the simplest to implement. Examples of dipole potential data for typical phospholipids on 10 mM potassium phosphate buffer, 100 mM NaC1 at pH 6.6 and 24°C are shown in Fig. 2. The lipids, for which surface pressure-area isotherms are also shown, are 1-palmitoyl-2oleoyl-sn-glycero-3-phosphocholine (POPC), -phosphoethanolamine (POPE), phosphoserine (POPS) and phosphate (POPA). Note that the zwitteronic lipids, POPC and POPE, exhibit a more negative potential toward the water than do the negatively-charged lipids, POPS and POPA, assuming that the identical aliphatic moieties make equal contributions to AV. Values of AV obtained with both methods are generally reported to be reproducible within 1-10 mV, a sensitivity much less than either type of instrument is capable of. Limiting the reproducibility of AV measurements are stray potentials, which can be minimized with shielding, and the purity of the materials with which the experiment is performed. As noted by many authors, minimization of organic contaminants is a challenge for all physical measurements involving

2.2. Measurement o f / i V using bilayer membranes In contrast to monolayers, bilayer lipid membranes do not lend themselves to the direct, potentiometric measurement of AV. This is a consequence not only of the physical problems imposed by the thinness of the bilayer but also of theoretical constraints [3,4]. Instead, AV is estimated from the kinetics of transport of large, hydrophobic anions and cations across black lipid membranes [43] and bilayer vesicles [44]. The basis for these measurements is the energy barrier to ion transport posed by the potential in the center of the membrane relative to the bulk phase. This produces differences in transport rate constants for anions and cations which can differ by 105 [45]. Because hydrophobic anions are transported across membranes of typical phospholipids faster than equivalent cations, the potential in the center of these membranes must be positive relative to the bulk phase. If all other factors are assumed constant and the partitioning of the ions between the aqueous and membrane phases is measured independently, such binding and transport data can be modeled to yield AV [43,44]. As with monolayers, the value of AV obtained with charged lipids is the sum of the surface and dipole potentials. The former can be measured or eliminated [46] to obtain the dipole potential difference between the interface and the center of the membrane. Differences in AV between membranes with different lipid compositions or in the presence and absence of perturbants can be determined directly from the ratio of ion conductance in the two membranes [44].

H. Brockman/Chent Phys.Lipids 73 (1994)57-79

A qualitative method of assessing changes in AV in bilayers has been introduced by Seelig and co-workers [47]. Measured are changes in the quadrapolar splitting for phospholipid molecules specifically labeled with 2H in the polar head group. These arise as a consequence of the orientation of the head group dipole moment being perturbed by changes in the dipole potential. As with monolayer techniques for measuring AV the method reports not only dipole perturbation [48] but also changes in surface charge induced by ion binding [49,50].

2.3. Comparison of AV values of monolayers and bilayers Despite the considerable differences in the methods by which they are determined, AV values obtained from monolayers and bilayers should be the same when normalized for lipid packing density. However, they are not. A recent estimate of AV for egg phosphatidylcholine bilayers is 280 mV [44]. In monolayers AV is obtained as a function of molecular area and the relationship of the collapse area of a lipid to bilayer packing density is not completely understood. If a liquidexpanded monolayer at collapse is considered to represent a packing density equal to that of bilayers, 1-palmitoyl-2-oleoyl-phosphatidylcholine bilayers should have a AV of 411 mV, which is considerably higher [51]. For this difference to be eliminated on the basis of packing density alone, it would be necessary for bilayer packing density of egg phosphatidylcholine to be 112 A 2 per molecule! This is a clearly unrealistic value compared to value of 60 A 2 determined for dioleoylylphosphatidylcholine in bilayers (e.g., [52]). One possible explanation for this difference is the lipid packing-independent component of AV measured in monolayers [51] which will be discussed more fully below (Section 3.3).

63

both seem to arise from water a n d / o r lipid dipoles, they reflect different properties of the system. Hence, their origins and analysis will be considered separately. Because emphasis is on dipole potentials, the properties of uncharged lipids will be emphasized. As opposed to charged lipids, their AV values are essentially independent of the ionic composition of the medium [53].

3.1. Lipid packing-dependentpotential: the surface dipole moment 3.1.1. The capacitor model of the interface In the simplest analogy, the presence of dipole-containing lipid molecules at an air- or oil-water interface can be viewed as a simple capacitor between aqueous and air or hydrocarbon phases. If it is assumed that the dielectric constant of the medium is 1, then, as expressed by Gaines [54], the classical Helmholz equation becomes, AV = 127r/z ± / A

(1)

where AV is the dipole 2otential in mV, A is the lipid molecular area in A2/molecule O/lipid concentration) and ~z is the 'surface dipole moment' expressed in milliDebye (mD) units [54]. The surface dipole moment ostensibly represents the component of lipid molecular dipoles perpendicular to the interface. Attempts have been made to correlate these dipole moments to specific orientations and 'bond moments' of lipid functional groups at the interface (e.g., [55]), but this approach has been abandoned for lack of consistency of the results with known dipole moments of functional groups determined by other means. Two major shortcomings to this approach were recognized early on. One is the assumption of a value of I for the dielectric constant of the interfacial phase and the second is the lack of recognition of the polarization of the dipolar water molecules by the lipid head groups.

