Biochimica et Biophysica Acta 906 (1987) 353-404

Elsevier BBA 85315

353

Lipid intermolecular hydrogen bonding: influence on structural organization and membrane function

Joan M. Boggs
Department of Biochemistry, Hospital for Sick Children, and Department of Clinical Biochemistry, University of Toronto, Toronto (Canada)
(Received 9 December 1986) (Revised manuscript received 15 May 1987)

Contents
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354 355 355 357 358 358 370 374 378 381 382 383 383 383 383 385 386 388 389

II. Evidence for lipid intermolecular hydrogen bonding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. General considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Fatty acid-anion complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Properties of glycerol-based lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Phase-transition temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Other properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Hexagonal-phase formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Sphingolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Intermolecular hydrogen bonding between two different lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Hydrogen bonding of cholesterol with other lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Influence of lipid intermolecular hydrogen bonding on membrane structure and function . . . . . . . . . . . . . . . . . . . . . . . A, Lamellar to non-lamellar phase transitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Phase separation or domain formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Lipid miscibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Asymmetric distribution in small unilamellar vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Preferential association of cholesterol with different lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Interactions with proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D, Other roles of lipid hydrogen bonding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Abbreviations: L, lauroyl; M, myristoyl; P, palmitoyl; S, stearoyl; E, elaidoyl; O, oleoyl; T, tetradecyl; H, hexadecyl; PC, phosphatidylcholine; PE, phosphatidylethanolamine; MePE, monomethylphosphatidylethanolamine; Me2PE, dimethylphosphatidylethanolamine; PS, phosphatidylserine; PA, phosphatidic acid; PG, phosphatidylglycerol; PM, phosphatidylmethanol; PI, phosphatidylinositol; lysylPG, lysylphosphatidylglycerol; MGDG, monoglucosyl- or monogalactosyldiacylglycerol; DGDG, diglucosyl- or digalactosyldiacylglycerol; DXPR, DXMGDG, DXDGDG, MeDXPE, Me2DXPE , and XYPR, dihydrocarbon chain form and mixed chain form of

diacylglycerol or phospholipid where PR = PC, PE, PS, PA, PM, PG, and X, Y = L, M, P, S, E, O, T, and H as defined above; Tc or To, gel to liquid crystalline phase-transition temperature; TCR, crystalline to liquid-crystalline phase-transition temperature; TH, lamellar to hexagonal phase-transition temperature; DSC, differential scanning calorimetry; SUV, small unilamellar vesicle. Correspondence: J.M. Boggs, Department of Biochemistry, Hospital for Sick Children, 555 University Avenue, Toronto, M5G 1X8, Canada.

0304-4157/87/$03.50 1987 Elsevier Science Publishers B.V. (Biomedical Division)

354 IV. Control mechanisms for membrane function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Regulation of hydrogen bonding by change in environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Regulation of hydrogen bonding by enzymatic alteration of lipid composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Response to changes in growth conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Response to signals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389 390 392 392 395 396 397 398

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

!. Introduction

The great variety of lipids present in membranes and the specificity of the lipid composition of different types of membrane suggest that many of these lipids have specific roles to play [1]. In mammalian membranes this variety is achieved through a number of structural modifications in the hydrocarbon chains and the polar head-group joined to the glycerol or sphingosine base of the common phospho- and sphingo-lipids. Further structural modifications occur in the type of linkage (ether or ester) of the hydrocarbon chains to glycerol and the presence of free hydroxyl groups on the sphingosine base and fatty acid chain of sphingolipids. Rapid in situ modifications to lipid head groups and hydrocarbon chains can also occur in response to various stimuli. These modifications may affect the structural organization and fluidity of the membrane and the interactions of lipids with proteins, altering their behavior. They may also allow the lipid bilayer to respond dynamically to changes in its environment and to carry out certain dynamic functions of the membrane. Examination of the physical properties and phase behavior of different lipids has helped in the understanding of the molecular forces which control lipid behavior and the contribution of different structural modifications to these forces. Various forces which have been considered to control lipid behavior include repulsive or attractive forces between lipid head groups [1-8], the molecular shape and ratio between the volumes of the head group and hydrocarbon region [9-11] and hydration forces [12,13,113]. While these forces are all important for the behavior and properties of lipids and are interrelated, they are not identical. This author considers that the repulsive

or attractive forces between lipid head groups are the most important property of lipids, other than their amphipathic character. These forces are partly responsible for the molecular shapes of the lipids and hydration forces, but go further in stabilizing the specific molecular organization taken up by a particular lipid under different conditions. Thus, consideration of these forces has greater predictive value for the effect of a particular structural modification of a lipid molecule on its properties. The repulsive forces are primarily the electrostatic repulsive forces between similarly charged lipids while the attractive forces are electrostatic interactions between oppositely charged groups and intermolecular hydrogen bonding interactions between charged or neutral lipids which have hydrogen-donating and -accepting groups. Some lipids are less hydrated than others and many authors attribute the physical properties and phase behavior of these lipids to their reduced hydration. However, those lipids which are less hydrated are generally those with hydrogen bonddonating and -accepting groups and it seems reasonable to conclude that the reduced hydration is usually caused by the participation of the lipid head groups in intermolecular hydrogen bonding with each other rather than with water. Therefore, these interactions must ultimately be responsible for the phase behavior and other properties of these lipids which accompany the reduced hydration. However, some structural modifications to lipids which make them less polar or more hydrophobic may also cause increased dehydration, which can in turn, contribute to an increase in the strength or probability of hydrogen bonding interactions. There is, of course, no absolute proof that intermolecular hydrogen bonding occurs between the head groups of lipids when in the presence of

355 water. NMR, infrared, or Raman spectroscopy might be able, eventually, to provide such proof but have not yet done so. However, there is a correlation between the physical properties of different lipids and the presence or absence of potential hydrogen-donating and -accepting groups. Historically, it was the unusual physical properties of water and alcohols which suggested that they are associated, leading to the concept of hydrogen bonding [15]. The purpose of this review is to demonstrate this correlation between the physical properties of lipids and their molecular structure, updating two earlier reviews on this subject [1,6], and to discuss the significance of intermolecular hydrogen bonding interactions for lipid organization and dynamic function. A special mention should be made of the work of Tr~iuble, Eibl, Pascher and their collaborators, on which this review heavily depends. for a head-on view in Fig. 1. In cerebroside, the sugar points sideways away from the ceramide part because of intramolecular hydrogen bonding between the amide N - H group and the oxygens of the glycosidic linkage and the fatty acid hydroxyl group (Fig. 2, bottom). This allows it to interact with neighboring molecules, forming an intermolecular hydrogen bonding network via the amide group and hydroxyls on the sugar, fatty acid chain and sphingosine chain (Fig. 2, top). However, it is often questioned whether intermolecular hydrogen bonding between lipid head groups could take place in the presence of water. Jencks [21] points out that it would require that the hydrogen bonds of each of the participating groups with water be broken, as indicated in Eqn. 1. A-H.
-OH 2

+B..HOH --, A-H..B+HOH-

.OH 2

(1)

1I. Evidence for lipid intermolecular hydrogen bonding

H-A. General considerations Hydrogen-donating groups on lipid molecules include NH~-, NH2, POH, COH, COOH and HNC--O, while hydrogen accepting groups include some of these as well as PO-, C O O - , OC=O and COC. The strength of hydrogen bonds generally increases in the order of electronegativity of the donor, S < N < O, and, for the acceptor, in the order ethers < carbonyls < amines [15]. Two common groups in phospholipids, the PO 4 and NH~-, might interact as either ion pairs or proton transfer complexes. However, the latter are appreciably more stable than true ion pairs and have smaller dissociation constants due to hydrogen bonding between the cationic and anionic moieties [15]. X-ray diffraction studies of single crystals of the phospholipid, dilauroyl-oL-phosphatidylethanolamine:acetic acid [16-19], and the sphingolipid, cerebroside (fl-D-galactosyl-N-(2-o-hydroxyoctadecanoyl)-o-dihydrosphingosine) [20], have shown that the molecules are packed in such a way that intermolecular hydrogen bonding can occur. In PE, each amine and phosphate can participate in two hydrogen bonds with intermolecular N - O distances of 2.74 and 2.86 .A, as shown

where A - H is the hydrogen-donating group and B is the hydrogen-accepting group on two lipid molecules. The stability of such hydrogen bonds depends on the differences in the stabilities of the bonds on the two sides of Eqn. 1, not on the

Fig. 1. Head group packing of 1,2-dilauroyl-DL-phosphatidylethanolamine : acetic acid determined from single crystal X-ray diffraction analysis by Elder, Hitchcock, Mason and Shipley [17], viewed perpendicular to the bilayer interface. Dashed lines show the intermolecular hydrogen bonds between the nitrogen of one moleculeand O-13 and O-14 of two neighboring molecules.The acetic acid moleculeshave been omitted for clarity. Reproducedfrom Ref. 17 with permission of the author and The Royal Society.

356
B~ A Ba A2

,~

) O-

U Fig. 2. Conformation and bilayer packing of the head group of cerebroside ( fl-D-galactosyl-N-(2-D-hydroxyoctadecanoyl)-Ddihydrosphingosine) as determined by Pascher and Sundell [20] using single crystal X-ray diffraction analysis. Upper part, the head groups of molecules in one half of the bilayer are viewed parallel to the bilayer. There are two independent molecules A and B in the unit. The A molecules are half a unit edge above the B molecules. The dotted line indicates the intra- and intermolecular hydrogen bond systems between molecules B> A, B 3 and A 2. One system originates at the amide N-H group of molecule A (A-N) and proceeds via the hydroxyl groups A-O2' to B1-O4" to A-O2" to B3-O2 to A2-Ol'. The other hydrogen bond system starts at B3-O6" and continues via A-O3" to A2-O1'. The lengths of the hydrogen bonds are 2.63-2.77 A except for that from B1-O4'' to A-O2" which is 3.02 ~,. Each cerebroside molecule participates in eight hydrogen bonds, forming a tight two-dimensional network of hydrogen bonds throughout the polar boundaries of each layer. An intramolecular hydrogen bond between A-N and A-O1 is also shown. The intramolecular hydrogen bonds between the amide N-H and the oxygens of the glycosidic linkage (O1) and the fatty acid hydroxyl group (02') are shown more clearly in the lower part. Reproduced from Ref. 20 with permission from the authors and Elsevier Scientific Publishers Ireland, Ltd.

absolute strength of the b o n d in A - H - B [21]. A detailed analysis of all enthalpy and entropy contributions from b o n d breaking and b o n d forming, to the free energy of formation of a lipid bilayer

would be necessary in order to evaluate the stability of A - H B. This has not been done and will not be attempted here. However, some properties of a lipid bilayer which would favor intermolecular hydrogen b o n d ing between appropriate lipids can be pointed out. The lipid molecules are already intermolecularly associated as a result of h y d r o p h o b i c effects and the Van der Waal's interactions between the acyl chains. The lipid head groups m a y already be correctly positioned for intermolecular hydrogen b o n d i n g to take place. Correct orientation m a y be facilitated by electrostatic interactions between the head groups Thus, the decrease in entropy which disfavors intermolecular association of two molecules in solution would not be a factor. Smith and T a n f o r d [22] have suggested that intermolecular hydrogen b o n d i n g can be extraordinarily stable when the participating groups have long alkyl chains Thus, the lipid bilayer can be thought of as a large molecule or polymer in which 'intramolecular' hydrogen bonding or intersubunit hydrogen b o n d i n g takes place rather than conventional intermolecular hydrogen bonding. There is m u c h more evidence that the former can occur in water, as in proteins, oligonucleotides and complex carbohydrates, than the latter. However, it should be mentioned that intermolecular hydrogen bonding appears to occur between ionized and unionized species of orthophosphoric acid in water [23]. A n o t h e r factor which m a y help stabilize A - H - B between bilayer lipids is the release of hydrogen-bonded water from the surface of the bilayer which would contribute to an increase in the entropy and help compensate for the energy necessary to break the hydrogen bonds between the lipid and water. The relatively rapid axial rotation of PE and other lipids for which intermolecular hydrogen b o n d i n g has been postulated, and the motion of the head groups, even in the gel phase [24,25], indicate that intermolecular hydrogen b o n d s between the lipid head groups cannot be long lived. Deuterium and 13C-NMR studies show that in the gel phase of PE axial diffusion occurs at a rate of 105-106 s -1 [26,27]. In the gel phase of cerebroside, however, axial diffusion is slower, 10 2 s -1 [281. Vinogradov and Linnell [15] point out that in

357

order to be considered structurally significant the average lifetime of a given arrangement of molecules must exceed, by a factor of about 10, the vibrational period of the bonds which form the structure, The period of an O - H stretching vibration is about 10 -14 S while that of an intermolecular hydrogen bond which absorbs at 200 cm-1 is about 2.10-13 s. The lifetime of hydrogen bonds in liquid water is about 10 -11 s. It seems likely that the lifetime of intermolecular hydrogen bonds between lipids in a bilayer could be of these orders of magnitude. Thus, individual hydrogen bonds between two lipid molecules need not be 'strong' and fixed for long periods of time; they are probably easily broken but then reform with another molecule. They are probably not of a fixed stoichiometry. Each proton and acceptor may be involved in several hydrogen bonds simultaneously. Thus, bilayers may be stabilized over a fairly wide range of ratios of donor to acceptor. Protons may be exchanged from one molecule to another and be transported laterally along the bilayer [29]. Protons can diffuse along the surface of a lipid monolayer of PE 20-times faster than in the bulk phase [30]. This ready availability in the lipid bilayer of other molecules for interchange of protons and rapid reformation of hydrogen bonds may also contribute to the stability of intermolecular hydrogen bonding in the lipid bilayer. Thus, although these bonds may not be long lasting, they stabilize a particular arrangement of lipid molecules in which the bonds can readily reform. Theoretical studies show that it is necessary to include intermolecular hydrogen bonding forces between lipid head groups in order to account for the high transition temperature of PE and PA [4,31-33]. They show further that these forces increase the enthalpy by only a small percentage. The enthalpy of the transition is determined primarily by changes in the Van der Waals' attractive forces and the increase in rotational isomeric energy during the phase transition. Other theoretical studies do not take the intermolecular forces into account specifically, but do make use of experimentally determined parameters, such as the smaller head group volume or the closer packing of PE, which are probably a result of the intermolecular interactions [9,14]. Nagle [32] has estimated that only one hydrogen bond of length 2.8

for every 40 molecules is necessary to increase the transition temperature by 10-12 Cdeg (relative to the neutral state). This is consistent with transient and/or weak hydrogen bonds and the known rotational and translational mobility of PE, but is enough to inhibit lateral expansion of the lipid and stabilize the gel phase.

H-B. Fatty acid-anion complexes

Complex formation between fatty acids and their soaps in water [34-36] may be stabilized by intermolecular hydrogen bonding, and represent the simplest example of the effects of such hydrogen bonding [29]. Fatty acids are in a state of partial dissociation over a wide pH range, both below and above their transition temperatures [29,36,37], as shown by Haines and Heller in Fig. 3 for oleic acid. The pH titration curve of oleic acid has two plateaus [29,36]. At the first one, at pH 9.5, where the fatty acid becomes partially protonated, a phase transition from micelles of fatty acid to acid-anion liposomes occurs. At the second plateau, at pH 7.5, where the fatty acid is protonated by more than 80%, a phase transition to oil droplets occurs. Oligolamellar liquid-crystalline phase liposomes, which are relatively imper-

100 90 80 70 ~- 60 --q 50
....

o~o

0 10 20 ~, 30 i

Liposomes

50
6o 70

~.~

~ g
:~

30

~_~H ~ e lc lh a ~ n g e ~p iih a s e o,~ 6 7 8 pH | 9

80 90

~0

;1

i'~ 100

Fig. 3. Titration curve (e) of aqueous dispersion of oleic acid (50 mM), labeled with [1-13C]lauric acid, from pH 12 to 6 and percent dissociation (A) as determined from the chemical shift using 13C-NMR by T. Haines and M. Heller [29]. The higher inflection point in the titration curve is associated with a transition from anion micelles to acid-anion liposomes. The lower inflection point reflects a phase change from liposomes to oil droplets of the acid form. Reprinted from Ref. 29 with permission from the author.

358

meable to small molecules, are formed only over the pH range where significant percentages of both the anion and acid are present. Below the transition temperature, the acid-anion complex forms crystals over this pH range [37]. Intermolecular hydrogen bonding between the acid and anion is undoubtedly involved in crystal formation below the transition temperature. The formation of liposomes and stabilization of the acid and anion over a wide pH range in the liquid-crystalline phase suggests that hydrogen bonding occurs in this phase also, as concluded by Haines [29]. However, the formation of liquid-crystalline phase bilayers over this pH range has also been attributed to reduction of the surface charge density and repulsive forces when a certain percentage of the fatty acid is in the protonated state [38]. Evidence that formation of bilayers stabilizes both the anion and acid, i.e., that it both lowers the intrinsic pK (first plateau) and raises it (second plateau), would help to support the conclusion that intermolecular hydrogen bonding occurs. The occurrence of intramolecular hydrogen bonding between the acid and anion of the cis form of a dicarboxylic acid, maleic acid, was inferred from the lower pK 1 for dissociation of the free acid to form the monoanion and the higher pK 2 for dissociation of the monoanion to the dianion, compared to the trans form, fumaric acid, in which intramolecular hydrogen bonding is not possible [21]. It is difficult to distinguish the effects of electrostatic repulsion from intramolecular hydrogen bonding on the pK values of simple dicarboxylic acids, but studies of more complex alkylated succinic acids support the involvement of intramolecular hydrogen bonding [45]. Although the apparent pK1 of a long chain fatty acid in a bilayer is considerably higher than for a carboxylic acid monomer in solution (pK 4), this is due partly to the high negative surface charge density of the bilayer resulting in a surface pH which is lower than the bulk pH. The intrinsic pK values are not known. However, the pH dependence of dissociation of the fatty acid-anion bilayer can be compared to that of the fatty acid in a bilayer of similar surface charge density where hydrogen bonding cannot occur, or where it occurs exclusively between another compound and either the anion form or the acid form of the fatty

acid. Some data are available for the latter situation on a fatty acid/alcohol mixture (dodecanoic acid and dodecanol) and a fatty acid/phospholipid mixture (palmitic acid and DPPC). The dodecanoate/dodecanol mixture also forms bilayers at pH values where the fatty acid is ionized and could hydrogen bond with the alcohol [36]. The pK of the fatty acid in the acid/alcohol mixture is 7 and only one plateau occurs. Thus, the fatty acid becomes completely ionized at a lower pH than in the acid/anion mixture suggesting that the anion form is stabilized by hydrogen bonding with the alcohol. In DPPC on the other hand, where hydrogen bonding could only take place between the protonated form of the fatty acid and the phospholipid, the pH at which the palmitic acid begins to dissociate is higher, about pH 8 [39] compared to about 7 for the acid anion mixture [29]. The pK of the fatty acid in DPPC was reported as 10.2, but insufficient data were given to determine whether the titration curve exhibits two or only one plateau. However, the higher pH at which dissociation begins suggests that the acid form is stabilized by intermolecular hydrogen bonding with the anionic phosphate of DPPC. Comparison with the fatty acid/anion mixture then suggests that hydrogen bonding between the acid and the anion stabilizes both forms allowing them to persist over a wider pH range than in the absence of hydrogen bonding.