3. Origin of the dipole potential As noted above there is evidence that there are two separable components to AV measured in monolayers, one which depends on the lipid packing density and one which does not. Although

3.1.2. Partial molecular dipole models Davies and Rideal recognized that AV has multiple components. They considered them to be water polarization, the lipid polar head groups and the terminal C-H bonds of the lipid aliphatic

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H. Brockman / Chem. Phys. Lipids 73 (1994) 57-79

chains [56]. Because these are arranged approximately in series, they modeled the interface as a three-layer capacitor with voltages additive normal to the interface. Following the nomenclature of Eq. (1), AV = 12~r(/x 1 +/x 2 + l.~3)/A

(2)

where /z 1, /z2 and #3 are the apparent partial dipole moments due to water polarization by the lipid polar head groups, the head groups themselves and the chain termini. More recently, this approach has been generalized to consider the monolayer as foliated planes of dipoles and image charges [57]. The resulting model, however, can be simplified to the original Helmholz equation. The same is true for a smeared-dipole approach which attempts to compensate for discrete nature of the lipid dipoles [58]. Implicit in the partial dipole approach is the assumption that the CH 2 groups of the acyl chains of lipids do not contribute significantly to AV. This is based on the geometry of chains in which the C-H dipoles of successive positions perpendicular to the surface cancel such that only the terminal CH 3 group makes a contribution to ~ ±. In what are the best data reported to date for AV measured at the oil-water interface, Mingins et al. [41] have shown that dimyristoyl, dipalmitoyl and distearoylylphosphatidylcholines exhibit AV-A isotherms which are identical within experimental error. Measurements at the air-water interface show that at comparable area per molecule, there is only a 9 m V / C H 2 dependence of /z± on aliphatic chain length (e.g., [59]), although this is not always observed [60]. A value of 9 m V / C H 2 is small compared to the typical value of AV of ~ 400 mV for 1-palmitoyl-2-oleoylylphosphatidylcholine at physiological packing density (e.g., [51]). In theory, this variation could be due to chain-length-dependent changes in the time-averaged tilt angle of the aliphatic chains in the air-water monolayers. This point was addressed by Evans and Ulman [61] who also observed a 9 mV increment for each CH 2 group between 6 and 22 carbon atoms for alkyl-thiol monolayers chemisorbed to gold. However, they independently demonstrated that the tilt angle of the chains was constant at about 30°C. To explain the

result, it was argued that the dielectric constant of the monolayers was dependent on distance from the gold surface, which causes the contribution of the CH 3 dipole to vary with chain length. The partial dipole approach was applied in a different way by Smaby and Brockman [51] for AV-A data obtained with biologically-relevant lipids in the liquid-expanded state. It had been noted earlier [62] that there are similarities in the AV behavior of lipids having the glycerol-ester backbone in common. Values of /z~_ calculated for six species which shared the glycerol-ester backbone were analysed by combining the contributions to the dipole moment of the glycerol ester and terminal methyl group of each acyl chain into one term and assigning the remainder of /z ± to the water of polarization together with the contribution made by polar groups, like the phosphocholine group of diacylylphosphatidylcholine. With knowledge of the non-equivalence of acyl moieties in such lipids from NMR and crystal structure studies, the acyl groups of each of six species were assigned to one of two classes, extended or kinked. The values of/~ ± were then analysed using multiple linear regression to find the best values of partial dipole moments for the two types of acyl chains. Substantially different values of 132 + 50 and 252 + 27 mD were obtained for the extended and kinked acyl chains, respectively. Assuming that the terminal methyl group of each type of chain is identically oriented at the interface, the difference of about 120 mD is attributable to the difference in orientation and eorfformation of the glycerol ester groups. The direction of the difference is consistent with the carbonyl group of the kinked chain, such as that found in the sn-2 position of phospholipids, being more perpendicular to the interface than that of the kinked chain in the sn-2 position [63,64]. Another variation of the Helmholz model was the introduction by Demchak and Fort [65] of an effective dielectric constant, D i, for each layer of the three-layer capacitor. Thus, AV = 127r( iXl/D1 + iz2/D 2 + ~3/D3)/A

(3)

They measured AV-A isotherms for the methyl ester of 4-carboxy-p-terphenyl and the a - oJ bifunctional dimethyl ester of 4,4'-dicarboxy-p-

l-l. Brodonan/ Che~ Phys.Lipids 73 (1994)57-79

65

terphenyl and, further, assumed the correct con-

The main difference is that in the mixed

formations of the carboxymethyl groups at the

monolayer there is an additional chain-terminal

air-lipid and lipid-water interfaces. In theory, this equates the dipole moments with their literature values determined by other means. Knowing these for the carbonyl and methoxy groups allowed them to determine lzl/D1, D 2 and D 3 b y solution of simultaneous equations. The values they obtained were 40, 7.6 and 5.3 mD. This approach involves a number of questionable assumptions noted by Demchak and Fort, among which are that D 2 and D 3 are independent of the film forming molecules and that the effect of different lipid head groups on txl/D 1 is the same. For long-chain aliphatic lipids, it has been shown that values of 65, 6.4 and 2.8 mD for I.h/D1, D2, and D 3 provided good agreement with the data [66]. In particular, D 3 = 2.8 seems a more realistic value for the relative dielectric constant of the hydrocarbon chain terminus because of its similarity to the commonly used value of 2 for pure aliphatic hydrocarbons. These values were subsequently applied to AV data obtained with more biologically-relevant phospholipids [67]. This analysis gave values of 2.23 D for the h e a d g r o u p dipole m o m e n t of dipalmitoylethanolamine, 2.44 D for dipalmitoylylphosphatidylcholine, 1.66 D for dipalmitoylylphosphatidic acid and 1.48 D for dipalmitoylylphosphatidylcholine, evaluated at close-packed areas of 37-38 A,2. The combined dipole moment of the two chain termini was 0.236 D. Thus, by this analysis the glycerol-ester headgroup region of the phospholipid molecules is the primary determinant of their AV at the air-water interface. Vogel and Mfbius addressed the capacitor model in a clever way in order to estimate the value of iz3/D 3 for the terminus of the hydrocarbon chain [68]. They compared monolayers of stearic acid with those of a 1:1 mixture of octadecane and octadecyl-malonic acid and found their surface pressure-area characteristics to be nearly identical on a per chain basis. This indicates that the octadecane is residing in the film, presumably aligned with the aliphatic chain of octadecylmalonic acid. Comparison of the two systems near monolayer collapse shows that in each monolayer there is one carboxyl group per aliphatic chain.

dipole aligned opposite to that of the normal chain termini, i.e., it points toward the water instead of the air. The surface potential-area characteristics of these two systems were quite different. To analyse this difference it was assumed that the component of AV/A contributed by water and the polar portion of each monolayer was identical. Hence the differences in AV/A were due to one of the two chain terminal dipoles pointed toward air being compensated by the additional dipole of opposite sign in the mixed monolayer. From this it was concluded that the value of 1~3/D3 for a chain terminus was +0.35 D. Building on knowledge of the dipole moment of the methyl group pointed toward air, they next compared stearic acid, methyl stearate and monomethyloctadecanedioate in which the chain terminal methyl group of stearic acid is replaced by a methyl ester functionality [69]. Comparison of the AV-A characteristics of the lipids with knowledge of the dipole moment of the methyl group allowed them to estimate that the dielectric constant of the methyl ester group adjacent to the water phase is about 5-7. This is a reasonable value and compares well with the value of 6.4 determined by the Demchak and Fort approach. Of interest here is the application of this approach to biologically relevant lipids. These include free fatty acids, dioleoylylphosphatidylcholine, dipalmitoylylphosphatidylethanolamine, dipalmitoylylphosphatidyl serine, dioleoylylphosphatidic acid and gangliosides [70]. Analysis by the method of Vogel and Mfbius indicated that the contribution of water and polar head groups, i.tlfD1 + /z2//D2, to the total dipole moment of the zwitterionic phospholipids was quite small and, for dipalmitoylylphosphatidylethanolamine it was slightly negative. Thus, according to this method of analysis, the value of AV for these phospholipids is determined almost exclusively by the methyl groups of the aliphatic chains.

3.1.3. Summary The results of Vogel and M6bius clearly differ from those obtained by the Demchak and Fort

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H. Broclanan / Chem. Phys. Lipids 73 (1994) 57-79

approach with respect to the origin of AV. That two similar approaches could yield such different results points out the major flaw in all analyses of AV results, the assumptions and the oversimplifications. Each approach is in some way tied to questionable implicit or explicit assumptions about interfacial conformations of functional groups or dielectric constants. For example, Taylor et al. in a detailed critique [67] have pointed out that the assumption that the interfacial orientations of the carboxyl groups of octadecylmalonic acid are identical and the same as that of stearic acid is likely flawed. Also, the substantial variation of the dielectric constant with distance from the interface reported by Evans and Ulman [61] contradicts the assumption that both ends of octadecane experience the same dielectric environment and, hence, the same absolute contribution to AV. It is also admitted by all authors (e.g., [57]) that the Helmholz model, on which these interpretations are based is undoubtedly a gross oversimplification of AV origins. In summary, there is currently no reliable way to gain microscopic structural or dielectric information about the lipid-water interface using AV measurements. However, on a comparative basis changes in AV upon changing lipid molecular area or the structure of groups which contribute little to AV can indicate changes in hydration, conformation and/or orientation of the lipid molecules has occurred. 3.2. The importance of water In all of the studies and analytical approaches described above, the polarization of water was considered to play a relatively minor role in determining the AV of the lipid-water interface. However, quantitative measurements of the effects of ions on AV show that ions like CI- have little effect on AV but that chaotropic ions, like SCN-, in molar concentrations can produce changes of - 2 0 0 mV at the lipid-flee air-water interface [71]. Moreover, qualitative measurements with the 2H-NMR technique applied to phosphatidylcholine bilayers shows the same relative effect [50]. Depending on the method of analysis, values of/J,1/Ol a r e generally estimated

to be at most 50-100 mD, compared to several hundred for the rest of the molecule (e.g., [51]). The minimal role assigned to head groups and water is, however, changing. The impetus for change comes from efforts to understand the nature of the repulsive hydration force which is exhibited when surfaces, like membranes of phosphatidylcholines, approach each other. This force, Ph, is observed for uncharged as well as charged membranes and at distances less than 20 A, it dwarfs coulombic electrostatic forces of charged membranes ([72,73] and references therein). The hydration force decays exponentially over short distances from the surface such that: eh = eo e - d / x