H-C. Properties of glycerol-based lipids II-C1. Phase-transition temperatures One of the most important properties of lipids which is suggestive of intermolecular hydrogen bonding in the gel phase is the high gel to liquidcrystalline phase-transition temperature of lipids with compatible hydrogen bond-donating and -accepting groups. An increase in the phase-transition temperature indicates stabilization of the gel phase. Intermolecular hydrogen bonding interactions which are stronger in the gel phase where closer lipid packing occurs, would stabilize this phase. If the intermolecular hydrogen bonding interactions can occur equally well in either phase, as may occur for some lipids with large head groups, it would have no effect on the transition temperature, although it might have effects on

359

other lipid properties. A charged head group on the other hand would cause repulsion of the lipid molecules and would stabilize the liquid-crystalline phase where the lipids are less closely packed, resulting in a decrease in the transition temperature. If the phase-transition temperatures of only a few lipids with different polar head groups are compared it may be difficult to distinguish the possible effects of head group size or other factors from that of their intermolecular hydrogen-bonding potential. However, if a large variety of lipids, including a number of synthetic lipid analogues, with different head groups in different ionization states are considered, the conclusion that intermolecular hydrogen bonding is involved seems inescapable. The phase-transition temperatures of a number of synthetic glycerol-based lipids containing palmitic acid at neutral pH are shown in Table I. The ionization state of each lipid at this pH is indicated by the charges present on the head

TABLE I E F F E C T O F T H E P O L A R H E A D G R O U P ON T H E TEMP E R A T U R E OF T H E G E L TO L I Q U I D - C R Y S T A L L I N E P H A S E T R A N S I T I O N , To, A T N E U T R A L p H Lipid DPPA DHMGDG b DPPE DPPS DPPG DPPM DPPC Cardiolipin Dipalmitin Charge a (- ) (0) (- + ) (- - +) (- ) (-) (-, +) (_ ) c (0) Tc ( o C) 65 63.6 63 54 41 44 41 40 50, 63 d Ref. 40 41 40 42, 43 44 8 73, 55 179 47

a N u m b e r and type of charged groups, not net charge. b This fipid has not been prepared with palmitoyl chains. However, comparison of the diacyl and dialkyl forms containing ClS chains suggests that the ether linkage m a y increase the Tc by only 3.5 Cdeg [41,48], similar to its effect on PE. c Charge per two chains. Contains four palmitoyl chains. a The first transition is due to a metastable phase. The second is that of a stable crystalline to isotropic liquid transition [47]. Thus, it is not strictly comparable to the gel to liquidcrystalline phase transitions of the other lipids listed. The enthalpy of the second transition is twice that of the first (Boggs, unpublished data).

group, e.g. PE ( - + ). There does not seem to be any relationship between the size or net charge on the head group and the phase-transition temperature since, of the three lipids with the smallest negatively charged head groups, one, DPPA, has the highest transition temperature, while the other two, DPPM and cardiolipin, are among those having the lowest. The transition temperature of DPPA with its negative charge is similar to those of three lipids with varying head group size and charge, DPPE with a net neutral charge but two ionized groups, and dipalmitin and DHMGDG, with neutral and unionized head groups. The transition temperature of DPPC with a large head group and net neutral charge is similar to those of the negatively charged DPPG, cardiolipin and DPPM. Examination of the transition temperatures of a number of synthetic analogue lipids with different head groups, shown in Table II, indicates that the size of the head group has little effect on the transition temperature as pointed out by Eibl [7,8]. Although there is a decrease in the transition temperature of lipids with long alkyl chains in the head group, this is probably caused by penetration of the chain into the bilayer causing a fluidizing effect. It is more pronounced at low pH values. This fluidizing effect does not occur if there is a charged group at the end of the chain, since insertion of up to ten methylenes between the phosphate and the quaternary amine of PC decreases the transition temperature by only a few degrees. 14N- and 31p-NMR spectra of such PC analogues indicate that an increasing number of methylene groups changes the average orientation of the C - N bond and its dynamics [52]. Monolayer studies indicate that lipids with large head groups have a larger molecular area at low surface pressures than lipids with small head groups, but all can be compressed to a similar molecular area at high surface pressures. The large alkyl head groups can change conformation and extend perpendicularly away from the bilayer into the aqueous phase, if necessary, to fit into the area available to them in a closely packed monolayer or bilayer, as indicated by a 2H-NMR study [49]. The transition temperatures of most of the lipids indicated in Table II, are similar to those of the lowest melting lipids given in Table I. Their simi-

360 T A B L E II E F F E C T OF M O D I F I C A T I O N S TO P O L A R H E A D G R O U P O F S Y N T H E T I C LIPID A N A L O G U E S ON T H E T R A N S I T I O N T E M P E R A T U R E OF T H E G E L TO L I Q U I D C R Y S T A L L I N E P H A S E T R A N S I T I O N , Tc a CH2-OR

RO-CH CH 2 - X Head group X pH 7 charge PO4- - C H 3 (DPPM) PO 4 - C H 2 - C H 3 PO4- - ( C H 2 ) 2 - C H 3 PO4- - ( C H 2 ) 3 - C H 3 PO4- -(CH2)5 - C H 3 PO 4 - ( C H 2) 7 - CH 3 PO4- - ( C H 2 ) 2 - N ( C H 3 ) ~ - (DPPC) PO 4 - ( C H 2) 5 - N ( C H 3 ) f PO 4 - ( C H 2 ) 8 - N ( C H 3 ) ~ PO 4 -(CH2)lo - N ( C H 3 ) ~PO4--(CH2)2-N(CH3) ~ -(CH2)2-N(CH3) ~ PO 4 - ( C H 2 ) 2 - - C(CH 3) 3 N(CH3) ~ PO 4 - C H 2 - C H O H - C H 2OH (DPPG) PO~- - C H 2 - C H 2OH PO 4 - C H 2 - C H 2 - C H 2 O H (((((((((((((+ ((() ) ) ) ) ) + + + + + ) ) ) ) ) T~ ( o C) 44 41 40-42 39-42 33 22 42 40 40 38 40 42 42 41 41 41 ~ pH 1 T~ ( o C) 53 51 38 34 28 28 49 59 8,49 7,49 7,49 7,49 7 7 7,50 b 7,50 7,50 7,50 8 7 8 46,44 46 46 Ref.

) ) ) ) +)

61

In the presence of NaC1. R, palmitic acid. C o m m o n n a m e is indicated in parentheses, if there is one. b Transition temperatures for other odd and even values of n also reported in these Refs. By analogy with the similarity of Tc for a lipid containing this head group and two myristoyl chains to D M P G .

larity regardless of head group size and net charge, which varies from - 1 to + 1, indicates that the low transition temperature of these lipids is due to charge repulsion and not head group size. The fact that DPPC and its longer chain analogues with a net neutral charge also have this low transition temperature indicates that they behave as lipids with a net repulsive charge [7]. Thus, the quaternary ammonium group does not help to reduce the lateral repulsive effect of the negatively charged phosphate, although it does reduce the surface charge of PC bilayers as seen from the aqueous phase. This is also supported by the similarity in transition temperature of the PC analogue with a C(CH3) 3 group and net charge of - 1 to that of PC with the N(CH3) ~- group and net charge of 0. Protonation of the negatively charged phosphate of these lipids raises the transition temperature to 53-61C as a result of loss of charge repulsion. The PC analogue with five methylene

groups has a similar transition temperature at low pH indicating that the large head group, now with a net positive charge, can be accommodated in the gel phase without destabilizing it. The transition temperature of DPPC at low pH is not as high. This may be because the positive charge on the head group of DPPC causes more repulsion than for the analogue with five methylene groups, or because of incomplete protonation of DPPC. The nearness of the positively charged quaternary ammonium group to the phosphate of PC may lower the intrinsic pK of the phosphate, or the increase in positive charge of the bilayer surface as the phosphate becomes protonated may repel protons resulting in a higher surface pH and a decrease in the apparent pK. Addition of palmitic acid to DPPC at pH 1 causes phase separation of two populations of DPPC, (i) a complex of fatty acid and DPPC ( - + ) with a transition temperature of 65 C (see subsection II-E), and (ii) another lipid domain which melted at 61.4C [53]. This latter

361 population may be a population of DPPC (+) w h i c h is c o m p l e t e l y p r o t o n a t e d , e i t h e r p u r e o r m i x e d w i t h p a l m i t i c acid. T h e o c c u r r e n c e o f t w o populations of DPPC supports the conclusion that D P P C is n o t c o m p l e t e l y p r o t o n a t e d at l o w p H . T h i s result also suggests t h a t t h e t r a n s i t i o n t e m p e r a t u r e o f D P P C ( + ) m a y b e close to 6 1 C , s i m i l a r to t h a t o f t h e P C a n a l o g u e w i t h five m e t h y l e n e s at l o w p H . I n t e r m o l e c u l a r h y d r o g e n b o n d i n g has l o n g b e e n h e l d r e s p o n s i b l e f o r the h i g h t r a n s i t i o n t e m p e r a t u r e o f P E at n e u t r a l p H . T h e p H d e p e n d e n c e o f t h e p h a s e - t r a n s i t i o n t e m p e r a t u r e s o f P E a n d PS are s h o w n in Figs. 4 a n d 5. T h e s e w e r e d e t e r m i n e d b y S e d d o n et al. [85] a n d C e v c et al. [42], r e s p e c -

tively. Transition temperatures at selected pH values for these lipids in different ionization states and containing palmitoyl chains are given in Table III. At pH values up to 9 for PE and 4 for PS, under the conditions used, these two lipids each have a hydrogen bond donating NH~ group and a hydrogen bond accepting PO4- group. In these pH ranges, their phase-transition temperatures are at a maximum (Figs 4 and 5A, respectively), and

TABLE III PHASE-TRANSITION TEMPERATURES, To, OF REPULSIVELY CHARGED AND INTERACTIVE STATES OF GLYCEROL-BASED PHOSPHOLIPIDS WITH C16 CHAINS Lipid Charge Tc ( C) 42 41 41 45 44 32 40 64 66 55 69 ~ 65 ~62 d 71 73 61 61 64 50,63 g pH >3 12 >4 11 >6 13 7 7 1 7 1 7 4 2 4 Refs. 73,55 7 44 54 8 42 179 40 4,7,5658,85 42, 43 42 54 54 44,60 8 41 47

160
14(3

tt~ ]

DDPE
2 -tM NoCI

Ha
120 T

Repulsively charged states DPPC ( - +) DPPE (-) DPPG (- ) DPPA (- -) DPPM (- ) DPPS (- -) cardiolipin (_) a Interactive states DPPE ( - +) DPPE ( - , + 1) b or (0) DPPS (_ _ + ) c DPPS ( - +) DPPA DPPA DPPG DPPM DHMGDG Dipalmitin (- ) ( - ) ( - ) or (0) e ( - ) (0) f (0)

A 80
1 " L~

~ 60,

[*C)20t' ~~~~~~L 0" ~

o
lb 12
pH Fig. 4. pH dependence of the gel to liquid-crystalline () and lamellar to hexagonal ([2) phase-transition temperatures of didodecylphosphatidylethanolarnine dispersions in 2.4 M NaCI, determined by Seddon, Cevc and Marsh [85]. All temperatures were determined by DSC except the data point in parentheses at pH 9.28 which was determined from an X-ray continuous temperature scan. The dashed line indicates the appearance of additional diffraction lines in the region of the lamellar to hexagonal transition. The TH values lying above the solid line at pH values from 4 to 7 were obtained with phosphate buffer. Some buffers yielded a constant value of Tr~ over this range while acetic and formic acid buffers abolished the lameUar to hexagonal calorimetric transition. Reproduced from Ref. 85 with permission from the authors and the American Chemical Society.

a See Table I, footnote c. b By extrapolation from measured values for POPE, DHPE and DDPE. Complete protonation may not be achieved. c Partially repulsive and partially interactive. d Temperature decreases with increasing hydration. A T~ of 72 o C was reported for this charge state by MacDonald et al. [43]. e It has been assumed that DPPG was in the neutral state at low pH. However, as discussed in the text, it may be in the ( - ) state. Even if in the neutral state, it should be able to interact intermolecularly by hydrogen bonding. r See Table I, footnote b. g See Table I, footnote d.

362

3o
50 t DMPS --

~7or

2O
qrr:* *~ G :-/ I !' ] "FTI'"!~-

- ** 4 ~ - ~ , . . . . . ;_
' J ~ ' T ~ , ~ T4-T~J,_I1"F "T"r'

!
DMPS

',,.

1
J

.1- t- q-

3O
0 1 2 3 pH z. 5 6

pH

Fig. 5. pH dependence of the gel to liquid-crystalline phase-transition temperature of DMPS (A) at acidic pH and (B) at alkaline pH, determined by Cevc, Watts, and Marsh [42]. The ionic strength was constant throughout at J = 0.1. Transition temperatures were determined by monitoring the height of the ESR spectrum of a partitioning spin label. In (A) transition temperatures (O) were seen only on the first heating scan and probably reflect a less hydrated or crystalline state. The intermediate set of temperatures was observed on subsequent scans of dispersions which were not incubated at high temperature. The lowest set of transition temperatures indicated at low pH ( t ) were obtained for dispersions incubated at 90 o C before scanning and probably reflect greater hydration of the sample. In (B) at pH 11.5-14, (o) corresponds to the main transition temperature and (O) to the pretransition temperature, only observed for PS in the ( - - ) state. Reproduced from Ref. 42 with permission from the authors and the American Chemical Society.

providing they have the same fatty acid chain lengths, are similar to each other (Table III) and to the other lipids having high phase-transition temperatures in Table I. The transition temperatures of both are at a minimum at high pH where the amine is deprotonated and they have acquired a net negative charge ( - 1 for PE and - 2 for PS) (Figs. 4 and 5B, respectively). DPPE in the ( - ) state has a similar transition temperature to the other negatively charged lipids in Table II while DPPS with two negatively charged groups has a lower transition temperature. Although these two lipids at high pH still have a hydrogen bond donating NH 2 group, the net negative charge on the lipid causes repulsion so that the stability of the lipid in the gel phase decreases. Intramolecular hydrogen bonding between NH 2 and PO4 (or COO- for PS) probably occurs instead of intermolecular hydrogen bonding. Addition of hexadecyl amine to DPPE, in a 2:1 molar ratio, at a high pH value where the hexadecyl amine is neutral and the PE has a net negative charge, restores the high transition temperature, indicating that the NH 2 group of a neutral molecule can hydrogen bond intermolecularly with the PO4- of PE ( - ) , although the NH2

group of PE ( - ) in pure PE cannot [53]. These results indicate that charge repulsion between hydrogen-donating and accepting molecules may inhibit intermolecular hydrogen bonding between them, even if suitable hydrogen-donating and -accepting groups are available. Alternatively, the NH 2 of PE ( - ) may no longer be favorably oriented to interact with the PO4- of a neighboring molecule if there is no longer any electrostatic attraction between them. The transition temperature of PS decreases at pH 4-7 where the carboxyl becomes deprotonated and the lipid is in its ( - - + ) state with a net charge of - 1 [42,43]. The fact that the transition temperature of PS ( - - + ) is intermediate between that of the ( - + ) states of PE and PS and the ( - ) state of PE (Table III) indicates that the negatively charged carboxyl weakens the hydrogen bonding interactions rather than abolishing them completely. Shielding of the negative charge of PS ( - - + ) at high ionic strength increases its transition temperature to a similar value as for the ( - + ) state [42] indicating that when the lateral repulsion is decreased, the hydrogen bonding interactions are strengthened and the stability of the gel phase increases.

363

The low phase-transition temperature of DPPG at neutral pH, similar to DPPM and other repulsively charged lipids in Table II, suggests that it does not interact intermolecularly even though it has hydrogen bond-donating and -accepting groups. In an X-ray crystallography study of sodium 1,2-dimyristoyl-sn-glycero-phospho-racglycerol, Pascher et al. [61] have recently demonstrated that intermolecular hydrogen bonds of length 2.7 and 2.9 ,~ occur between the glycerol O-15 and O-16 and the phosphate O-13 as shown in Fig. 6B. The phosphoglycerol head group extends parallel to the bilayer surface (Fig. 6A) as shown by N M R for the hydrated gel phase [62]. However, in excess water, the repulsive forces must dominate over the hydrogen bonding attractive forces as for PE ( - ) so that the stability of the gel phase decreases. Intramolecular hydrogen bonding may take place instead when PG is hydrated. Screening of the charge of PG ( - ) at high ionic strength raises the transition temperature by only 6 Cdeg [69], much less than the 18-20 Cdeg

increase found on lowering the pH [44,63,64]. This suggests that PG ( - ) does not interact intermolecularly by hydrogen bonding even when the repulsive charge is screened. It might be argued instead that intermolecular hydrogen bonding does occur, but to an equal extent in both the gel and liquidcrystalline phases of PG ( - ) and, therefore, it does not stabilize one phase relative to the other. However, the fact that lowering the pH raises the transition temperature by 18-20 Cdeg indicates that the gel phase of PG at low pH is stabilized by intermolecular hydrogen bonding considerably more than the gel phase of PG ( - ). The monosodium form of PA also has a negative charge but a much higher transition temperature than PG. X-ray diffraction of single crystals of the monosodium form of DMPA shows that the head groups are interdigitated into adjacent bilayers (Fig. 7) with the phosphate groups linked alternately from one bilayer to another by short hydrogen bonds of length 2.5 A into linear strands laterally separated by rows of Na + (Fig. 8) [65]. In

10.4 .~

12

IS I

A)

B)

Fig. 6. Packing and interactions of the phosphoglycerol headgroups of DMPG viewed (a) parallel and (b) perpendicular to the bilayer interface. Determined by single crystal X-ray diffraction analysis of sodium 1,2-dimyristoyl-sn-glycero-phospho-rac-glycerolby Pascher, Sundell, Harlos and Eibl [61]. There are two independent molecules (A and B) in the unit which are mirror images with respect to the configuration and conformation of their glycerol head groups. The polar groups interact laterally by an extensive network of hydrogen, ionic and coordination bonds with the sodium ions. Intermolecular hydrogen bonds are indicated by dotted lines and ionic and coordination bonds are indicated by broken lines. The contact distances are given in ,~. A sequence of two hydrogen bonds starts at the glycerol hydroxyl oxygen O(15) of molecule A. The bonds are directed via hydroxyl oxygen 0(16) of molecule B and terminated at a phosphate oxygen 0(13) of another A molecule. A corresponding sequence of hydrogen bonds, but with opposite orientation in the bilayer plane, runs between molecules B - A - B . Reproduced from Ref. 61 with permission from the authors and Elsevier Science Publishers.

364

A)

Fig. 7. Molecular packing and conformation of monosodium DMPA seen along two different axes parallel to the bilayer as determined by single crystal X-ray diffraction analysis by Harlos, Eibl, Pascher and Sundell [65]. The phosphate groups of two apposing bilayers project alternately into the bilayer interface and form a single phosphate group layer common to both bilayers. Thus, two sodium phosphate groups of oppositely oriented molecules determine the molecular cross section (43.3 ~2) of one DMPA molecule in the plane of the bilayer. Reproduced from Ref. 65 with permission from the authors and Elsevier Scientific Publishers, Ireland, Ltd.

the monohydrate form of monosodium dilauroyl phosphatidate, however, the phosphate head groups are no longer interdigitated (Pascher and Sundell, personal communication). Hydrogen bonds of 2.5 A then link the phosphates of molecules in the same bilayer into linear rows separated by rows of Na + and water molecules. The high phase-transition temperature of PA in the ( - ) state suggests that intermolecular hydrogen bonding also occurs in excess water despite the presence of the negative charge, as concluded by Jacobson and Papahadjopoulos [66] and Eibl and Blume [54,67]. Interestingly, a thioanalog of DPPA (1,2-dipalmitoyl-sn-glycero-3-thionphosphate) at neutral pH had a transition temperature similar to that of DPPG ( - ) and DPPC [348]. This was attributed to weaker hydrogen bonding between molecules of the thioanalog, since S-H hydrogen

bonds are weaker than O - H [15]. The behavior of PA ( - ) is different from that of PG ( - ) and PE ( - ) , since PA ( - ) apparently can participate in intermolecular hydrogen bonding in spite of its negative charge. However, intramolecular hydrogen bonding is impossible for PA, unlike the latter two lipids. Furthermore, the number of possible orientations of the head group is more limited for PA so that it may not be necessary to have electrostatic attraction of two groups as in PE ( - + ) in order to achieve the optimal orientation for hydrogen bonding. However, it seems unlikely that PA is organized into as regular an array in the hydrated gel phase as in the crystal. Rotational motion of the lipid may occur in the gel phase. Transient hydrogen bonding may occur whenever two or more molecules are correctly oriented. Such hydrogen bonding,

365
b

A)

80o U

?oo
/ / / O-L ~o ~o /

"' ""

B)

~ 60,,, !
I--

o_
z n,-

/1.2

- H H - GP

y - TT- GP

PROTON CONCENTRATION(pH)

Fig. 8. Packing and interactions of the polar part of DMPA viewed (A) parallel and (B) perpendicular to the bilayer interface, as determined by Harlos, Eibl, Pascher and SundeU [65]. The orientation in (A) is identical to that in Fig. 7A except that the polar region of two bilayers is shown. In (B) the phosphates of two apposing bilayers are shown, the plane of one bilayer above that of the other. Intermolecular hydrogen bonds are indicated by dotted lines and ionic bonds by broken lines. Hydrogen bonds form between phosphate oxygens O(13) and O(14) of molecules in apposing bilayers and link the phosphate groups of apposing layers alternately into a zig-zag ribbon extending along the bilayer interface in the b direction. These rows of phosphate ribbons are separated by rows of sodium ions in the a direction. Reproduced from Ref. 65 with permission from the authors and Elsevier Scientific Publishers Ireland, Ltd.

Fig. 9. pH dependence of the gel to liquid-crystallinephasetransition temperature of DTPA (O) and DHPA (O) determined by Eibl [8]. The lipid was in distilled water at a concentration of 1 mg/ml. The pH was adjusted by the addition of dilute NaOH. The dotted lines indicate regions where two transitions were sometimesobserved,a lower and an upper transition. Reproduced from Ref. 8 with permission from the author and Academic Press, Inc. and hydrogen bond-donating and -accepting groups are present (see Fig. 10). When the lipid is completely ionized the temperature drops by about 14-22 Cdeg, and becomes similar to that of the repulsively negatively charged lipids in Table II if the chain length is similar (see Table III for comparison of lipids with palmitoyl chains). When the lipid is completely protonated, the temperature drops by 6-14 Cdeg (depending on species of PA). However, the lipid is then in an anhydrous crystalline form and the transition temperature may no longer be comparable to that of hydrated gel phase lipids. The fact that it becomes dehydrated when completely protonated may be due to greater intermolecular hydrogen bonding in this form than when partially ionized. The maximum in the transition temperature of

however, should stabilize the gel phase, where it has a greater probability of occurring than in the liquid-crystalline phase. The effect of p H on the transition temperature of PA [8,54,67], shown in Fig. 9, shows that the relative stabilization of the gel phase over the liquid-crystaUine phase is greater for PA ( - ) than for the completely ionized form, PA ( - - ) . The transition temperature is high over the pH range 4-10 where the state of dissociation is 0.5 to 1.5

366

CHARGE
H 0 0.0
I

PHOSPHATIDIC ACID
H O
I

H O
I

H O
I

H O
I

PO-OR PO-OR PO-OR PO-OR PO-OR

&

&

0''" H'"8"" H'"O

I I I
I

H O
I
I

0"".H
i
I

0.5
H"

PO-OR PO-OR PO-OR PO-OR PO-OR

I I

O.HOO~%HoO

O"
I

..H.

e "'0
I

0""
I

.H.

e "'O
I

O"
I

..H

1.0

PO-OR PO-OR PO-OR PO-OR PO-OR

H'"

.o

o...H...o
.

o.......o
8 I
i o

8-'"" I
1

"'-8i o..
0
I

8-'" I
..o
I

PO-OR PO-OR PO-OR PO-OR PO-OR

...o

o
0

" A
0

O I
2.0
I

0 i
I

0 i
I

O I
I

O I
I

PO-OR PO-OR PO-OR PO-OR PO-OR

Fig. 10. Depiction of the intermolecular hydrogen bonding interactions in the polar region of different ionization states of PA by Eibl [8]. When completelydeprotonated the hydrogen bonds are broken and stabilization is lost. Reproduced from Ref. 8 with permission from the author and Academic Press, Inc.

PA occurs at a state of dissociation of 0.5. The small drop in transition temperature from pH 4-10 is due to the increasing concentration of negatively charged lipid as the state of dissociation increases to 1.5. At p H 4 (the pK1) the ratio of negatively charged-to-neutral species is 1 : 2 and most of the negatively charged molecules will be constantly involved in hydrogen bonding even if these hydrogen bonds are continually being broken and reformed. However, at higher pH, this ratio increases and the probability of non-participation of some negatively charged lipid in hydrogen bonding at any one moment increases. This negatively charged lipid will cause charge repulsion

and will destabilize the gel phase lowering the transition temperature. A similar gradual drop of a few degrees in the transition temperature of PE occurs as the pH is increased from low p H to pH 3 at high ionic strength (Fig. 4), or pH 11 at low ionic strength (Fig. 11). This illustrates the ability of even a small amount of charged lipid, which is temporarily not involved in hydrogen bonding, to destabilize the gel phase and lower the transition temperature. The much larger drop in transition temperature which occurs for PS when the carboxyl becomes deprotonated (Fig. 5A) reflects the presence of one negatively charged group per molecule which either does not participate at all in hydrogen bonding or else shares the hydrogen bond donors with the other negatively charged group. In either case, the charge repulsion is greater and destabilizes the gel phase more. The difference in transition temperature between the putative hydrogen bonding states of PE ( - + ), PA ( - ) , and PS ( - - + ) and their repulsively charged states, PE ( - ) , PA ( - - ) and PS ( - - ) , is 22-25 Cdeg (Table III) [7,42,44,54,67]. These large differences cannot be entirely accounted for by electrostatic repulsion [4,8]. Screening of the charge at high ionic strength does not raise the temperature to those of PE ( - + ), PA ( - ) and PS ( - - + ), consistent with the idea that these ionization states are involved in intermolecular hydrogen bonding [42]. A large increase in transition temperature of PG and PM of 17-20 Cdeg (Table III) on lowering the pH to 4 in the case of PM [7,68] and even lower for PG [44,63,60], also cannot be accounted for by electrostatic effects [4,8,69], suggesting that these lipids must also be involved in hydrogen bonding at low pH. Eibl and collaborators [4,8] investigated the effect of lower pH on PM using low ionic strength solutions and found that a further decrease in pH below 4 causes the transition temperature to drop by 7-9 Cdeg (Fig. 11) similar to the effect of low p H on PA (Fig. 9). They suggested that at a pH where the transition temperature of PM is at a maximum, the lipid is in a state of dissociation of 0.5 and intermolecular hydrogen bonding occurs between the protonated and ionized forms of the lipid. When the lipid is completely protonated the transition temperature falls. Thus, the difference in transition temperature between the neutral,

367

80-

"~
"

~0--"0-..--0~ O0

/
t2-1-tH-6PE

~60-

**,

_oz
z =
r

.~

\12-PP-GPMe I

/I 40"
I

k
7/O'O IO ~t2-MM-GPMe

3 6 9 12 PROTONCONCENTRATION (pH)
Fig. 11. pH dependence of the gel to liquid-crystalline phasetransition temperature of DHPE (), DPPM ( * ) and DMPM (O) determined by Eibl [8]. Reproduced from Ref. 8 with permission from the author and Academic Press, Inc.

non-hydrogen bonding states and the repulsively singly charged states was concluded to be only 8-12 Cdeg, a difference which can more easily be accounted for by electrostatic repulsion [4,8]. However, when completely protonated PA and possibly also PM are in a crystalline dehydrated state in which hydrogen bonding may still occur. It may not be valid to compare the transition temperature of this crystalline state to that of the more hydrated state when partially ionized. Thus it is difficult to know what the transition temperature of the neutral, non-hydrogen bonding species would be if it were in the same kind of bilayer phase as when ionized. Cevc et al. [69] showed that screening of the charge of PG ( - ) at high ionic strength raised the transition temperature by only 5.5-6.5 Cdeg, indicating that the electrostatic contribution is of this magnitude. If hydrogen bonding occurs in addition to elimination of electrostatic repulsion, it raises the transition tempera-

ture by an additional 8-16 Cdeg. Thus, mere elimination of repulsion does not stabilize the gel state over the liquid-crystalline state as much as introduction of hydrogen bonding interactions. The maximum transition temperatures reported for PG, PE, and PS at low pH may also reflect hydrogen bonding between phosphate acid and anion forms. A drop in transition temperature on lowering the pH further might be expected to occur for these lipids as for PM and PA but has not yet been reported. It may be impossible to completely protonate PG, PE, and PS due to the low intrinsic pK values of their phosphate groups or to the decreased attraction of protons to the bilayer surface as the lipids become more protonated, and in the case of PE and PS, more positively charged. Phosphate acid-anion hydrogen bonding for these lipids also may lower their intrinsic pK values as it appears to do for fatty carboxylic acid-anion mixtures (see subsection IIB). Alternatively, these lipids, or at least PG, which is uncharged in its completely protonated state, may be completely protonated at low pH but continues to interact by intermolecular hydrogen bonding. Thus, the transition temperature of PG at low pH, which is similar to that of PE ( - + ) and PA ( - ) in their interactive states, may reflect the completely protonated form which hydrogen bonds intermolecularly via the glycerol OH and P-OH groups. In support of this, addition of palmitic acid to DPPG at pH 1 does not cause phase separation of two populations of DPPG as it does for DPPC, suggesting that only one population, the completely protonated form, is present [53]. Studies with neutral unionized glycolipids indicate that it is not necessary for the lipid to have ionizable groups in order to participate in intermolecular hydrogen bonding. The high transition temperature of the glycerol-based glycolipid MGDG, which is similar to that of PE ( - + ) and PA ( - ) of identical hydrocarbon chain composition (Table I), indicates that intermolecular hydrogen bonding occurs between the sugar hydroxyls [41,48,70,71]. Intermolecular hydrogen bonding between neighboring molecules of the neutral lipid dipalmitin may be responsible for its high transition temperature also and for its formation of a dehydrated crystalline phase in water (Table I).

368 Single crystal analysis of 2,3-dilauroyl-D-glycerol showed that it forms bilayers with intermolecular hydrogen bonds of length 2.8 ,~ between the hydroxyl head group and the v-carbonyl oxygen of the ester linkage of adjacent molecules [72]. Thus, it is reasonable to suppose that PG may also interact by intermolecular hydrogen bonding in its completely protonated state. The various lipids with different head groups and in different ionization states, can be classified into two categories on the basis of their phase transition temperatures (Table III). There is no correlation between head group size or charge in these categories but there is a correlation with their capacity for intermolecular hydrogen bonding interactions. Those which have a phase-transition temperature of 41-45 C (also including those lipid analogues listed in Table II) are dominated by repulsive forces which stabilize the liquid crystalline phase. Those which have a phase transition temperature of 61-73 C are dominated by intermolecular hydrogen bonding interactions which stabilize the gel phase. Van Dijck et al. [73] noted that the large difference in transition temperature between PE and PC disappears if there is a cis unsaturated chain in both the 1 and 2 positions. This suggests that the large molecular volume taken up by the hydrocarbon chains may cause too much lateral separation for hydrogen bonding to occur for PE even in the gel phase. Branched chains may have a similar effect. Diisopalmitoyl PE goes into two phases, one of which has a similar transition temperature as the corresponding type of PC [74]. The other, in which interbilayer hydrogen bonding may take place, since it occurs more when the lipid is concentrated, has a transition temperature only 13 Cdeg higher than PC. Thus, although isobranched chains do not have a large disordering effect on the gel phase, this result suggests that they may inhibit lateral hydrogen bonding for PE. More natural, 1-saturated, 2-unsaturated forms of PE still have transition temperatures 15-25 Cdeg higher than the corresponding PC, however, as determined on POPE and POPC, SOPE and SOPC, and on egg PC and the PE prepared from it by transphosphatidylation [4,56,75,76]. PE and some of the other hydrogen bonding lipids go into other phases in which intermolecular hydrogen bonding is also undoubtedly involved. If water is added to PE at a temperature below its phase transition temperature it forms a crystalline, dehydrated or nearly dehydrated phase whose transition to the liquid-crystal phase occurs at a higher temperature and with a higher enthalpy than the gel to liquid-crystal phase transition [59,77-79]. Incubation of a previously heated gel phase sample at 2C for prolonged periods can allow the crystal phase to reform [80,81]. The less hydrated phase of PS ( - + ) with the highest transition temperature (Fig. 5A) may also be a similar crystalline phase [82]. Saturated forms of MGDG and DGDG in which the chains are ester-linked also undergo a transition from a metastable phase to a stable gel phase which has a higher phase-transition temperature and enthalpy than the metastable phase [48,83]. The stable phase may be less hydrated than the metastable phase. Intermolecular hydrogen bonding must occur in the less hydrated stable.phases of these lipids also and would be expected to be stronger than that for the more hydrated gel phase. Intermolecular hydrogen bonding in these less hydrated phases may be interlamellar rather than intralamellar. The effect of modification of the head group of PE on the transition temperatures of its gel and crystalline phases, TG and TcR, respectively, has been determined in a number of studies [80,81,84-87]. The head group was modified in ways which affect its size, hydrophobicity, and ability to hydrogen bond. Data from one of the most comprehensive of these studies [80] is given in Table IV. Methylation of the amine decreases T~ but in a non-linear way with increasing number of methyl groups, suggesting that increasing head group size is not responsible. Data from a number of studies show that one methyl group decreases the temperature by 24-28% of the difference between PE and PC, while two methyl groups decreases it by 61-76% [80,81,84-87]. The effect of an ethyl group is between that of one and two methyls [80,81]. Alkylation of the amine would be expected to decrease the probability of hydrogen bonding interactions due to steric hindrance, with one small group having less effect than two groups or one larger group. Interestingly, X-ray diffraction analysis of a single crystal of the dimethyl form of

369 TABLE IV EFFECT OF MODIFICATIONS TO E T H A N O L A M I N E TYPE HEAD G R O U P ON TRANSITION TEMPERATURES a CH2-OR

I
RO-CH O

II I
O-

CH 2 - O - P - O - X

Group X
C H 2 - C H 2 - N H ~- (DMPE) C H 2 - C H 2 - N H 2 ( C H 3 ) + (MeDMPE) C H 2 - C H 2 - N H ( C H 3 ) ~- (Me2DMPE) C H E - C H E - N ( C H 3 ) ~- (DMPC) CH2-CH2-NH2(CH2-CH3) + CH 3 CH 2 - C H - N H ~ CH3

TG b (C)
50.1 42.7 31.4 23 d 37.7

TCR b (C)
57.3 c c c 59.3

(43.4) e 64.4, 73.2

I I

CH2-C-NH ~

(37.5) e

80, 81

CH3
CH 2 - c a 3

I
CH2-CH-NH~ (CH2)3-NH ~ (CH2)4-NH~ (34.2) e 62.0 41.9 34.4 52.5 51.6

a R is myristic acid. Charge on head group is ( - + ). Common name is given if there is one. All data from Ref. 80 except where noted. b T~ is temperature of gel to liquid crystal phase transition, TCR is temperature of high enthalpy crystalline to liquidcrystal phase transition. c Crystalline phase does not form. d From Ref. 55. e Very low enthalpy peaks, often preceded or followed by exothermic transitions (see text).