where P0 is the force at the plane of origin and A is its decay length, typically 1-2 A. Of the several explanations proposed for its origin, that proposed by Cevc and co-workers [6,74,75] explains a large body of calorimetric and X-ray diffraction data on phospholipid phase transitions. The key concept of that model is that polar group dipoles and excess local charge densities on atoms of the lipid head group that have access to water create non-coulombic electrostatic fields which polarize interracial water. This 'hydration potential', ~bh, is related to P0 by: Po = [ 2 e o ( 8 - 1)/e](~bhO/,~) 2 where s 0 is the permittivity of free space and 6 is the bulk dielectric constant of the solvent. Thus, for a given lipid the hydration force is, at all distances, proportional to the square of the hydration potential. Values calculated for the ~bh0 of phospholipids are typically 400-600 mV [75]. Noting that this is similar to AV for monolayers and bilayers of phospholipids, Simon, McIntosh and co-workers have tested the hypothesis that qJh0----AV. This work is reported in a series of papers in which AV, P0 and A were measured for different lipids and lipid mixtures. In one study liquid-crystalline, gel-phase and interdigitated bilayers were compared with the result that h was independent of physical state but P0 and, hence, ~b~0 was linearly

H. Brocionan / Cherrt Phys. Lipids 73 (1994)57-79

related t o (ha) -2 where A is the molecular area of the lipid in the particular phase [76]. This shows that P0, like AV, is lipid concentration dependent. By changing the solvent both P0 and A were changed, as would be expected from AV measurements in monolayers [77,78] if hydration phenomena were important. The values of P0 increased with AV2, though not in a linear fashion [79]. Altering the concentration of cholesterol in the membrane again changed AV2 and P0 in a consistent manner [80], as did replacing phosphatidylcholine with sphingomyelin [81]. More recently 6-ketocholestanol has been used to perturb the dipole potential of bilayer membranes and monolayers. This additive does not produce the same condensing effect in monolayers as cholesterol [82] but produces a 300 mV greater value of AV measured in monolayers with equimolar phosphatidylcholine [83]. It also increases P0 as would be expected if P0 is determined by AV. Energy calculations, X-ray data and compressibility measurements suggest that both the hydroxy and keto functions of 6-ketocholestanol reside at the hydrocarbon-water interface, removed from the phosphorylcholine head groups. This supports the idea that the dipolar potential, per se, generates the fields which polarize water. In the series of papers described above the correspondence of AV2 and P0 was qualitative. Absolute values of P0 predicted by substituting AV for ffh0 are typically 4-fold too high. This can occur for several reasons. One is that the relationship between P0 and ~h0 derived from nonlocal electrostatic theory relies on a number of assumptions, as does any simple explanation for a complex phenomenon. A second, more practical problem is that the value of P0 calculated from experimental data depends strongly on the placement of the plane of origin of the force [83,84]. With respect to zlV, it was noted above that values obtained with monolayers and bilayers usually differ by over 100 inV. Moreover, it has been suggested that there may be contributions to AV measured in monolayers, such as the hydrocarbon-air interface, which may not contribute to the hydration potential [84]. Thus, within reasonable error the studies described above make a

67

strong case for a role for AV in determining the hydration properties of membranes and for controlling the electric field close to the membrane surface. The notion that AV is synonymous with ~0h0 is not, however, without problems. Gawrisch et al. [85] were aware, from earlier work by Paltauf and collaborators [86,87], that lipids in which ester linkages to the aliphatic chains are replaced by ether or vinyl ether linkages exhibit AVs in monolayers which are about 100 mV lower than those of the corresponding ester lipids. This effect presumably arises from the absence of the carbonyl dipole, ~ 2.5 D, the normal component of which could easily contribute 100 mV to AV. That difference was confirmed when AVs were determined for black lipid membranes of dipalmitoylylphosphatidylcholine and dihexadecylylphosphatidylcholine on the basis of hydrophobic ion conductance [85]. Again, however, the absolute potentials were lower than those measured in monolayers. Importantly, no difference in either P0 or A could be detected from the X-ray analysis of bilayer spacing as a function of osmotic pressure. Nor could this lack of difference be attributed to any other differences, e.g., bilayer thickness, between the systems. This led to the conclusion that the dipolar contributions of the phosphorylcholine head group and water must be the primary determinants of the hydration pressure. Moreover, because the overall sign of the dipole potential is positive toward the inside of the membrane and the normal component of the phosphorylcholine group dipole is negative toward the inside, it was argued that water dipoles must be the primary determinant of AV and, hence, P0. Thus, this analysis, in contrast to the more classical models described in Section 3.1., ascribes AV primarily to i z l / D 1 in the classical nomenclature. The contradictions among the different models for analysing AV measurements indicate that the correct model has yet to be devised. It seems clear, however, that the importance of water was underestimated in the past and that hydration is an important contributor to AV. This view is also supported by simulations which show that water

68

H. Broclaman / Chent Phys. L~oids 73 (1994) 57-79

molecules are highly oriented near biological interfaces ([6,75] and references therein) and that lipid dipoles alone cannot explain measured AV values (e.g., [88]). It has also been pointed out that measured values of AV may contain constant components which are not properly being accounted for [84,85] and that for monolayer AV measurements, the air-water interface may not be the best reference [85].