2,3-dilauroyl-rac-glycero-l-phospho-N, N-dirnethylethanolamine showed that intermolecular hydrogen bonding occurs between the phosphate oxygen of one lipid and the single N - H of another [88]. However, the head group is extended perpendicu-

lar to the bilayer plane and is interdigitated into the head group layer of the adjacent bilayer. Thus, the phosphate of a molecule in one bilayer hydrogen bonds with the amine of a molecule in the adjacent bilayer. In contrast, PC, PE and PG have their head groups oriented parallel to the bilayer plane in both the crystal and the hydrated gel phase [16,19,61,62]. The fact that Me2PE is different suggests that a perpendicular orientation significantly increases the probability of its hydrogen bonding with another molecule. Thus, the methylated forms of PE can hydrogen bond intermolecularly if a suitable orientation of the head groups can be achieved; however, a parallel orientation may not be very suitable, particularly for the dimethylated form. However, it is unlikely that a perpendicular orientation occurs for Me2PE in the presence of water. Indeed, both mono- and di-N-methylation appear to prevent formation of the crystalline phase, in which interbilayer hydrogen bonding probably occurs [80,81,87]. In contrast, monoethylation of PE does not prevent formation of the crystalline phase. In fact it raises the temperature of its transition to the liquid-crystal phase (Table IV) indicating a stabilizing effect on the crystalline phase. Perhaps, because of its greater hydrophobicity or steric factors, the monoethyl head group is more likely to be extended perpendicular to the bilayer in the presence of water, so that it can hydrogen bond with lipid in the apposing bilayer and become dehydrated. The less hydrophobic and smaller monomethyl form may have the more usual parallel orientation and hydrogen bond only laterally with molecules in the same bilayer. Lateral hydrogen bonding of the dimethyl form must be greatly weakened by the suboptimal orientation and steric effects. An increasing number of methylene groups between the phosphate and amine also decreases the transition temperature, although with four methylenes it is still higher than that of PC [80,81,85]. The fact that the crystalline phase still forms suggests that these head groups can still participate in interbilayer hydrogen bonding. The most likely reason for the decrease in TG is that these longer head groups can hydrogen bond laterally with neighboring lipids nearly as well in the liquid-crystal phase as in the gel phase and, thus,

370 do not stabilize the gel phase as much as ethanolamine does. Addition of more than one sugar to the head group of glycolipids may have a similar effect. DSDGDG has a transition temperature 20-30 Cdeg below that of the corresponding MGDG and lower than DSPC [48,70]. Although the sugar hydroxyls of DGDG should also be able to participate in intermolecular hydrogen bonding, this hydrogen bonding may be able to occur equally well in the liquid-crystal phase, because of the larger size of the head group, and, hence, may not stabilize one phase relative to the other. However, two synthetic diglycosyldihexadecylglyceridesconraining maltose and cellobiose have transition temperatures which are 11-15 Cdeg higher than the similar chain length DPPC, suggestive of hydrogen bonding which does stabilize the gel phase in these cases [89]. C-2 alkylation of the ethanolamine head group was found to decrease TG and increase TCR, sometimes greatly as for C-2 dimethylation [80,81] (Table IV). This led to the conclusion that head group size and hydrophobicity are more important in determining the transition temperatures than hydrogen bonding. However, the enthalpies of the gel to liquid-crystal phase transitions observed for C-2 alkylated lipids are very small and are usually preceded or followed by exothermic transitions and, at a higher temperature, by the large enthalpy crystalline to liquid-crystal phase transition. It is not necessary to incubate these samples at low temperatures to convert them to the crystalline phase. Raman spectroscopy showed that during these exothermic transitions a decrease in the number of gauche conformers and an increase in chain packing density occurs [81]. Thus, the small lower temperature endothermic and exothermic transitions observed for these lipids involve transitions to the crystalline phase, either directly from the gel phase or from the liquid-crystal phase; the latter exists only transiently. The very rapid conversion of the liquid-crystal phase to the crystalline phase suggests that the liquid-crystal phase of the C-2 alkylated PE's may be less hydrated and more involved in hydrogen bonding than that of unmodified PE, due to the greater hydrophobicity of the C-2 alkylated forms. Thus, the liquid-crystal phase is more stable relative to the gel phase than is the case for unmodified PE and the transition temperature is lower. The combined factors of intermolecular hydrogen bonding and decreased hydration forces caused by the greater by_ drophobicity of the head groups also stabilize the crystalline phase relative to the liquid-crystal phase of the C-2 alkylated PE's, thus raising TCR. Thus, hydrogen bonding interactions must be one of the determining factors in the behavior of the C-2 alkylated forms of PE, even though they have a lower TG than unmodified PE.

H-C2. Other properties

Other properties of glycerolipids which can be correlated with the repulsive or attractive forces between their head groups are their molecular areas in monolayers, bilayer permeability, their degree of hydration, the change in enthalpy and entropy during the gel to liquid-crystalline phase transition, and their molecular motion and conformation determined by use of spectroscopic techniques. Lack of reactivity of the amino group of pure PE and PS with 2,4,6-trinitrobenzenesulfonic acid and increased reactivity of PE when mixed with PC led to the early suggestion that the amine is involved in inter- or intramolecular hydrogen bonding [90]. The permeability of PE vesicles is less than that of PC and does not depend on acyl chain length, in contrast to PC, indicating that the reduced permeability of PE is controlled by its head group interactions [91]. The permeability of PE vesicles and hydrolysis of PE by phospholipase A 2 do not increase at the phase-transition temperature, in contrast to PC [91,92], suggesting that intermolecular hydrogen bonding of PE may persist in the liquid-crystalline state. The hydrogen bonding lipids PE ( - + ) and PA ( - ) pack more closely in monolayers than PC [93,94]. MGDG forms a more condensed monolayer than DGDG [95-98]. However, the collapse pressure is higher for the distearoyl form of DGDG which was attributed to its greater number of hydrogen bonding groups [97]. Hydrogen bonding by DGDG may occur but does not cause it to form a closely packed monolayer because of the larger size of the head group. An X-ray diffraction study of MGDG indicated that the head group could reorient to reduce steric hindrance in the

371

more closely packed stable crystalline phase [357]. A similar reorientation may allow it to pack closely in monolayers. The diglycosyldiacylglycerals containing maltose and cellobiose precipitate spontaneously in water and are considerably more ordered at the surface as monitored with dansylhexadecylamine, in both the gel and liquidcrystalline phases, than DPPC, consistent with their higher phase-transition temperatures, suggesting that intermolecular hydrogen bonding by these diglycosyl groups does cause closer packing of the lipid [89]. Incorporation of hydroxyl groups into the lipid head group of anionic phospholipids was found to have a condensing effect in monolayers, leading to the suggestion that charged lipids such as PG interact intermolecularly by hydrogen bonding [46]. However, this condensing effect occurs more at pH 5.5 where the lipid may be partially protonated, than at pH 7. Condensation occurs at pH 7 only at high surface pressures, indicating that if PG is compressed, intermolecular hydrogen bonding interactions can overcome the repulsive forces between the negatively charged head groups. However, as discussed earlier, the low transition temperature of PG suggests that this does not occur at the surface pressure of a gel-phase bilayer; rather, the repulsive forces predominate. Head group interactions also decrease the critical micelle concentration, determined using spinlabeled phospholipids, while repulsion between the head groups increases it [356]. The critical micelle concentration increased in the order PE < PC < PA ( - ) < P G < P S ( - - + ) < < P A ( - - ) . Thus, head group interactions can influence lipid self-assembly and the transfer of lipid between vesicles. The occurrence of tilting of the lipid molecules in the gel phase, the presence of an endothermic premelt transition in which the lipid molecules become less tilted [99], and the observation of a ridge pattern on the fracture surface of the bilayer by freeze-fracture electron microscopy can be correlated with the absence of intermolecular hydrogen bonding interactions between the head groups. Lipids with a repulsively charged head group such as PC are tilted at an angle to the bilayer normal and have a premelt transition [99], while lipids with the capacity for intermolecular hydrogen bonding such as PE are not tilted and do not have

a premelt transition [100]. The tilt allows maximum interaction between the acyl chains and maximum separation of the repulsively charged head group. At the premelt transition more water may penetrate into the polar head group region lessening the repulsive effect and allowing a decrease in tilt. This change in tilt causes a rippling of the bilayer which can be observed by freezefracture electron microscopy as a distinctive ridge pattern on the fracture surface of the bilayer at temperatures between the premelt and main transition [101]. The occurrence of tilting a n d / o r a premelt transition and freeze-fracture ridge pattern have been observed for DPPG ( - ) but not when protonated, for DTPA ( - - ) (at 2 M K ) and DHPA ( - - ) but not in their ( - ) states, for DHPE ( - ) (in 1 M Na ) but not in the ( - + ) state [57,64,66,103-105]. A low enthalpy endotherm resembling a premelt transition a few degrees below the main transition of DMPS ( - - ) and DPPS (--) has been observed [42] (and see Fig. 5B) although this lipid has not yet been studied at high pH by freeze-fracture electron microscopy or X-ray diffraction. Saturated forms of PS in the ( - - + ) state may be slightly tilted in the gel phase [82]. No pretransition has been detected for the mono- and dimethylated forms of DPPE but infrared spectroscopy suggests there may be a progressive increase in tilt in the gel phase with increasing methylation of PE [87]. Thus, it appears that in general, lipids with repulsively charged head groups are tilted in the gel phase to allow maximum separation of their head groups, while lipids with head groups which can interact intermolecularly by hydrogen bonding are not tilted because their attractive interactions resist head group separation. However, there may be exceptions to this since synthetic PE's containing chain lengths longer than 18 carbons have been recently reported to form a more hydrated tilted gel phase and show less tendency to form the crystalline phase than shorter chain length PE's [106]. This is surprising and difficult to reconcile with the other results mentioned. The limited hydration of PE ( - + ), PS ( - + ), MGDG and DGDG, compared to repulsively charged lipids such as PC, PG, PM and the (--+) state of PS [5,42,48,71,82,83,105,107,

372

109,355] is also consistent with intermolecular hydrogen bonding, rather than hydrogen bonding of the ionized groups in these lipids to water. The principal values of the 3~p-chemical shielding tensors of hydrated PE are similar to those of anhydrous PE and PS and monohydrate PC, and smaller than in hydrated PC. This suggests that the amine interacts with the phosphate in PE in place of water in contrast to PC [110,111]. Infrared dichroism spectroscopy has also provided evidence for hydrogen bonding between the amine and phosphate in DPPE both in the anhydrous form and in the hydrated gel state [112]. Bilayers of PE in the gel phase are separated by only 5 A, the thickness of two water molecules, in contrast to PC bilayers which are separated by a distance corresponding to the thickness of five water molecules [113]. This distance does not increase in the liquid-crystalline phase of PE or upon addition of cholesterol. The repeat distance of bilayers of methylated forms of DMPE is greater than for unmodified DMPE [86] suggesting that the methylated forms are more hydrated, consistent with their weaker hydrogen bonding interactions. The small amount of water between bilayers of PE suggests that attractive interbilayer forces such as electrostatic interactions and hydrogen bonding must compensate for the repulsive hydration forces between the bilayers and that these interactions are not solely in the plane of the bilayer [113]. These interbilayer forces probably lead to formation of the less hydrated crystalline phase of PE after prolonged incubation and prevent complete hydration when water is added to PE below its transition temperature. There is little variation in the transition enthalpies of different glycerolipids at neutral pH. In order to judge if small differences are significant, it is necessary to compare values for different lipids obtained in the same laboratory as much as possible in order to eliminate differences in instrument calibration or systematic errors in quantitation of lipid. Blume [114,40] has found using several species of PA, PC and PE of varying chain length, that the enthalpy of PC is similar to or somewhat greater than that of PE ( - + ) while PA ( - ) has a definitely lower enthalpy (Table V). Others also have found no difference in enthalpies of PC and PE [77], while some studies report a

TABLE V E F F E C T OF D I F F E R E N T H E A D G R O U P S A N D IONIZAT I O N STATES ON T H E R M O D Y N A M I C P A R A M E T E R S OF G E L TO L I Q U I D - C R Y S T A L L I N E PHASE TRANSITION T~ ( C) Ester-linked lipids DPPC (- +) DPPE (- +) DPPA (- ) (- -) DPPG (- ) DPPS (- + ) (- - + ) Ether-linked lipids DHPC (- + ) DHPE (- +) (- ) DHPA (- ) (- -) Increasing methylation DMPE (- +) MeDMPE (- +) Me/DMPE (- +) DMPC (- +) AH (kcal/ mob 8.7 8.6 7.9 5.7 8.3 8.1 9.1 AS (cal/ mol deg) 27.7 25.5 23.4 17.8 26.4 23.8 27.8 Ref.

41.5 63.9 65.0 43.1 41 67 54

114 114 114 114 115 82 82

43.5 68.5 45.6 73.3 53.8

8.5 7.6 9.2 7.1 5.8

26.7 22.3 28.9 20.5 17.8

114 114 56 114 114

50.1 42.7 31.4 23

5.9_+0.6 8.0_+0.4 7.2_+0.3 5.7_+0.4

18.2 25.3 26.2 19.2

77 77 77 77

somewhat smaller enthalpy for PE compared to PC [73,86]. The enthalpies of PS and PG are also similar to PC, PE and PA, indicating that it is differences in the transition entropies which are responsible for the variations in transition temperature of these lipids. The transition entropies of PC, PG and PS ( - - + ) are larger than those of PE ( - + ) and PA ( - ) (Table V). The differences between PC and PE ( - + ) and PA ( - ) are greater for the ether-linked lipids than the ester (Table V). The smaller transition entropies of PE ( - + ) and PA ( - ) could be a result of greater disorder in the gel state or greater order in the liquid-crystalline state relative to PC. In view of the ability of PE ( - + ) and PA ( - ) to participate in intermolecular hydrogen bonding, the latter is more likely. Consistent with this, the change in volume of D M P E during the phase transition was determined by dilatometry to be 28% less than that of DMPC, indicating restricted lateral expansion in PE above the phase transition temperature [116].