3.3. The packing-independent dipole potential of monolayers 3.3.1. Characteristics If the conformation and orientation of lipid molecules and polarized water remain constant as lipid molecular area is changed, the calculated value o f / z ±, the 'surface dipole moment' should be a constant (Eq. 1), regardless of the partitioning of its constituent parts. Such behavior is observed for particular lipids within a given lipid phase (e.g., [60]). More often, however, /z± as calculated at different areas using the Helmholz equation is not constant, but varies as a function of lipid area within a homogeneous phase. Examples include monolayers at the gas-liquid interface of myristic acid in the liquid-expanded state at low pH [89], condensed films of fatty alcohols [59] and a number of glycerol ester based lipids in the liquid-expanded state [51]. Variable/z ~_ values have also been reported at the oil-water interface [90]. One way to deal with this apparent violation of the Helmholz equation is to report or compare ~ values at one or a limited number of molecular areas (e.g., [68]). As reviewed by Schuhmann, other explanations for this result are a changing distribution of molecular conformations, changing dipole orientation due to steric hindrances or interactions and variation of the local dielectric constant as area is changed [57]. Using data obtained for 38 lipid species representing 19 chemical classes, Smaby and Brockman approached this question in a more empirical manner for films in the liquid-expanded state [51]. The impetus for this study was analysis of surface pressure-area isotherms for lipids in the liquid-expanded state which shows that such monolayers can be modeled by assuming that the surface is

occupied by lipid and water molecules in a quasitwo-dimensional manner ([91-93] and Feng, S., Brockman, H.L. and MacDonald, R.C., submitted). An advantage to studying the area dependence o f / z ± in the liquid-expanded state, which is comparable to the liquid-crystalline state of bilayers, is that molecular area can be varied over a large relative range, 2-3-fold, within a one-phase region. It was observed that area-dependent /z ± values calculated using [Eq. 1] were the rule rather than the exception [51]. One possible artifactual origin of area-dependent /z± values was subphase contamination by surface active species. However, contamination was shown to be minimal [94] as was perturbation of the signal by the non-compensated measuring circuit (Jones, C.M. and Brockman, H.L., unpublished). Although AV was not proportional to lipid concentration, plots of AV as a function of lipid concentration were, with few exceptions, linear. This means that for uncharged lipids the variation of AV with A can be reasonably described by the simple relationship, analogous to Eq. 1, A V = A V 0 + 121rtz±/A where AV0 is an area-independent contribution to AV. For charged lipids the surface potential would contribute a second area-independent tenn. Values of AV0 and /z± calculated from the data given in Fig. 2B are summarized in Table 1. Also in the table are the values of the three characteristic parameters obtained from analysis of the surface pressure-area isotherms shown in Fig. 1A. Note that the values of the parameter ~o0' the molecular area extrapolated to infinite surface pressure, are nearly identical. This implies that packing of all four species is ultimately limited by the cross-sectional area of their acyl chains. Also similar, but not identical are the values of/~ ~_, which range from 490-441 mD. On the other hand the values of AV0, which include surface potential for the charged lipids, vary by over 120 mV, with the zwitterionic lipids, POPE and POPC having the more negative values. Interestingly, if the data of Fig. 2B were corrected for AV0 and replotted, the curves would be almost superimposable over the range of the liquid-expanded state.

1t. Brockraan/ Chert Phys.Lipids73(1994)57-79

69

Table 1 Interracial parameters for model phospholipids Phospholipid

oj° (/~2)

fl

q

/~ ± (roD)

AV0 (mV)

POPC POPE POPS POPA

39.3 39.8 38.3 39.8

1.16 1.21 1.16 1.19

3.14 2.03 2.62 1.94

473 490 452 441

124 93 4 2

One finding of this analysis was that the values of AV0 were lipid-class dependent. For fatty acids and alcohols they were generally < 50 mV but for phospholipids, like diacylylphosphatidylcholines, they were generally 100-125 mV. This is particularly interesting in light of the observation, discussed above, that AV values measured in monolayers of diacylylphosphatidylcholines are higher than bilayer values by this same amount.

Based on this observation, it was suggested [51] that AV0 was the constant potential term alluded to [84,85] in studies of the relationship of AV to @h0.Also, subtracting AV0 from AV in calculating P0 brings the calculated values more into line with measured values but does not make them identical (Brockman, H.L., unpublished). Yokoyama and K6zdy also observed AV0 with phosphatidylcholine monolayers but attributed it

5o

•

-~-- " ~" x \ I " \ ' \ 40

""'~...

30 n

20

8

_

_

A

\\ "\ "\~ "".,,

POPG . . . . . . . POPS . . . . .

~"'.,, "". N~",.,,..'.,.

POPE

...........

POPA--

lo •.,.

o

,oo

I

I

I

'"'""'"'"...

,~, L ~ . ~

• . ....

~ ~ ' , ~ . ~

~

I

..,..

a

""°..,~..~. ,...

.,~ • °. .~. ",....

~.,~.

~,,~

Q.

°.....,

,

°~

a

100

I 40

I

I 60

I

I 80

I 100

M O L E C U L A R AREA, ~ Fig. 2. Surface pressure- and dipole potential-molecular area isotherms of phospholipid monolayers. The phospholipids were 1-palmitoyl-2-oleoyl-phosphatidylcholine (POP(]), phosphatidylserine (POPS), phosphatidylethanolamine (POPE) and phosphatidic acid (POPA). Surface pressure (A) and dipole potential (B) were recorded at 24°C under an argon atmosphere using an aqueous subphase of 0.01 M potassium phosphate buffer, 0.1 M NaCI, pH 6.6.

70

H. Broclonan / Chem. Phys. Lipids 73 0994) 57-79

to the potential of the hydrocarbon-water interface. This explanation is, however, inconsistent with the head-group specificity of AV0 values observed in the more extensive study of lipids with similar aliphatic moieties. 3.3.2. Relation to bilayer dipole potentials Although the possibility exists that AV0 is some as yet unidentified experimental artifact, its reproducibility and species specificity suggest otherwise. One possibility is that AV0 relates to the reference point in the measurement of AV in monolayers, the air-water interface [51]. The potential of the empty interface is determined by water structure which involves networks of hydrogen bonds. As described above, it is believed that membrane constituents, like diacylylphosphatidylcholines, possess polar groups which interact strongly with water, structuring it and giving rise to the q'h0 and, hence, P0. When such lipids are initially spread at large molecular areas at the air-water interface, each molecule in this gaseous monolayer state presumably structures only the water in its immediate vicinity. As lipid packing density is increased the liquid-expanded state coexists with the gaseous state. The lipids in each state should have different values o f / z +, just as they do in the liquid-expanded to liquid-condensed coexistence region [60]. However, for monolayers in the gaseous-liquid-expanded transition region, AV does not vary continuously with molecular area. Instead, at an area well removed from the end of the transition region, AV abruptly increases to values comparable to those at the end of the transition when the monolayer exists solely in the liquid-expanded state [51]. This discontinuous transition is not limited to AV. Morgan et al. [95,96] have observed that the jump in surface potential is accompanied by an abrupt increase in monolayer lateral proton conduction. This, they propose, reflects the formation of a hydrogen bonding network involving water and the lipid polar head groups at a critical packing density. Also, Ohara and Nakajima [97] measured large displacement currents for lipids in the transition region. It should be noted that all the studies described here involved phosphatidylcholines, which do not hydrogen bond intermolec-

ularly, in addition to species which do hydrogen bond, like phosphatidylethanolamines and phosphatidic acids. Yet another technique, surface viscosity, exhibits a small, discontinuous decrease as phosphatidylcholine monolayers are compressed in the gaseous to liquid-expanded coexistence region [98]. It has been suggested that this reflects the transition between the liquid-expanded phase being continuous or discontinuous, as has been revealed by fluorescence microscopy of monolayers in the transition region [99-101]. With respect to the diffusion of the lipid molecules themselves, no discontinuity is observed in the liquid-expanded to gaseous transition region. Rather, diffusion constants measured in didodecanoylylphosphatidylcholine monolayers vary continuously with molecular area in a manner consistent with continuum theory [101]. Phase compositional transitions of the type described above during monolayer compression or expansion would not, a priori, be expected to cause a discontinuous change in the AV because the density of lipid dipoles changes continuously. However, at some critical density, perhaps corresponding to that sensed by most of the other techniques noted above, the organizing effect of the lipid head groups on water structure may propagate through hydrogen bonding and reorganize water molecules over the entire interface. In effect, this would change the reference point for AV measurements in monolayers, giving rise to the AV0 measured experimentally. Because AV values in bilayers are measured differentially in preexisting bilayers, values of AV measured with bilayers would be lower than monolayer AV values by AV0, as is observed. Thus, the suggestion [51] that AV0 reflects a reorganization of water structure over the entire interface remains an attractive one.

4. Significance of dipole potentials 4.1. Membrane structure Cevc has noted that membrane hydration is probably the single most important determinant of membrane structure [6]. This hydration arises from direct, quantum-mechanical water binding

H. Brockman / Chem. Phys. Lipida 73 (1994)57-79

to head group constituents of membrane lipids. For uncharged lipids this binding is driven by strong local interactions arising from atomic and dipolar fields near the head group atoms. The measured potential in the vicinity of the membrane-water interface reflects these noncoulombic charge, dipole and hydration effects, but specific models relating AV to structure and conformation in the near interracial region are yet to be developed. Because of this intimate relationship between hydration and dipole potential, any action or agent which perturbs dipole potentials will regulate the conformation and hydration of the lipids [48] and the macroscopic phase behavior of the system [102]. An example of such a perturbation in monolayers is the compression of a-to functionalized surfactants which undergo a compression-dependent reorganization [1031. Dipole potentials can also be major determinants of the macroscopic lateral organization of lipids in membranes. The use of fluorescent microscopy to study lateral phase distribution in monolayers [104] has made it possible to determine how the size, shape and dynamics of thermotropically phase-separated lipid domains are regulated. This includes liquid-liquid as well as the more frequently studied liquid-expanded to liquid-condensed phase separation. A most interesting finding from these studies is that domains of one phase which form in a continuum of another exhibit regular shapes and sizes and that these domains can be regularly spaced, generating superstructure phases (e.g., [105]). That dipole fields are important in regulating the properties of domains is simply revealed by noting the effects of an externally applied field on such domains. Placing a point electrode above a monolayer with solid domains in a liquid medium causes the domains to be attracted under the source or repulsed, depending on the polarity of the potential applied [33,60]. It has also been demonstrated experimentally with the scanning AV microscope [25] and the scanning Maxwell stress microscope [35] that there is a difference in AV between liquid and solid phases. For supported monolayers of dimyristoylylphosphatidyl-