373 Greater differences in transition enthalpies and entropies occur for different ionization states of particular lipids (Table V). The transition enthalpies and entropies are less for PA in its non-hydrogen bonding ionization state ( - - ) than for PA ( - ) , regardless of chain length or type of linkage [66,67,114], suggesting that either PA ( - ) is much more ordered in the gel state or more disordered in the liquid-crystalline state than PA ( - - ). Because of the greater repulsive charge on PA ( - - ) which would favor a more expanded state with greater freedom of motion in the liquid crystalline state, the former is more likely. This does not rule out the possibility that the liquid crystalline phase of PA ( - ) may also be more ordered than that of PA ( - - ). A particularly low transition enthalpy and entropy was found for DHPA in the ( - 1 / 2 ) state [67], suggesting that for this ionization state at least, the liquid crystalline phase is also more ordered than that of DHPA ( - - ). The transition enthalpies and entropies are greater for PE ( - ) in its non-hydrogen bonding state and PS ( - - + ) in its weaker hydrogen bonding state than for PE and PS in their strong hydrogen bonding states ( - + ) [42,56,57,82], suggesting that the ( - + ) states are more ordered in the liquid crystalline phase than the more negatively charged states. Mono- and dimethylation of PE were found to significantly increase the transition enthalpy and entropy in a number of studies [77,80,81,86] (see Table V), although not in one study [87]. This greater entropy suggests that methylation increases the disorder in the liquid crystalline phase. It probably also increases the disorder in the gel phase as indicated by infrared studies of DPPE, DPPC and the mono- and dimethylated forms [87]. Thus, the differences in transition enthalpies and entropies of PA ( - ) , PE ( - + ) and PS ( - + ) compared to their repulsively charged states, to other repulsively charged phospholipids such as PG and PC, and to the weaker hydrogen bonding methylated forms of PE, are consistent with intermolecular hydrogen bonding in the gel state. They also suggest that weaker intermolecular hydrogen bonding may persist in the liquidcrystalline phase. Consistent with this, the deuterium order parameter of the chains, deuterated at the second carbon, in the liquid crystalline phases of DPPS ( - - + ) and DPPE ( - + ) is a little greater than for DPPC [117]. N M R studies also showed that the acyl chains of the liquidcrystalline phase of DPPE are subject to greater conformation constraints than in DPPC [26,27]. Proton and 3~p-NMR studies of natural forms of PE and PC in their liquid-crystalline phases also showed that the molecular motion of the head group of PE ( - + ) at neutral pH is less than that of PC and of PE ( - ) at high pH [118]. The motion of spin labels in natural PE in the liquidcrystalline phase also increases at high pH [119]. An N M R study showed that the motion of the head groups of PE and monomethyl PE are restricted to a similar extent [108], supporting the involvement of monomethyl PE in hydrogen bonding. However, an N M R comparison of various saturated lipids with head groups deuterated at several positions found that in the liquid crystalline phase, the ethanolamine and choline head groups have similar motion, as deduced from the 3~p-chemical shift anisotropy and deuterium quadrupole splittings, while only the serine ( - - + ) group has much less motion [62,120]. Dilution of PS with PC causes an increase in motion of the serine head group. On the other hand, an 15N-NMR study showed that the activation energy for the motion of the C - N bond was 1.6-times greater for DPPE than DPPC [348]. Furthermore, the rates of head group motion as monitored from deuterium N M R spin-lattice (T1) relaxation times, are less for the head groups of PE and PS than for PC or a lipid containing the repulsively charged phosphopropanol head group [121]. The T1 value for serine ( - - + ) is equal to that of serine methyl ester ( - + ). In the propyl and choline head groups, the rate of motion of the r-segment is greater than that of the a-segment, while the ethanolamine and serine head groups have identical rates of motion at all positions, indicating greater flexibility of the repulsively charged head groups than the interactive ones. These results indicate increasing head group flexibility in the liquid crystalline phase in the order PS < PE < PC, which is consistent with inter- or intramolecular hydrogen bonding in PE and PS. Deuterium T1 values of anionic hydroxyl contain-

374 ing head groups indicated that addition of hydroxyl groups to a phosphopropanol head group substantially reduces the rates of motion in both the liquid-crystalline and gel states and decreases the flexibility of the head group [46]. This was attributed to intra- and intermolecular hydrogen bonding. However, in view of the low transition temperature of these lipids, it probably indicates intramolecular hydrogen bonding. The motion of the sugar head group of several synthetic monoglycosylglycerolipids studied by 2H-NMR, was lower than that of the head groups of phospholipids [354]. Whether or not intermolecular hydrogen bonding persists in the liquid crystalline phase is an important question since most natural lipids are in this phase at physiological temperature. Although some of the results discussed above suggest that hydrogen bonding persists in the liquid-crystalline phase, the fact that it increases the transition temperature indicates that it must be weaker in the liquid-crystalline phase than in the gel phase. lndeed, Elamrani and Blume have shown that participation of PA ( - ) in intermolecular hydrogen bonding in the gel phase decreases the rate of its phase transition by an order of magnitude compared to that of PC and PA ( - - ) [122], further suggesting that these interactions must be weakened in the liquid crystalline phase. Eibl and Woolley [4] argued that the transition temperature would be increased to the extent observed only by weakening of strong hydrogen bonds during the phase transition, and not by breaking of weak hydrogen bonds. There are a number of differences in behavior of PE, PS and PA relative to PG and PC in the liquid crystalline phase which can be explained by the persistence of intermolecular hydrogen bonding. One of these differences, which will be discussed next, since it is further evidence in support of intermolecular hydrogen bonding, is the phase behavior above the gel to liquid-crystalline phase transition temperature. Other differences which will be discussed in Section III are their tendency to form domains and their interactions with other lipids and with proteins. can undergo a transition to the reversed hexagonal HI~ or micellar M n phases from the liquid-crystalline phase [123-125], The liquid-crystalline to hexagonal phase transition was later shown to be endothermic, usually cooperative enough to be detected calorimetrically, with an enthalpy approx. 10% of that of the gel to liquid-crystalline phase transition [126,56]. More detailed studies using 31P-NMR to detect the H n or M u phases by Cullis and de Kruijff [10,126] showed that increasing unsaturation of the fatty acid chains lowers the temperature of this transition, indicating the importance of a large ratio of the molecular area of the hydrocarbon chains to that of the head group in destabilizing the lamellar phase. This was also demonstrated nicely using a series of compounds related to cardiolipin with 2-5 chains per molecule (in the absence of Ca 2 +) [128]. Mixtures of cholesterol and PC containing two polyunsaturated fatty acid chains have been shown to go into a phase with an isotropic 31P-NMR spectrum at temperatures above 60 C, in contrast to similar forms of PE which do so below 0 C and in the absence of cholesterol [129]. Unsaturated chains are not required for the transition of PE to the hexagonal phase, however. Saturated forms of PE can go into the hexagonal phase at high temperatures. The temperature of the lamellar to hexagonal phase transition, T H, of saturated forms of PE decreases with an increase in ionic strength and with an increase in the fatty acid chain length [130]. PE with ether-linked saturated chains can go into the hexagonal phase at considerably lower temperatures than when these chains are ester-linked [56,58,130,131]. T H values for PE with ether-linked chains at low ionic strength are similar to those for PE with ester-linked chains at high ionic strength. In contrast to PE, PC with saturated chains cannot go into the hexagonal phase even at high ionic strength nor when the chains are ether-linked. Cullis and de Kruijff [10,1261 also showed that a pH at which the polar head group is in the ( - + ) state, is required for a transition of PE to the Nil phase (also see Fig. 4). This requirement for the interactive ( - + ) state suggests that intermolecular hydrogen bonding interactions between the head groups are also involved and help stabilize the hexagonal phase relative to the liquid

II-C3. Hexagonalphase formation

Early X-ray diffraction studies showed that PE

375 crystalline lamellar phase. These interactions would tend to inhibit lateral separation of the head groups as the molecular volume of the hydrocarbon region increases with increasing temperature and/or unsaturation. The only way to accommodate a large molecular volume of the hydrocarbon chains and allow close interactions of the head groups is in the HII or M~I phases. Other lipids with hydrogen bonding capacity, unsaturated forms of PS ( - + ) and saturated or unsaturated forms of PA ( - 1 / 2 ) , can also go into the hexagonal phase at pH values where these interactions are strongest, as inferred from the maxima in their phase-transition temperatures [58,132,133]. However, DSPS in the ( - - + ) state has also been found to go into the hexagonal phase by X-ray diffraction [82]. The 2 : 1 (mol/mol) complex of palmitic acid with DPPC, which is probably stabilized by intermolecular hydrogen bonding [39,53] (see subsection II-E), also forms the hexagonal phase just above its gel to liquid-crystalline phase transition temperature [134]. MGDG with ester-linked chains forms the hexagonal phase if the chains are unsaturated [71,109], but not if they are saturated [83,96]. However, the ether-linked form with saturated chains, DTMGDG, forms the hexagonal phase [136]. The ether linkage also lowers TH for PA ( - 1 / 2 ) [130] as it does for PE. Thus, the ether linkage stabilizes the hexagonal phase of saturated forms of a number of hydrogen bonding lipids. The effects of high salt concentration and the ether linkage in stabilizing the hexagonal phase relative to the lamellar may reflect an increase in the strength or probability of intermolecular hydrogen bonding. A high cation concentration shields the repulsive charge of a small amount of PE in the ( - ) state at neutral pH or the repulsive effects of PE ( - + ) molecules not involved in hydrogen bonding at any one moment. This may allow closer head group packing, increased participation in hydrogen bonding and greater dehydration of the head groups. Tn of DHPE increases and the transition enthalpy decreases with increase in pH until pH 8.5, well below the pK of the amine, where the transition disappears (Sen, A., Isac, T.V., Boggs, J.M. and Hui, S.W., unpublished data) suggesting that a small percentage of negatively charged lipid greatly inhibits formation of the hexagonal phase. This is also known from the inhibitory effect of PS ( - - + ) on the hexagonal phase transition of PE [137,138]. At very low pH, where the gel to liquid-crystalline phase transition temperature, TG, of PE increases by a few degrees, TH is greatly decreased [85] (Fig. 4). This can probably be explained by the same mechanism used to account for the small increase in TG (see subsection II-C1). Even at low pH PE is probably not completely protonated and hydrogen bonding occurs between P-OH of protonated species and P - O - of unprotonated species, as well as with NH~-. As the ratio of protonated hydrogen donating to hydrogen accepting forms of PE increases, the number of molecules of PE in the ( - + ) state not involved in hydrogen bonding at any one moment is minimized and their repulsive effect is decreased. This is probably also the explanation for why PA goes into the hexagonal phase only in the ( - 1/2) state. The ether linkage increases TG of PC, PE, and PA by a few degrees [56,67,84,104] indicating that the dialkyl and 1-alkyl,2-acyl forms are more stable in the gel phase or less stable in the liquidcrystalline phase. This may reflect closer packing or decreased hydration of the ether-linked forms. These effects may increase the strength or probability of hydrogen bonding interactions in the gel phase of ether-linked forms of PE and PA relative to the ester-linked forms. The ether linkage does not result in a detectable decrease in fluidity [139] or molecular area in monolayers for PE [140] but decreases the molecular area for PC [141]. Greater orientational order of the head group of dialkyl PC relative to diacyl PC has been found using 14N-NMR [142]. The ether linkage has little effect on the enthalpy and entropy of the gel to liquidcrystalline phase transition of PC but decreases them for PE and PA [40,58,84,104,114] (Table V). The ether linkage also decreases the rate of the phase transition of PA and causes the phase transition to be more cooperative [122]. The increase in TG and decreased rate of transition suggest that the ether linkage stabilizes the gel phase allowing increased hydrogen bonding interactions while the decreased transition entropy suggests that these interactions persist in the liquid-crystalline phase more strongly for the ether-linked forms than the ester-linked forms. This increased strength or in-

376

creased probability of forming intermolecular hydrogen bonds must then account for the significant reduction of TH and stabilization of the hexagonal phase of ether-linked forms of P E ( - + ),PA ( - 1/2) and MGDG. A decrease in hydrogen bonding in the liquidcrystalline phase could also lower TH by stabilizing the hexagonal phase relative to the liquidcrystalline phase. By forming the hexagonal phase the stabilization from intermolecular hydrogen bonding can be regained. This may be the case with the more common form of ether-linked lipids, plasmalogens containing an alk-l-enyl chain in the 1-position and an acyl chain in the 2-position. The vinyl ether linkage of plasmalogens decreases T~ by a few degrees compared to the ether-linked form, presumably because of a small disordering effect of the cis double bond between the first and second carbons of the alkenyl chain [131], although it has no effect on the packing properties of these lipids in monolayers [141]. However, the vinyl ether linkage significantly lowers TH even more than the ether linkage does [131]. The difference between T6 and TH for PE's without the vinyl linkage is 23-49 Cdeg, using temperatures given in Refs. 4, 73, 77, 81, 131 for ester-linked DOPE and POPE, and the 1-ether,2-ester-linked alkyl,oleoylPE. However, the difference for ethanolamine plasmalogen containing an oleoyl chain in the 2-position and 16:0 and 18:0 alk-1enyl chains in the 1-position is only 4 Cdeg [131]. The ethanolamine plasmalogen of myelin also goes into the hexagonal phase only a few degrees above completion of its gel to liquid-crystalline phase transition, unlike natural forms of diacylPE [56]. Thus, the vinyl ether linkage stabilizes the hexagonal phase relative to the liquid-crystalline phase considerably more than it destabilizes the gel phase relative to the liquid-crystalline phase [131]. This may be due to the combined factors of stronger hydrogen bonding in the hexagonal phase contributed by the ether linkage, a lower probability of hydrogen bonding in the liquid-crystalline phase as a result of lateral expansion caused by the alkenyl double bond, and the increased molecular volume of the hydrocarbon region in the hexagonal phase contributed by this double bond [131,56]. Involvement of intermolecular hydrogen bonding in stabilization of the hexagonal phase is fur-

ther suggested by the fact that head group modifications such as N-methylation, which weaken hydrogen bonding, increase TH (Refs. 77, 81, 85 and D. Vaughan, personal communication) (Table VI). These modifications also increase head group size. However, modifications which increase head group size but do not affect hydrogen bonding, such as alkylation of the C-2 carbon of the head group, decrease TH [77,81] (Table VI). DOPE is an exception to this since C-2-alkylation increases its TH. However, this occurs because C-2-alkylation stabilizes the crystalline phase and the lipid cannot go into the hexagonal phase until the crystalline phase melts. In fact, formation of the hexagonal phase for C-2-alkylated forms of PE may occur immediately after melting of the crystalline phase rather than from the liquid-crystal phase [81]. Hydrogen bonding stabilizes the hexagonal phase relative to the liquid-crystal phase where these interactions are weaker, but does not stabilize the hexagonal phase relative to the crystallinephase, where these interactions are strong. Modifications which do not affect hydrogen bonding but which do not stabilize close packing, such as an increase in the number of methylene groups between the phosphate and amine, also increase TH [77,81,85] (Table VI). Addition of a second sugar to MGDG to form DGDG prevents hexagonal-phase formation for this lipid [109,71]. The greater molecular area of the hydrocarbon chains above the transition temperature can be accommodated for these lipids in the lamellar liquid-crystalline phase without causing such great separation of the head groups that hydrogen bonding is prevented and, thus, hydrogen bonding of these lipids does not stabilize the hexagonal phase relative to the lamellar phase. Thus, a number of factors, such as high ionic strength, low pH and substitution of the ether linkage for the ester, which increase stabilization of the gel phase relative to the liquid-crystalline phase, also increase the stabilization of the hexagonal phase relative to the lamellar liquid-crystalline phase. Factors such as alkylation of the amine and increasing length of the head group, which decrease stabilization of the gel phase relative to the liquid crystalline phase, also decrease stabilization of the hexagonal phase. Factors such as C-alkylation which increase stabilization of the crystal-

377 TABLE VI EFFECT OF MODIFICATIONS TO ETHANOLAMINE-TYPE HEAD GROUP ON TEMPERATURE (TH) OF THE LAMELLAR TO HEXAGONAL PHASE TRANSITION Determined by temperature of endothermic transition by DSC or appearance of 31p-NMR spectrum characteristic of H n or M n phase, as noted. 3ap-NMR usually detects these phases at considerably lower temperatures than the transition detected by DSC.

X a

TH (of)

DTPE DSC
(CH2) 2 - N H 3 ~ (CH2)2-NH2(CH3) + (CH2) 2 - N H ( C H 3 ) ~(CH2)2-NH2(CH2-CH3) + CH 2-CH-NHj-

DHPE DSC b 86.7 108.5 77.2 91.3

DPPE DSC b 122 c 93.3 117.1

POPE DSC 69

DEPE DSC 63.5 d a

DEPE 31P-NMR > 60 > 80

DOPE DSC 10 73 c 71.5 ~

DOPE 31P-NMR 8 > 40 > 80 > 20 > 20

96 120

62.3

57.3

I
CH 3

CH3

CH 2-C-NH3 ~

55.5

36.1

J
CH 3 CH 2 - C H 3

I
C H 2 - C H - N H ~CH2-(CH3)2

47.2

47.8 f

> 45

27.5 f

> 40

I
CH 2-CH-NH~( C H 2 ) 3 - N H ~( C H z ) 4 - N H ~-

62 f 111 121 [e] [e] [81] 88.5

d

> 65 > 80

48.7 f 35
~

(CHz)5-NH ~" Reference a b c d e f

> 20 > 60

[85]

[81,77]

[81,77]

As in Table IV. First set of measurements at low ionic strength (5 mM Tris) second set at high ionic strength (4 M NaCI). No lamellar to hexagonal phase transition detected by DSC. Not studied by 31p-NMR unless temperature given. No lamellar to hexagonal phase transition detected by DSC or 31P-NMR below 100 C. D. Vaughan, personal communication. A crystalline to hexagonal phase transition.

l i n e p h a s e r e l a t i v e to t h e gel p h a s e a n d t h e l i q u i d c r y s t a l l i n e p h a s e also m a y i n c r e a s e s t a b i l i z a t i o n o f t h e h e x a g o n a l p h a s e r e l a t i v e to t h e l i q u i d - c r y s t a l p h a s e , b u t n o t r e l a t i v e to t h e c r y s t a l l i n e p h a s e . T h e s e results s u p p o r t t h e c o n c e p t t h a t i n t e r m o l e c u l a r h y d r o g e n b o n d i n g is i n v o l v e d in s t a b i l i z a t i o n o f t h e gel, h e x a g o n a l a n d c r y s t a l l i n e p h a s e s a n d t h a t w e a k e n i n g o f t h e s e i n t e r a c t i o n s stabilizes the l i q u i d - c r y s t a l l i n e phase. I n t e r e s t i n g l y , c h a o t r o p i c

a g e n t s such as g u a n i d i n e h y d r o c h l o r i d e , u r e a a n d N a S C N i n c r e a s e d T H o f soy P E b y 60 C d e g [350]. T h i s was a t t r i b u t e d to d i s r u p t i o n o f w a t e r structure, b u t m i g h t , i n s t e a d , i n d i c a t e d i s r u p t i o n o f hydrogen bonding between the PE head groups. T h e effects o f these a g e n t s o n TG a n d TCR o f P E has n o t b e e n r e p o r t e d . T h e e m p h a s i s a b o v e o n t h e i n v o l v e m e n t of i n t e r m o l e c u l a r h y d r o g e n b o n d i n g is n o t to suggest

378

that head group interactions are absolutely necessary for formation of the hexagonal phase or that the ratio of head group to hydrocarbon chain volume does not also play a role. The fact that cardiolipin derivatives with five chains per molecule and mixtures of cholesterol with highly unsaturated PC go into the hexagonal phase shows that a large hydrocarbon volume can induce this phase, probably in the absence of head group interactions (although this has not been established). Certainly, a large hydrocarbon volume lowers the temperature at which a transition to the hexagonal phase occurs. However, strong head group interactions can induce hexagonal phase formation in the absence of a large hydrocarbon chain volume, as indicated by the ability of saturated forms of PE to form this phase. Involvement of hydrogen bonding in formation of the hexagonal phase suggests that hydrogen bonding is generally only weakened and not entirely inhibited in the liquid-crystalline phase. In support of this, an infrared study of egg PE found that the bands originating from the phosphate and amine groups are invariant during its gel to liquid-crystalline and lamellar to hexagonal phase transitions, while the bands originating from the methyl and methylene groups of the acyl chains shifted to higher frequency during both transitions. The invariance of the phosphate and amine bands with temperature was attributed to a similar degree of hydrogen bonding in all phases and throughout both transitions [143]. Interestingly, the shift in frequency and bandwidth of the C=O stretching band at the lamellar to hexagonal phase transition was in the opposite direction to that which occurred during the gel to liquid-crystalline phase transition. This suggests that some structural feature or constraint on this region of the molecule, which is present in the gel phase, is lost in the liquid-crystalline phase and regained in the hexagonal phase. For lipids with two polyunsaturated chains, hydrogen bonding interactions might be completely lost in the liquid-crystalline phase due to the large molecular volume of the hydrocarbon region. Since this would be energetically unfavorable, such lipids go into the hexagonal phase at a low temperature, allowing these bonds to reform.