71

choline the magnitude of this difference was 90 mV when measured by scanning Kelvin microscopy [106] and 50-100 mV by scanning Maxwell stress microscopy [36]. For dipalmitoylylphosphatidylcholine monolayers at the air-water interface a value of 260 mV was estimated on the basis of domain dynamics [107,108]. The difference in AV values between phases is consistent with the difference in lipid and, hence, dipole density between the coexisting phases, but the reason for the high value estimated by the analysis of domain dynamics is unclear. As shown in the elegant mathematical analyses of Andelman and McConnell, differences in AV between phases are directly responsible for determining the size, shape and arrangement of the domains [109,110]. The key to understanding these shapes and distributions is the balance of two forces, dipole (and charge) repulsion and line tension. The former is long range and repulsive in the plane of the monolayer [111]. It arises because the dipoles are forced by virtue of the amphipathic character of membrane-forming lipids to assume a parallel arrangement. Opposing the dipolar repulsion is the line tension at the interface between the domain and the continuum which tends to make the domains assume a compact shape [112]. The net result is that domain size, shape and arrangement can be equilibrium properties of the system, i.e., that the system will not undergo unchecked domain growth leading to total, macroscopic phase separation [110,113-117]. The existence of size-stable domains in the plane of a membrane is not only of interest for understanding surface physics but may have consequences for chemical and enzymecatalysed processes occurring at the interface [118]. 4.Z Membrane function

Although the importance of membrane hydration is acknowledged, there are only two well documented cases of membrane-dependent phenomenon which appear to be regulated directly by AV. One is the apparent relation to the hydration pressure, P0, described above. The

72

H. Brocknmn / Chem. Phys. Lipids 73 (1994) 57-79

magnitude of P0 presumably determines the repulsive force between two approaching membranes. Thus, ff AV is decreased by biochemical events at the interface, the membranes may be more likely to fuse or allow adsorption of a partitular protein. For example, it has been suggested that the activation of a membrane-bound phospholipase C is a local event which may generate a transient domain rich in diacylglycerol [119]. If the substrate for the enzyme were phosphatidyleholine the local AV would increase by about 40 inV. In contrast, ff the substrate were phosphatidylmositol the local AV, including the change in surface potential, would decrease about 130 mV [51]. This transient, local change could trigger or facilitate the translocation of an enzyme like protein kinase C to the interface. Also of interest with respect to protein adsorption is the lateral distribution of AV in the plane of a mixed-lipid interface at equilibrium. The lateral distribution of, for example, a fatty acid in a phosphatidylcholine membrane is governed both by statistics and by the degree of immiscibility between the species (e.g., [120]). The differences in dipole density between the species should contribute to this distribution and, hence, to the size distribution of clusters of the minority species in the continuum of the other. Measurements of lipase adsorption to such mixed interfaces shows that adsorption is not regulated by average interfacial AV [121] but this does not rule out the possibility that AV contributes to lipid species differences in lipase adsorption through its effect on species cluster size. Lastly, the protein is not a passive participant in the adsorption process. The value of /z ± for cytochrome C in dioleoylphosphatidylcholine monolayers at the air-water interface is about 6.5 D and is aligned the same way as that of the phosphatidylcholine [122]. For concanavalin A tetramers in mixed monolayers with dipalmitoylylphosphatidylcholine, however, /~± is estimated at 200 D and is aligned opposite to the net /z± of the lipid [123]. The magnitude of these values might suggest that adsorbed proteins would totally dominate monolayer AV. It should be remembered, however, that protein concentrations are typically 10-100 times smaller than those of the lipids, lessening their impact (Eq. 1).

The other well documented phenomenon which depends on AV is the rate of transport of hydrophobic anions and cations. In addition to the differential transport rates of anions and cations, transport studies with black lipid membranes [124] and bilayer vesieles [44] as well as 2H-NMR measurements of headgroup conformation in vesicles, show that the effects of compounds like phloretin and 6-ketocholestanol can be correlated with their ability to alter AV as measured in monolayers [124]. Importantly, at effective levels, pb_loretin [48] and 6-ketocholestanol [125] do not alter membrane structure as probed by other physical techniques. Based on these data for ion transport, it has been suggested that any protein conformational change or translational movement that causes charges or dipoles to move through the membrane interface should be regulated by AV [1,44]. Particularly relevant examples are the stop-transfer sequences of membrane proteins which possess positively charged residues [44]. Related to diffusional ion transport is that occurring through channels. Although this might be expected to be very sensitive to AV, a difference of 120 mV in AV produces only a two-fold change in gramicidin B-mediated transport [126]. Mathematical modeling suggests that the reason for this small effect is that the dipole potential is shielded by water within the channel, just as it is at the membrane-water interface. Thus, AV should directly regulate channel-mediated conductance only if a pore is very narrow [127].

4.3. Analytical applications Because AV is sensitive to the concentration and dipolar properties of molecules at interfaces, it is useful for characterizing molecular assemblies at interfaces. It has been used to characterize the thermal behavior of organic films on solid substrates (e.g., [61,128-131]) as well as its more traditional use in the characterization of monolayers at the air-water interface (e.g., [51]). With respect to the latter, the ideal values of both /x_L and AV0 for mixed-lipid monolayers can be calculated from those for the individual components. These ideal and actual values can then be compared to help reveal non-idealities of lipid

H. Brockman/ Ch~ Phys.Liptds73 (I994)57-79 mixing [93]. Measurements of AV for an empty air-water interface can be a sensitive indicator of impurities in the aqueous subphases on which monolayers are spread [94]. Its sensitivity is greater than surface pressure for detecting impurities because AV shows jumps to relatively high values at molecular areas at which the surface pressure, as measured by all but exceptionally sensitive film balances (e.g., [132]), is effectively zero. Monitoring compositional changes at interfaces is another area in which AV has found application. One common example is the use of AV for detecting the partitioning of molecules from the bulk aqueous phase to an interface. Small organic molecules are examples [133,134]. Similarly, AV was used to demonstrate a correlation between the ability of n-alkanols to serve as local anesthetics and their ability to alter AV of egg phosphatidylcholine-cholesterol (2:1) mixed monolayers [135]. Protein adsorption can be monitored in the same way [122,123,136]. The last study is particularly interesting because it demonstrates that AV can detect protein adsorption in the absence of any change in surface pressure. A similar conclusion was reached in measurements of the adsorption of poloxamer, a non-ionic surfactant [137]. Such adsorption presumably occurs because of interaction of the adsorbing species with the hydration layer beneath the lipids. In contrast, surface pressure is more sensitive to protein insertion between the lipid molecules. The adsorption of metal ions and the ionization of monolayer functional groups can also be monitored using AV (e.g., [89,138-140]). These studies also involve measurement of surface potential in the case of charged or ionizable groups. For example, measurement of the dependence of AV on monovalent ion concentration under monolayers of phosphatidylglycerol showed the expected change of about 59 mV for each decade change in salt concentration as predicted from Gouy-Chapman theory [140]. In addition to being used to monitor protein adsorption, AV can provide a simple way to monitor rates of dynamic processes in monolayers. This potential was recognized long ago [55,141],

73

but has not been widely used. Most applications relate to hydrolysis of phospholipids ([142] and references therein and [143,144]). Under initial rate conditions the hydrolysis of long-chain phosphatidylcholines catalysed by phospholipases generates changes in AV which can be related to the extent of substrate hydrolysis. In one study utilizing a medium-chain substrate at constant surface pressure, it was claimed that not the soluble products of the reaction but the adsorption of catalytic levels of phospholipase A 2 during catalysis was responsible for changes in AV [144]. This seems unlikely, however, considering that the extremely low interfacial enzyme concentration necessary for catalysis generally does not change AV [142]. Rather, it is likely that the observed changes reflect the accumulation of steady-state levels of the medium-chain hydrolysis products in the interface [145]. In addition to enzymology and chemical reactions, AV has been used to monitor lateral proton conduction in monolayers [146]. With the availability of inexpensive, reliable instruments for monitoring AV in the uncompensated mode in monolayers, its use for monitoring interracial reactions should increase.

5. Perspective Measurements of AV in monolayers have been made for over 60 years and in bilayer membranes for over 20 years. However, the use of these measurements for probing the regulation of biological phenomena has been minimal. In part this stems from the lack of any successful, microscopic model of the origin of AV. This is largely a consequence of the large change .in dielectric constant over a distance of a few Angstroms at the interface and t "ertainties about the state of water molecules in le first few hydration layers adjacent to the interface. Thus, in the absence of any consistent framework in which to interpret the data, it can only be stated with certainty that something has changed, but not what or why. Slowly, this situation is improving, hut a quantitative model which allows AV changes to be related to specific changes in dipole orientation or head group hydration will not soon be available. One

74

H. Broclonan /Chem. Phys. Lipids 73 (1994) 57-79

promising area for improving interpretation is the correlation of AV changes with quadrapole splitting described above. Careful comparison of 2HN M R data with AV data obtained by the ion transport technique should provide insight into the role of head group dipole reorientation and hydration in determining AV values. A largely unexplored area which shows promise for the future is the role of AV in regulating protein conformation and enzyme activities in membranes. It has been demonstrated that surface potential [147] and transmembrane potential [8,148] can regulate enzyme activities at interfaces. Such studies have implications for the regulation of m e m b r a n e - m e d i a t e d signaling events important to cellular homeostasis. It is likely that AV, because of its intimate relationship to interfacial structure, can also regulate enzyme adsorption and catalysis. The recent improvement of techniques for measuring and controlling AV in unilamellar vesicles [44] will allow a range of other physical techniques to be used to study proteins under conditions of known and controllable AV. In mixed-lipid systems or those at phase boundaries, these will, however, address only the average AV of the system. The lateral distribution of AV in membranes comprised of miscible species remains largely unexplored. An exciting possibility is the application of the scanning Maxwell stress microscope [35] to characterize lipid domain and cluster structure in mixed lipid surfaces under water at high resolution. Evidence suggesting the existence of lateral organization in mixed-lipid, fluid phases and biological m e m branes has been reviewed by Kinnunen [149]. Ultimately, it should be possible to correlate specific protein adsorption sites and regulation of catalysis with the potentiometric topography of the interface.

Acknowledgements I wish to thank Drs. R.E. Brown, C.M. Jones, R.C. MacDonald, R.A. Uphaus and G. Zografi for taking the time to read this work and contributing their comments. The financial support of U S P H S grant H L 49180 and the H o r m e l Foundation is gratefully acknowledged.

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