II-D. Sphingolipids
Sphingolipids and glycosphingolipids possess additional chemical groups which can participate in intermolecular hydrogen bonding. Both the sphingosine base and the fatty acid acylated to it can have free hydroxyl groups. These free hydroxyls, the amide N - H and C=O moieties, and the sugar hydroxyls of cerebroside can participate in an intra- and intermolecular hydrogen bonding network in the crystal as discussed earlier (Fig. 2) [3,20,145]. Intra-molecular hydrogen bonding in glycosphingolipids may alter the conformation of the sugar head group. Glucose bound to ceramide is less extended away from the bilayer and has a smaller orientational order parameter than when bound to diacylglycerol as detected by 2H-NMR [136]. Intramolecular hydrogen bonding between the amide and glycosidic linkage in cerebroside is not possible in MGDG. This may account for differences in the reactivity of diacylglycerol- and ceramide-bound sugars, both sulfated and nonsulfated, with enzymes and antibodies [146-149]. Hydrated sphingolipids, synthetic forms of sphingomyelin [150-152], natural and synthetic forms of cerebroside [153-159] and synthetic forms of cerebroside sulfate [160-162], exhibit complex calorimetric behavior and metastability, particularly when the fatty acid chain is longer than the sphingosine chain. Single crystal X-ray analysis of ceramide suggests that only the first 14 carbons of the sphingosine chain of sphingolipids would penetrate into the bilayer [163]. Since natural sphingolipids generally have long chain fatty acids, their hydrocarbon region is quite asymmetric. The metastability may be related to this asymmetry which may cause interdigitation of the fatty acid chains, and/or to differences in the degree of hydrogen bonding and hydration [164-168,151154]. Measurements of the amount of unfreezable water of Gaucher's spleen glucocerebroside and palmitoylgalactosyl cerebroside, both in the stable state, indicate that the amount of bound water is four molecules per molecule lipid while that for unfractionated bovine brain galactosylcerebroside, in the metastable state, is eight or nine molecules/ molecule lipid [169,170]. Glycosphingolipids have high transition tem-

379

T A B L E VII T R A N S I T I O N T E M P E R A T U R E S A N D E N T H A L P I E S OF S P H I N G O L I P I D S Fatty acid a Sphingomyelin

Tc AH

Galactosyl cerebroside Ref. T~ ( C) 65 c 69 70 56 66.5

AH

Galactosyl cerebroside sulfate b Ref.

( o C) Metastable state 18:0 24 : 0 Hydroxy 18 : 0 Hydroxy 24 : 0 Kerasin Phrenosin Stable state 16:0 18:0 24 : 0 Hydroxy 16 : 0 Hydroxy 18 : 0 Hydroxy 24 : 0 Kerasin 46 42.6

(kcal/mol) 7.0 1.9 151 150

(kcal/mol) 4.9 6.0 5.7 7.0 7.7 156 d 157 156 d 155 155

T~ ( o C)
61.9 62,65 67 67,70

AH (kcal/mol)
8.3 7.6 6.9 7.6

Re f.

161 161 161 e

41.3 57 48.6

6.8 20 15.3

150 151 150

82 83 82 73 72 71.8

17.5 9.9 15 12 12.4 15.8

154 156 d 157 156 d 156 d 155

64.7 63.9 69.0 75.6 83.4 81.5

9.1 13.8 15.5 9.9 14.3 17.4

161 161 161 161 161 e

a Fatty acid N-acylated to sphingosine base of synthetic forms or relatively homogeneous forms isolated from natural lipid, except for kerasin and phrenosin which are the non-hydroxy and hydroxy fatty acid containing fractions of bovine brain galactosylcerebroside, respectively. b In 2 M KCI. c From cooling scan at 5 C d e g / m i n . 4 Cdeg added on to correct for instrumental hysteresis so that temperature can be compared to that reported for hydroxy 18 : 0 form (obtained on heating). d Isolated from natural cerebroside. Contains 99% fatty acid indicated except hydroxy 2 4 : 0 which is 86% hydroxy 24:0, 9.5% hydroxy 24 : 1, and 4.3% hydroxy 22 : 0. e Boggs, J.M., Koshy, K.M. and Rangaraj, G., unpublished data.

peratures as indicated in Table VII suggesting that intermolecular hydrogen bonding occurs in the presence of water and stabilizes the gel phase relative to the liquid-crystalline phase. The 2HNMR spectra of N-palmitoylgalactosyl sphingosine indicates restricted motion of the C-6 hydroxymethyl group of the galactose consistent with its participation in a hydrogen bonding network [135]. Infrared spectroscopy of the non-hydroxy form of natural cerebroside, kerasin, has provided evidence for intra- or intermolecular hydrogen bonding involving the amide group [171]. Conversion of the metastable form to the stable form increases the involvement of the amide C=O group in hydrogen bonding. Gel formation in water by single chain amphiphiles with one or two amide groups and a number of hydroxyl groups also has been attributed to intermolecular hydrogen bonding involving these groups [172]. Methylation of

the amide increases the water solubility suggesting weakening of the hydrogen bonding interactions. Natural sphingolipids also have high phasetransition temperatures so that most are in the gel phase at physiological temperature. Thus factors which affect the intermolecular hydrogen bonding properties of these lipids may be particularly significant for biological membranes. The transition temperatures and enthalpies do not depend significantly or in a regular way on chain length for cerebroside [156] and cerebroside sulfate [161,162] suggesting that hydrogen bonding interactions between the head groups may be stronger than the Van der Waals' interactions between the chains for these lipids. Cerebroside sulfate with non-hydroxy fatty acids has a lower transition temperature than cerebroside, especially in the stable state, indicating that the negatively charged sulfate contributes a repulsive force which weakens the head

380 group interactions. It also forms more expanded monolayers than cerebroside [173]. An increase in ionic strength significantly increases the transition temperature of cerebroside sulfate indicating that added cations shield this repulsive charge and allow stronger hydrogen bonding [161]. The transition temperature of sphingomyelin, however, is not elevated much above that of PC's of similar chain length. Palmitoyldihydrosphingosinephosphorylcholine (without the 4,5-trans double bond usually present in sphingomyelin and the other sphingolipids listed in Table VII) has a transition temperature about 7 Cdeg above that of DPPC [150], while palmitoylsphingosinephosphorylcholine has a similar transition temperature to DPPC (Table VII). Other synthetic choline-containing lipids with a potential hydrogen bonding carbamyloxy group have similar transition temperatures to PC's of comparable chain lengths [174]. However, like synthetic forms of sphingomyelin and other sphingolipids, they have metastable phase behavior and the stable state has a considerably greater transition enthalpy and entropy than PC [174,175]. Synthetic choline-containing lipids with one chain linked by an amide linkage and the other by an ester or ether linkage, also have transition temperatures similar to PC [176]. Since no other structural information is available about these latter synthetic lipids, the reasons for their similarities and differences to sphingomyelin and PC are not understood. However, the results do suggest that the amide linkage in these lipids does not contribute significantly to an increase in the transition temperature or stabilization of the gel phase. This may be because of the choline head group which behaves as a repulsively charged head group as in PC and may counteract the tendency of the amide linkage to be involved in intermolecular hydrogen bonding. Intramolecular hydrogen bonding may occur instead. A 14N-NMR study of sphingomyelin and DPPC indicated that there are differences in the orientational order of the choline head group in the liquid-crystalline phase [177]. These may be caused by intramolecular hydrogen bonding between the sphingosine amide, hydroxyl and the head group phosphate in sphingomyelin. Alternatively, a similar degree of hydrogen bonding may occur in both the gel and liquidcrystalline phases of sphingomyelin so that neither phase is stabilized relative to the other. A deuterium NMR study of DPPC and N-palmitoylsphingosinephosphorylcholine labeled with deuterium at C-10 showed that the latter is more ordered than DPPC in the liquid-crystalline phase [178]. Cerebroside and sphingomyelin labeled with 13C at the amide moiety both have slow axial diffusion in the gel phase (no greater than 10 2 s 1) [179]. The role of the hydroxyl groups on the sphingosine base or the acyl chain has been investigated in a number of studies. The 2D-hydroxyl group on the acyl chain and the 4D-hydroxyl group on the sphingosine base of synthetic forms of ceramide both promote condensation in monolayers [3,145]. The hydroxyl group on the acyl chain significantly increases the phase-transition temperature of cerebroside sulfate, particularly of the stable state, suggesting that it contributes to the hydrogen bonding network and stabilizes the gel phase [161]. In fact the transition temperature of hydroxy 24:0 cerebroside sulfate in the presence of 2 M K + is similar to that of 24 : 0 cerebroside (Table VII). The hydroxyl group has little effect on the transition enthalpy. It inhibits but does not prevent formation of the stable state, particularly for shorter fatty acid chains [160,161]. It was earlier thought that hydroxylation of the fatty acid in cerebroside completely prevents formation of a stable state with a higher transition temperature and enthalpy [155,158,168]. However, it has recently been reported that, as for cerebroside sulfate, the fatty acid hydroxyl group only inhibits formation of the stable state [156]. The stable state of hydroxy fatty acid forms of cerebroside has a high transition enthalpy like that of the non-hydroxy fatty acid forms, but its transition temperature is 10 Cdeg less. Thus, the hydroxy fatty acid decreases the transition temperature of the stable state of cerebroside, in contrast to cerebroside sulfate. However, as for cerebroside sulfate, the hydroxy fatty acid in cerebroside increases the transition temperature of its metastable form (Table VII). This is true both for the 18 : 0 forms and for the natural mixtures, phrenosin and kerasin. The latter two lipids also differ in unsaturation, making it difficult to compare them. However, Raman

381 spectroscopy has shown that in the stable state of kerasin a high percentage of the 24:1 chains are all trans in the region between the double bond and the methyl [168]. This is consistent with resuits of Pascher and collaborators [3,145] who showed that the 15-cis double bond of 24:1 ceramide does not cause expansion of the monolayer, unlike cis double bonds closer to the polar head group. The 15-cis double bond may penetrate further into the bilayer than the terminal methyl of the sphingosine base and, thus, does not interfere greatly with chain packing. However, it does lower the transition temperature of cerebroside sulfate (Boggs, J.M., Koshy, K.M. and Rangaraj, G., unpublished data) and cerebroside [156]. The lowering of the transition temperature of the stable state of cerebroside by hydroxylation of the fatty acid was attributed to disruption of this phase [156]. However, it can be argued instead that it may indicate an increase in stabilization of the liquid-crystalline phase relative to the gel phase. The hydroxy fatty acid may strengthen the intermolecular hydrogen bonding interactions of cerebroside in the liquid-crystalline phase more than those in the gel phase. Hydrogen bonding of cerebroside may already be so strong in the stable gel phase that an extra hydroxyl group does not contribute to its strength, even though it may participate. In the less highly organized metastable phase, however, it does contribute to the strength of these interactions, as inferred from the increase in the transition temperature. Thus, it may do the same in the liquid-crystalline phase. Most other studies of the effect of the hydroxy fatty acid on cerebroside compared the metastable state of phrenosin to the stable state of kerasin, although it was not known at the time that these fractions were in different states. Raman spectroscopy indicated that the stable state of kerasin is more ordered than the metastable state of phrenosin. The stable state of phrenosin was not studied. However, in their anhydrous crystalline solids, a form in which they may be more comparable, phrenosin is more highly ordered than kerasin [168]. The amide vibrational band of phrenosin is shifted to a lower wavelength than that of kerasin, also consistent with stronger hydrogen bonding in the hydroxy fatty acid form. The molecular areas of kerasin and phrenosin in monolayers are similar [181]. H-E. Intermolecular hydrogen bonding between two different lipids Intermolecular hydrogen bonding interactions can also occur between two different lipids with suitable donor and acceptor groups. Eibl and Woolley [4] have shown that equimolar mixtures of DMPM ( - ) and dimyristin or an amino lipid consisting of dimyristin with the glycerol 3-OH replaced by NH(CH3)~-, have transition temperatures considerably higher than those of the pure components. A mixture of DMPM ( - ) and a similar lipid with the glycerol 3-OH replaced by N(CH3)~, which cannot participate in hydrogen bonding, does not have an elevated transition temperature. This suggests that intermolecular hydrogen bonding rather than reduction of the surface charge density or electrostatic interactions is responsible for the elevated transition temperature. Addition of palmitic acid to DPPC at a molar ratio of 2:1 increases the transition temperature to a value similar to that of DPPE [8]. The thermotropic peak is sharp at this ratio indicating a cooperative transition while lower and higher concentrations of palmitic acid broaden the transition. This suggests specific complex formation between DPPC and palmitic acid. The effect on the transition temperature was attributed to relief of the crowding of the large choline head groups due to a spacer effect. However, long chain compounds which cannot interact with PC by hydrogen bonding such as sodium palmitate, hexadecane and the methyl ester of palmitic acid, have a much smaller effect on the transition temperature, while other long chain compounds which can donate hydrogens, such as hexadecanol and hexadecylamine are able to increase the transition temperature to the same level as palmitic acid [53,182,184], suggesting that intermolecular hydrogen bonding is responsible for the high transition temperature. The hydrogen bonding compounds have a similar effect on DPPG [53]. They can be incorporated into the hydrogen bonding lipids, DPPE ( - + ) and DPPA ( - ) , but have little

382 effect on their transition temperatures. However, the non-hydrogen bonding compounds are partially insoluble in DPPE and DPPA and also decrease their transition temperatures, indicating that they break up the hydrogen bonding interactions of these lipids. Intermolecular hydrogen bonding was earlier invoked to explain the high transition temperatures of complexes of alkyl alcohols and alkyl sulfates [185]. A strong condensing interaction between DPPC and DMPA occurs in monolayers at a low surface pressure where the DMPA by itself forms an expanded film [94]. Condensation between PG and dimannosyldiacylglycerolin monolayers at pH 5.6 and between PG and PC at pH 2.9, but not at pH 5.6, where both are repulsively charged, has also been reported [190,191]. This condensation suggests intermolecular hydrogen bonding occurs between the different lipids involved. A similar intermolecular hydrogen bonding and electrostatic interaction occurs between PG and lysylphosphatidylglycerol (with a net positive charge and hydrogen donating amine), causing condensation of monolayers and an increase in the phase transition temperature above that of either of the two pure lipids [188,189]. The ~-amino group of this lipid, which has a pK of about 7, is involved in intermolecular hydrogen bonding with the phosphate of PG while the a-amino group with a pK of 10.5 interacts intramolecularly with the phosphate of lysylPG [189]. The intermolecular interaction of lysylPG with PG is stronger than that of either lipid with divalent cations or anions, even at high concentrations. Neither Ca 2+, which can bind to the phosphate of pure PG, nor MoO4:-, which can bind to the ~-amino of pure lysylPG, are able to bind to the mixture until the pH is increased above 7.5 [188]. acyl chain of egg PC at C-1 of the glycerol of a neighboring PC molecule. This allows a better fit between cholesterol and the unsaturated 2-chain of a PC molecule on the other side of the cholesterol [194]. In another model, hydrogen bonding of the cholesterol OH with the glycerol oxygen esterified to the 2-acyl chain, rather than the 1-carbonyl, has been proposed [195A96]. Molecular models suggest this may be a more favorable interaction, since the carbonyl oxygens point up away from the cholesterol OH. However, there is little direct evidence for hydrogen bonding of the cholesterol with either of the ester oxygens. The 3-r-OH is necessary for the well-known condensing effect of cholesterol on lipids [197] but the ester carbonyl of the phospholipid is not. Cholesterol has an equivalent condensing effect on lipids with the ether linkage [139,199] and it decreases the permeability of diester and diether lipids. More importantly, it also decreases the permeability of lipids in which the chains are linked directly to the head group via carbons and thus have no hydrogen accepting groups [200-203]. It could be argued that hydrogen bonding of the cholesterol OH to the lipid is not required for condensation and reduced permeability, but that hydrogen bonding nevertheless occurs, and could be important for interactions of cholesterol with specific lipids. Cholesterol caused a downfield shift of the PC carbonyl 13C-NMR resonance which could be consistent with hydrogen bonding [204]. However, no evidence for a hydrogen bond with the ester carbonyl of PC could be found using vibrational Raman and infrared spectroscopy [205,206]. In view of the tendency of hydrogen donating groups of lipids and long chain amphiphilic compounds to form hydrogen bonds with hydrogenacceptor groups of other lipids, as inferred from the high phase-transition temperatures of the mixtures, it seems unlikely that the cholesterol OH would not hydrogen bond with appropriate acceptor groups. Unlike long chain hydrogen bonding compounds, hydrogen bonding of cholesterol could not increase the transition temperature of lipids significantly because it disrupts the ordered packing of the gel state lipid chains. However, at lower concentrations it does induce a component with a broad phase transition at a higher temperature

lI-F. Hydrogen bonding of cholesterol with other lipids

Cholesterol has a hydrogen-donating group, the 3-B-OH, which could conceivably form a hydrogen bond with a hydrogen acceptor, such as the ester oxygens of glycerolipids and the amide oxygen of sphingolipids [192,193]. An intriguing model has been described in which cholesterol hydrogen bonds with the carbonyl of the saturated

383 than the pure lipid for PC and sphingomyelin [207-209]. When added to PE, however, it only reduces the transition temperature [73,211] which may be a result of lateral separation of PE molecules and breaking of hydrogen bonds between the PE head groups. Thus, in spite of the lack of direct evidence, it is difficult to discard models in which hydrogen bonding between the 3-fl-OH of cholesterol and other lipids occurs. A different approach may be necessary in order to determine whether such hydrogen bonding occurs.
III. Influence of lipid intermolecular hydrogen bonding on membrane structure and function III-A. Lamellar to non-lamellar phase transitions

The temperature of the lamellar to hexagonal phase transition of most naturally occurring hexagonal phase forming lipids such as PE and MGDG is below 37 o C, so that this phase could form in membranes at physiological temperature [71,126]. In lipid mixtures and biological membranes, the structure formed by these lipids is probably some kind of isotropic phase such as inverted micelles or lipidic particles sandwiched between the two monolayers of the bilayer [10]. A transition to a non-lamellar phase could be involved in various membrane functions such as fusion, exo- and endocytosis, transbilayer movement of lipids, transport of small molecules across the bilayer, compartmentalization of membranes, membrane flow and interorganelle communication (see Refs. 10 and 212 for excellent reviews). Intermolecular hydrogen bonding between lipids with small head groups stabilizes the close head group packing which occurs in the hexagonal or inverted micellar phases, as discussed in subsection II-C3. Thus, it significantly destabilizes the lamellar liquid-crystalline phase and stabilizes the hexagonal phase. Changes in membrane lipid composition which affect hydrogen bonding could thus have profound effects on membrane structure and function. Addition of other lipids can stabilize the lamellar phase so that membranes are maintained in a bilayer in the absence of any physiological perturbation. Cholesterol stabilizes the lamellar phase

of some types of PE and mixtures of PE with other lipids, but destabilizes others [126,137,138]. The former effect can be explained by interruption of intermolecular interactions or a condensing effect on the fatty acid chains of the PE, while the latter can be explained by the wedge shape of cholesterol making it compatible with the high degree of curvature of the H n or inverted micelle phase. PC, PS, PG and sphingomyelin stabilize the lamellar phase of PE probably by causing lateral separation of the PE molecules and disrupting their intermolecular interactions. The stabilizing effect of a small percentage of repulsively charged lipid on the lamellar phase has been discussed previously. Addition of Ca 2 to PS/PE and P G / P E mixtures abolishes the stabilizing effect of the anionic lipid, probably by causing its phase separation away from the PE a n d / o r by neutralizing the charge and dimerizing the anionic lipid, making it compatible with the H n phase of PE [137,138,213]. Even in pure PE at neutral pH, addition of Ca 2 lowers Tn by 2-3 Cdeg, which is attributed to charge neutralization and/or phase separation of a small percentage of PE ( - ) present at this pH [214]. Thus, a phase transition to a non-lamellar phase could be triggered in biological membranes at 37C by Ca 2+. In mixtures of lipids in biological membranes, the lamellar phase may be only slightly more stable than the hexagonal phase. Local changes in pH or divalent cation concentration may cause a transition to a non-lamellar phase. Other factors which alter the strength or probability of intermolecular hydrogen bonding between the lipids may have a similar effect.
III-B. Phase separation or domain formation III-B1. Lipid miscibility Intermolecular hydrogen bonding interactions may result in phase separation or domain formation in lipid mixtures. These interactions may occur between lipids of one type (homomolecular) or between several types (heteromolecular) with appropriate hydrogen-donating or -accepting groups in the mixture. PC does not mix ideally in the gel phase with interactive lipids such as PE [215-218], PS [219], sphingomyelin [183] and cerebroside [102,159,220-222]. The hydroxy fatty

384

acid fraction of cerebroside may be more associated in DPPC than the non-hydroxy fatty acid fraction, since it has less perturbing effect on the PC head group as detected by NMR [224]. Better mixing generally occurs in the liquid-crystalline phase but simulation of the phase diagrams suggests that mixing is less than ideal in this phase also [225-227]. However, N-palmitoylsphingomyelin was concluded to be thoroughly miscible with DMPC in the liquid-crystalline phase [228]. DSC thermograms of the liquid-crystalline to gel phase transition, obtained on cooling, indicated that glucosyl cerebroside from Gaucher's spleen, containing predominantly C22 and C24 fatty acid chains is less miscible with DPPC in the liquidcrystalline phase than N-palmitoylgalactosylcerebroside [227,228]. This may be related to the greater difference in fatty acid composition between the Gaucher's spleen cerebroside and DPPC. Use of a cross-linking reagent to investigate the mixing of DPPE ( - + ) and DPPS ( - - + ) showed that mixing is non-ideal in the gel phase but becomes even more non-ideal at the phasetransition temperature of the lower melting lipid when phase separation of liquid-crystalline phase (DPPS) and gel phase lipid (DPPE) occurs [229]. When both lipids are in the liquid-crystalline phase they are more miscible. 15N-NMR spectroscopy of DPPE at pH 11 where both the ( - + ) and ( - ) states are present, shows that the exchange between these two populations is slow suggesting that they are phase separated [349]. Thus, DPPE ( - + ) does not mix randomly even with its own negatively charged form. In spite of the above, there is no doubt that significant mixing of PC and other lipids occurs in the liquid-crystalline phase. Incorporation of PA or PE into PC vesicles destroys binding of 1anilino-8-naphthalenesulfonate to PC. Since this compound binds to PC in a molar ratio of 1:4 PC, this suggests that the other lipids mix with PC to the extent that individual PC molecules no longer have three other PC neighbors [230]. Addition of PC to PE results in greatly increased reactivity of PE with 2,4,6-trinitrobenzenesulfonic acid [90] and alteration of the 31P-NMR spectra of both PE and PC [231,232,118] indicating that some mixing occurs. Some heteromolecular hydrogen bonding of PE and PA with the phosphate of PC

may occur in the liquid crystalline phase, where homomolecular hydrogen bonding interactions are weakened. This is supported by the fact that equimolar PC/PE mixtures behave like PE and not PC with respect to leakage at the phase-transition temperature and hydrolysis by phospholipase A 2 [92]. However, in the gel phase of mixtures with PC, homomolecular hydrogen bonding is undoubtedly stronger. Hydrogen bonding between PE and PC in the gel phase would be inhibited by the repulsive charge of PC. Consistent with this, phospholipase A 2 is able to hydrolyze PC/PE mixtures in the gel phase, like pure PC and unlike pure PE, in support of phase separation of these two lipids in the gel phase. However, these results do not rule out clustering of these interactive lipids into microdomains in liquid-crystalline phase lipid mixtures. It is difficult to obtain evidence for this but there is one study that indicates that such clustering may occur. This study used dyes which bind to acidic lipid head groups in a 1:1 ratio to measure the number of dye dimers which occur when acidic lipids are mixed with PC [233]. One problem with this approach is that the bound cationic dye will alter the charge of the lipid polar head groups and, thus, may change the degree of association of the lipids, although arguments were made against this. In any case, the degree of association for the different natural unsaturated lipids used, decreased in the order PI > PA > PS > PG at neutral pH. This order for PA, PS and PG correlates with the decrease in their tendency to interact by hydrogen bonding and increase in their repulsive charge in the same order. The high degree of association found for PI suggests that this lipid might also be an interactive lipid. Its physical properties have not been studied by biophysical techniques but it might be able to interact intermolecularly by hydrogen bonds between the inositol hydroxyls, if these interactions can compensate for the electrostatic repulsion caused by the negatively charged phosphate. Intermolecular hydrogen bonding may contribute to enhanced domain formation in the presence of other agents which cause phase separation of acidic lipids such as Ca 2+ or proteins, even if it is not sufficient to cause domain formation by itself. For example, Ca 2+ causes a greater degree of

385 phase separation of PS from PE than from PC, a n d / o r is effective at a lower concentration for P S / P E mixtures than for P S / P C mixtures [234-237]. These studies included both natural lipids in the liquid-crystalline phase and synthetic lipids in the gel phase. However, if cholesterol is present, Ca 2+ is not able to separate PS from a P E / P S mixture although it does so from a P C / P S mixture, at similar Ca 2+ concentrations as in the absence of cholesterol [235]. This may be because insertion of cholesterol between PE molecules inhibits the stronger homomolecular interactions of PE and allows weaker heteromolecular interactions between PE and PS. Ca2+-induced phase separation of PA from PE is a little more complete than from PC [238]. The Ca 2+ concentration dependence of the phase separation of these two mixtures was not compared. III-B2. Asymmetric distribution in small unilamellar vesicles Other forces, such as a high radius of curvature in a section of membrane or small vesicle may also induce domain formation between interactive lipids. Asymmetric distribution of lipids in small unilamellar vesicles (SUVs), where the radius of curvature of the inside monolayer is greater than that of the outer monolayer, has been found in a number of studies. Some of these studies are contradictory which may be partly caused by differences in the pH used and by the methods of measurement of asymmetry. Many of these studies involve use of divalent or trivalent cationic reagents to shift N M R resonance positions, or positively charged dyes whose spectral properties depend on their degree of association. Binding of cations to anionic sites on lipids will affect their interactive or repulsive properties and may alter the bilayer distribution. Other methods involve chemical or enzymatic modifications of the head groups which may also affect their properties and cause redistribution. In spite of these problems, the general consensus seems to be that in mixtures with PC, interactive lipids such as PS, PE, PA and PI are preferentially localized in the inner monolayer at neutral pH [118,233,239-244]. The preference of both PS and PE for the inner monolayer decreases as the pH is raised [118,239] indicating that an ionization state which can interact by hydrogen bonding, rather than a repulsively charged one, is required. The tendency of PE to localize on the inner monolayer increases with the concentration of PE in the mixture indicating that there must be a certain probability of having nearest neighbors of PE to interact with in order to seek a highly curved surface. The preferential location of PE on the inner monolayer occurs only for SUVs and not large unilamellar vesicles, where its distribution is symmetrical [242]. This indicates that it is the highly curved surface of SUVs which causes the asymmetric distribution. Intermolecular interactions which cause closer packing of the head groups would allow the lipids to accommodate better to the highly curved inner monolayer while the repulsively charged PC could be better accommodated in the outer monolayer with its greater surface area, allowing a lower surface charge density. Insertion of an extra methylene group between the phosphate and the amine of the PE head group, causes the lipid to be located preferentially in the outer monolayer [243]. Thus, if close packing of the head group is not necessary for intermolecular hydrogen bonding to occur, the lipid will not be found preferentially in the inner monolayer. Modification of PE with ethyl, phenyl and benzyl groups on C-2 of the ethanolamine also causes the lipid to be preferentially located in the outer monolayer [243]. These modifications should not affect the hydrogen bonding interactions but may have other effects on the head group conformation which are not understood. P G / P C mixtures do not adopt a consistent non-random distribution [233,240,245-247], which is perhaps not surprising since both PG and PC behave as repulsively charged lipids. Although the effect of p H on the distribution of PA has not been investigated in detail, at p H 7 in the presence of shift reagents, PA is distributed symmetrically in P A / P C vesicles [248]. Using phospholipase D at pH 5.5 to convert PC in the outer monolayer to PA and shift reagents to monitor its redistribution, the same laboratory showed that the PA formed rapidly translocates to the inner monolayer [249]. Its final concentration in the inner monolayer is greater than in the outer. The asymmetric distribution at pH 5.5 in contrast to that at pH 7 may be due to lowering of the p K 2 of PA by

386

the divalent cationic shift reagent so that at pH 7 it is in the repulsive ( - - ) state while at pH 5.5 it is in the interactive ( - ) state. Only in the ( - ) state would it be expected to locate preferentially in the inner monolayer. Sphingomyelin and cerebroside have been found located preferentially in the outer monolayer in mixtures with PC [116,239,251]. With their large head groups and mostly saturated chains, they would not be expected to seek or form a highly curved surface. It is noteworthy that cerebroside does not form the hexagonal phase, unlike unsaturated forms of M G D G . The asymmetric distribution of lipids in SUVs is reminiscent of that which occurs in biological membranes. PE, PS, and PI are located preferentially on the cytoplasmic side while PC, sphingomyelin and cholesterol are located primarily on the extracellular side of plasma membranes [252-254]. The majority of the PE in erythrocyte ghosts is associated with itself while most of the PS is associated with either PE or proteins as shown by use of cross-linking reagents [255]. They may have a similar distribution in intracellular membranes, although this has been challenged [250]. However, the driving force for the asymmetric lipid distribution in plasma membranes must be different, since they are not likely to be highly curved except in localized regions. Thus, it cannot be concluded that it is the interactive properties of lipids which primarily determine the asymmetry of biological membranes, although they may contribute toward maintenance of the asymmetry. Spectrin may stabilize the asymmetric distribution of a substantial proportion of the PE and PS of red cells [256]. The important aspect of measurements of lipid distribution in SUVs is that they have been made primarily with natural lipids in the liquid-crystalline phase. Therefore, if the results are not artifactual, as a result of the methods used to determine them, they suggest that hydrogen bonding interactions between lipids must continue to occur in the liquid-crystalline phase, particularly if the lipid can find or form a sufficiently curved surface. The principle that hydrogen bonding between lipids in the liquid-crystalline phase will cause them to form a highly curved bilayer while a

repulsive negative charge will cause them to form a less curved bilayer was utilized by Haines and collaborators [257,258] to develop a method to prepare large unilamellar vesicles of selected sizes of acidic lipids such as PA, PG, PE and PS. The vesicles are formed from the protonated form of the lipids, which can hydrogen bond, and then the pH is raised so that they become ionized and charge repulsion occurs. The curvature of the vesicles, and consequently their size, is varied by changing the balance between attractive vs. repulsive interactions in the inner monolayer. This is done by such means as varying the acyl chain composition, the ionic strength of the medium, adding cholesterol or other lipids to the acidic lipid and varying the rate of change of pH.

III-B3. Preferential association of cholesterol with different lipids

Since cholesterol decreases the enthalpy of lipid domains with which it associates, Van Dijck and colleagues [73,259,260] were able to use DSC to show that cholesterol preferentially associates with certain lipids in ternary mixtures. In non-cocrystallizing mixtures of P E / P C , P E / P G , P E / s p h i n g omyelin, PS/sphingomyelin and PC/sphingomyelin, the order of preference of cholesterol for the different lipids is sphingomyelin >> PS =-PG > PC >> PE, regardless of whether the preferred lipid is the higher or lower-melting lipid of the mixture. This indicates that cholesterol does not just dissolve into the fluid phase of the lipid which melts first. Rather, it suggests that cholesterol interacts with SM domains better than with PS, PG and PC domains and that it is excluded from PE, if some other lipid is available for it to mix with, regardless of whether these lipids are in their gel or liquid-crystalline phases. However, in lipid mixtures of P E / P C or sphingomyelin/PC which cocrystallize or mix well enough to give only one transition, there is no evidence of preferential interaction with either of the lipids [211,261]. The preference of cholesterol for other lipids over PE in non-cocrystallizing mixtures can be explained by the interactive properties of PE. It would be energetically unfavorable to disrupt these interactions by insertion of cholesterol if there is another non-hydrogen bonding lipid available into which it can insert. However, in cocrystallizing

387 mixtures of PE with another lipid, these interactions are already disrupted and thus cholesterol should be able to interact equally well with either lipid. Preferential interaction of cholesterol with sphingomyelin over PC and other lipids in noncocrystallizing mixtures might be caused by greater hydrogen bonding of the 3-fl-OH of cholesterol with sphingomyelin, although as discussed earlier there is no direct evidence that this occurs. The results suggesting equal interaction of cholesterol with sphingomyelin in cocrystallizing mixtures may only indicate that cholesterol is not able to cause phase separation of sphingomyelin. Complexes of sphingomyelin and cholesterol which remain mixed with the PC may reduce the enthalpy of the PC also, in a cooperative manner, even if the cholesterol preferentially associates with the sphingomyelin. Alternatively, preferential interaction with sphingomyelin may occur only with a domain of several molecules of sphingomyelin and not with individual molecules, either because sphingomyelin in domains forms a different kind of bilayer structure than PC or because hydrogen bonding of cholesterol with sphingomyelin is enhanced if there are a number of acceptors available rather than individual molecules. Domains of sphingomyelin would only be available in noncocrystallizing mixtures. That preferential interaction of cholesterol may occur only with a domain of sphingomyelin and not individual molecules is suggested by a study in which the effect of different lipids on availability of cholesterol to cytochrome P-450 was monitored from the effect of cholesterol on the absorbance spectrum of the enzyme [262]. This study showed that at high concentrations sphingomyelin inhibits transfer of cholesterol to the enzyme more than PC, PE and PS, but not at low concentrations. The concentration dependence of inhibition by sphingomyelin is sigmoidal while that by the other lipids is not. This study is also important because it shows that lipid-lipid interactions can limit their availability to and interaction with enzymes. Using another approach to study interaction of cholesterol with different lipids, the uptake of cholesterol from red cell ghosts by SUVs of different lipids was measured and found to be similar for DPPC, bovine brain sphingomyelin and Npalmitoylsphingomyelin but greater for these three lipids than egg PC [263]. However, this approach does not indicate which lipid cholesterol would interact preferentially with if both were available. In measurements of the rates of exchange of cholesterol between vesicles of different lipids, palmitoylsphingomyelin was found to have a greater affinity for cholesterol than DPPC [264]. In a similar experiment cholesterol did not exchange from vesicles of bovine brain sphingomyelin into vesicles of POPC [351]. However, it preferred vesicles of POPC to vesicles containing high concentrations of POPE. No preference for a particular lipid was found in non-cocrystallizing mixtures of P S / P E , P S / P C , and P C / P G . Cholesterol interacts preferentially with the lower-melting lipid of the pair regardless of which lipid has the lower transition temperature [259,260]. In these mixtures, the characteristics of the two lipids of the pair are probably so similar that cholesterol can interact with either equally well. PC and PG are both repulsively charged while PS ( - - + ) is intermediate between a repulsively charged lipid and an interactive lipid like PE ( - + ). If any degree of domain formation of particular lipids occurs in biological membranes, either as a result of the properties of the lipids themselves or as a result of interactions of some of the lipids with divalent cations and proteins, these studies suggest that cholesterol should be found to the greatest extent in sphingomyelin domains and the least in PE domains. Demel et al. [259] have demonstrated an interesting correlation between the amount of cholesterol and sphingomyelin in a number of biological membranes. They also demonstrated an inverse correlation between the amount of cholesterol and the amount of PC and PE in membranes [259] (with the exception of red blood cells from some species which have high concentrations of both PC and cholesterol [1]). It is noteworthy that cholesterol is found in natural membranes on the opposite side of the bilayer from PE and PS and the same side as sphingomyelin and PC. 11I-C. Interactions with proteins Intermolecular interactions between lipids can also affect how they interact with proteins and

388 determine the conformation of the lipid-bound protein. Many water-soluble extrinsic membrane proteins, such as myelin basic protein, cytochrome c and spectrin have hydrophobic residues which may be able to penetrate partway into the bilayer even though most of the protein sits on the surface of the bilayer and interacts electrostatically with anionic lipid head groups [265]. Interaction of hydrophobic residues of extrinsic proteins with lipids has been investigated in great detail for myelin basic protein and has been reviewed recently [266]. Evidence suggestive of penetration of some residues of myelin basic protein partway into the bilayer consists of its perturbing effect on lipid bilayers and monolayers [115,265,267-271], increased labeling of the protein in lipid vesicles by a hydrophobic photolabel, 3-trifluoromethyl-3-(m-[ 125I]iodophenyl)diazirine (Boggs, J.M., Rangaraj, G. and Koshy, K.M., unpublished), and the ability of the lipid to protect certain sites on the protein from enzymatic hydrolysis [273,274] and antibody binding [275]. The degree of penetration of the protein has been found to vary with the type of lipid [273,274,276]. Using well-defined lipids the degree of penetration decreased in the order PA = PG > PS > cerebroside sulfate > PE, as inferred from the effects of the protein on these lipids and the effects of the lipids on the protein [162,268,269,275,277]. The secondary structure of the protein also depends on the type of lipid to which it is bound. The protein has no secondary structure in solution, as determined by circular dichroism and infrared spectroscopy [278-280], but acquires such structure on binding to lipid [278]. The degree of a-helical structure induced by lipids decreases in the order PG = PA > PS >> PE as detected by circular dichroism spectroscopy [279], although infrared spectroscopy of basic protein (BP) in D M P G indicated that it acquires primarily flstructure [280]. The relatively greater penetration of BP into PG, PA, and PS can be explained by the inhibition of intermolecular hydrogen bonding interactions when basic residues of BP bind to the hydrogen bond accepting phosphate sites. In contrast, binding of BP to the anionic sulfate of cerebroside sulfate would neutralize its repulsive effect and strengthen the intermolecular hydrogen bonding interactions between the sugar and other hydroxyl groups. Thus penetration of BP into cerebroside sulfate is restricted. This could happen with PS under some circumstances, depending on whether the protein binds to the carboxyl or the phosphate and which of these groups is most important for intermolecular hydrogen bonding. Studies of the interaction of BP with lipid monolayers showed that the protein penetrates less into PS monolayers than other acidic lipids [276]. Less BP binds to PE than to other acidic lipids indicating that the anionic phosphate group prefers to interact with the amine of a neighboring molecule of PE rather than with those of the protein. Thus, intermolecular hydrogen bonding between the lipids persists and that protein which is bound to PE is unable to penetrate into the bilayer. These conclusions were tested further by studying the interaction of BP with different ionization states of DPPA, achieved by varying the pH [281]. An increase in p H from 4 to 8 increases the hydrophobicity of the protein, since the histidines become deprotonated. This causes the protein to have a greater perturbing effect on DPPG, a lipid which is not affected by pH in this range, as the pH is increased [271]. In contrast, the perturbing effect of BP on DPPA decreases with increasing pH, until from pH 7.5-9 the protein has little effect on the lipid. These results were rationalized by considering the capacity for intermolecular hydrogen bonding of the protein bound lipid at different pH values [281]. At pH 7.5, in the presence of the protein, the second hydroxyl begins to dissociate so that there are some molecules of PA in the ( - - ) state as well as some in the ( - ) state. There are, thus, enough lipid hydrogen bond-accepting sites available that hydrogen bonding can continue between P - O H of PA ( - ) and P - O - of PA ( - - ), even if basic residues of the protein are bound to P - O sites on all or almost all of the lipid molecules. At pH values above 9, the lipid becomes more completely dissociated and there are no longer enough hydrogendonating groups for hydrogen bonding to continue. Above pH 9, the protein has a very great perturbing effect on the lipid and probably causes a transition to some other kind of structure, possibly micellar. It should be noted that while this explanation is consistent with the hydrogen bond-

389 ing properties of different ionization states of protein-bound PA, alternative explanations also may be possible. The degree of penetration of spectrin into monolayers of different lipids at pH 7.4, monitored from the increase in surface area produced by the protein, decreases in the order PG > PA >> PS > PE [282]. The results were attributed to the closer packing density of PS and PE in monolayers relative to that of PG, thus restricting penetration of the protein into PS and PE. However, PA also forms closely packed monolayers in the absence of any protein [94]. Consideration of hydrogen bonding interactions in the presence and absence of the protein as discussed above for myelin basic protein, can account for the close packing of PS, PE and PA and also for the greater degree of penetration into PA and PG. The effects of cytochrome c on the surface pressure of monolayers of several lipids have also been compared [283,284]. At a high initial surface pressure, 40 dynes/cm, the protein increases the surface pressure of PA and cardiolipin further, indicating penetration into the film of these two lipids, but not that of PE and PC. It increases the surface pressure of PE only at a low initial surface pressure, 24 dynes/cm. This is lower than the estimated surface pressure in SUVs and the PE may be too expanded to hydrogen bond significantly, thus allowing penetration. Myelin basic protein also has a greater effect on the surface pressure of monolayers of cerebroside sulfate at low initial surface pressure than at high surface pressure, where hydrogen bonding interactions may be stronger [285]. Cytochrome c causes a much greater increase in the surface pressure of PE at pH 9 where hydrogen bonding no longer occurs or is weakened, even though less protein was bound at this pH. lower dissociation constant than if only bound electrostatically to two molecules of acidic lipid. The lipid polar head groups surround the cation and allow solubilization in a hydrophobic medium [287]. If these lipids include PI, Brockerhoff has suggested that phosphorylation of the inositol could release the Ca 2 by breaking the hydrogen bonds between the two lipids [286]. Inhibition of lipid hydrogen bonding by anesthetics and other drugs has been suggested as a mechanism of anesthesia [288]. This is supported by a correlation of the anesthetic potency of four derivatives of n-octane with their hydrogen bonding capacity. A number of cationic drugs with a protonated amino group such as local anesthetics, fl-receptor blockers, or neuroleptic compounds, cause a decrease in the transition temperature of DPPA to the value for DPPG when complexed with these drugs [289]. This indicates that they disrupt the hydrogen bonding interactions between molecules of PA causing it to behave like PG. This could have a significantly greater effect on lipid structure and function than the small fluidizing effect of apolar drugs on non-hydrogen bonding lipids.
IV. Control mechanisms for membrane function

III-D. Other roles of lipid hydrogen bonding

Several other roles of lipid intermolecular hydrogen bonding in membranes have been suggested. It may allow lipids to participate in Ca 2+ storage and transport [286,287]. Hydrogen bonding between two lipids, at least one of which has a large polar head group, can form a cage in which a calcium ion can be entrapped and bound with a

In the previous section, we have discussed how intermolecular hydrogen bonding interactions between lipids may be one of the factors responsible for hexagonal-phase and inverted micelle formation, clustering or domain formation of certain lipids in the bilayer, maintenance of lipid asymmetry and protein conformation in the membrane. Events which affect the degree of hydrogen bonding could therefore control protein and membrane function. The degree of hydrogen bonding might be altered physiologically by changes in the lipid distribution, the state of ionization of the polar head groups, or the composition of the polar head groups. We will now discuss the last two mechanisms.

IV-A. Regulation of hydrogen bonding by change in environment

Hydrogen bonding interactions between phospholipids generally occur between ionizable groups

390 and therefore depend on the p H and on the p K values of these groups. Because of the high surface charge density of a bilayer of ionized lipids, which attracts protons to the bilayer surface, the apparent p K values of their ionizable groups are often considerably different than for similar groups on small molecules in aqueous solution (Table VIII) [2,5]. One of the most striking effects of the surface charge density on the p K is the difference in p K in the gel and liquid-crystalline phases. The p K values ( p K means the apparent p K value unless otherwise noted) of the carboxyl on D M P S and of the amine of D M P E are lower in the liquid-crystalline phase than the gel phase, due to the lower surface charge density of the former [43,291]. In fact, PS and PM release protons during the phase transition if the p H is near their p K values [2,43]. This leads to p r o n o u n c e d hysteresis between heating and cooling scans of the phase transition. The p K is likely also to depend on fatty acid composition of the lipid, since an unsaturated lipid will be more expanded and have a lower surface charge density in both the gel and liquid-crystalline phases than a lipid with saturated fatty acids. The p K values of lipids in a bilayer are, therefore, very sensitive to their environment - to the ionic strength of the aqueous phase, cations b o u n d to the bilayer, to the presence of other charged groups in the bilayer and to other factors which can alter the surface charge density [2,68]. The occurrence of hydrogen b o n d i n g m a y also alter their intrinsic p K value by stabilizing ionization states which function as hydrogen b o n d acceptors or donors. Indeed, as suggested by Haines [29], a particular ionizable group of a lipid m a y have two p K values if hydrogen b o n d i n g occurs between the acid and anion, as found for fatty acids in bilayers. This may be why it appears to be impossible to completely protonate the phosphate in lipids such as PC and PE and possibly P G (see subsection II-C1). The intrinsic p K of a lipid ionizable group can be affected by the presence of other ionizable groups on the same molecule. Thus, the p K of the carboxyl of PS is considerably less than that of a carboxylic fatty acid in monolayers due to the contribution of the positively charged amine in lowering the surface charge density. Similarly, the TABLE VIII pK VALUES OF IONIZED GROUPS OF PHOSPHOLIPIDS AND RELATED WATER-SOLUBLE COMPOUNDS ~ Compound PE Group phosphate pK ~' 2- 3 <2 8 10 11.1 11.25 9 9.6 b 2 3 <2 3-3.5 1.2 4-4.6 5.5 3.6 h 10-10.5 Ref.

PC PG PS

PM PA Fatty acid Sodium glycerophosphate Phosphoethanolamine Ethanolamine Phosphoserine

290 4 amine 291 290 349 42 5 292 phosphate 290 4 phosphate 44,64 phosphate 293 carboxyl 290, 43, 44 42 292 amine 290, 5, 44 11.5 42 9.8 b 292 phosphate 3.5 68 phosphate pK 1 3.5 54 phosphate pK 2 8-9 290, 44, 54, 294 carboxyl 7.5, 9.5 ~ 29 phosphate pK 2 phosphate pK 2 amine amine phosphate pK 1 phosphate pK 2 carboxyl amine 6.5 5.8 10.3 9.1 <1 5.9 2.6 10.0 1.2 4.8 51 295 295 349 42 42 42 42 296 5

Dimethylhydrogenphosphate phosphate Acetic acid carboxyl

a Apparent pK values unless otherwise noted, generally at a salt concentration of 100-200 mM NaCI. Variability in the values may be partly due to the method of measurement and whether the lipid is in the gel or liquid-crystalline phase. h Intrinsic pK. c See text (subsection II-B).

p K of the phosphate of PC, PE and PS is less than that of PG, PM or PA (pK 1). Both the apparent and the intrinsic p K a values of the amine of PS are a little higher than for PE due to the greater negative charge of PS. The surface charge density and the p K values of the lipid head groups can be altered by monoand divalent cations. Increasing the salt con-

391 centration from 0 to 0.5-1 M NaC1 has been reported to lower the pK 2 of PA from 9 to 7.5, the pK of PM from 7 to 3, and the pK of the carboxyl of PS from 6 to 3.7 [43,51,68,298]. Divalent cations lower the pK 2 of PA and the pK of the amine of PS as indicated by titration curves or by the displacement of protons from the phosphate of PA and from the carboxyl and amine of PS [290,293,297,299-303]. Furthermore, divalent cations can bind to the lipid in a 1:1 molar ratio (suggesting the lipid has a net charge of - 2 ) at pH values well below the pK values of these groups in the absence of cations. Binding of positively charged residues of proteins can also lower the pK of groups on the lipid. Myelin basic protein and polylysine lower the pK of the phosphate of PG, PS and PA (pK 1 and pK 2) and the carboxyl of PS, as detected from its effects on the transition temperatures of these lipids and from displacement of protons into the aqueous phase (Refs. 271,281,300 and Boggs, J.M. and Rangaraj, G., unpublished results). Polylysine was also found to lower the pK of the amine of PS indicating that the amino acids do not have to bind directly to the ionizable group of the lipid to affect its pK but can do so by altering the charge density in its environment [300]. Dilution of charged lipids with neutral or oppositely charged lipids will also decrease the surface charge density and affect the pK. The pK of a fatty acid incorporated at low concentrations into a PC bilayer has been reported as 7.2-7.4 [304], considerably less than that in a pure fatty acid monolayer or bilayer vesicles [29]. This decrease may be partly due to the loss of hydrogen bonding of the acid and anion forms. At much higher concentrations of fatty acid in PC where hydrogen bonding between the protonated form of the fatty acid and PC can occur (see subsection IE), the pK of the fatty acid may be considerably higher. The apparent pK of palmitic acid at a concentration of 12 mol% in DPPC has been reported to be 10.2 [39]. Incorporation of low concentrations of a fatty acid into positively charged micelles of stearylamine decreases its pK to 5 while incorporation into negatively charged micelles of sodium tetradecyl sulfate increases it to 7.6 [304]. Similarly the pK of long chain acylated amines incorporated into negatively charged micelles is 11.5, while incorporation into positively charged micelles decreases it to 8.5. Addition of PC to PE has been reported to lower the pK of the amine of PE [291], while an increase in the concentration of PC in PE/PC vesicles also lowers it [292,352]. This may be due to dilution of the effects of PE ( - ) on the surface charge density or disruption of PE hydrogen bonding. Similarly, addition of PC to PA lowers the pK of the phosphate of the latter [299]. Thus, the occurrence of hydrogen bonding could be regulated by changes in cation concentrations or changes in the lipid and protein environment of an ionizable lipid, as well as by changes in pH. This could result in lipid phase transitions, lipid domain formation or phase separation, hexagonalphase formation and conformational changes in proteins. A suggested mechanism for the regulation of lipid hydrogen bonding and its consequent effects on protein conformation and on hexagonal-phase formation by a change in cation concentration at constant pH has been presented previously [300]. Tokutomi et al. [298] demonstrated that a decrease in ionic strength at constant pH near the pK of the carboxyl could induce phase separation of PS in a PS/PC mixture by causing protonation of the PS to the ( - + ) form. Such an effect was predicted by Trauble et al. [68]. This phase separation may have been at least partly due to a transition of PS ( - + ) to the gel state at the temperature used, however, and not just to the greater hydrogen bonding of the ( - + ) form. Trauble [2] also demonstrated that a decrease in pH from 8 to 3 could cause phase separation of DMPM/DSPC mixtures. This is presumably due to intermolecular hydrogen bonding of DMPM in the ( - 1 / 2 ) state. In order for regulation of the ionization state by changes in cation concentration to be a physiologically relevant mechanism for regulating hydrogen bonding and membrane structure and function, the changes in cation concentration must be effective near physiological pH. The pH at the surface of the membrane is not the same as the bulk pH so a wide range of pH values may occur. The pK 2 of PA and the pK of the carboxyl of PS are close to neutral pH under some conditions. Although there is generally little PA in biological

392

membranes, its amount can rapidly increase by more than 10-times after exposure to secretory stimuli [305]. If clustered into its own domain or if next to other lipids with which it can hydrogen bond, changes in its ionization state could affect the membrane structure. The p K of the amine of the important hydrogen bonding lipids, PE and PS seems to be well above any p H which could occur physiologically. However, as discussed earlier, it may be lowered under some conditions so that the amino groups of a significant percentage of the lipid may be unprotonated at neutral pH. Intermolecular hydrogen bonding can also occur between unlike lipids and it is difficult to predict how these interactions in a complex lipid mixture would depend on the pH or cation content.

I V-B. Regulation of hydrogen bonding by enzymatic alteration of lipid composition

The percentage of interactive and repulsively charged lipids in membranes is undoubtedly related to the membrane function. For example, most plasma membranes have a relatively low PE content, while intracellular membranes which may undergo fusion or exocytosis, such as synaptosomal, chromaffin granule, and zymogen granule membranes, or membranes with special functions such as mitochondrial membranes, have a high PE content (reviewed in Ref. 1). Repulsively charged lipids will help to inhibit hydrogen bonding of PE and hexagonal-phase formation and other consequences of hydrogen bonding. However, they can be separated into their own domains or neutralized by binding of cations and proteins allowing a mechanism for regulation of hydrogen bonding.

IV-B1. Response to changes in growth conditions This balance between interactive lipids and repulsively charged lipids may be maintained under different growth conditions at constant temperature or for cell mutants with various deficiencies in lipid synthesizing enzymes [306-308]. One negatively charged lipid may be able to substitute for another and one interactive lipid for another. Escherichia coil mutants deficient in cardiolipin synthesis have an increased content of another repulsively charged lipid, PG [306]. Myoinositol1-phosphate phosphatase defective strains of Neu-

rospora crassa are able to substitute PS for PI when grown at low concentrations of inositol [308]. Interestingly, their levels of cardiolipin and PG do not change, however. This may be due to the fact that PI and PS may have both interactive and repulsive properties, while the latter two are only repulsive. Fibroblasts or mutants of N. crassa grown on different choline analogues could substitute the weak hydrogen bonding dimethylPE for PC or the stronger hydrogen bonding monomethylPE for PE [307,308]. The physical properties of the fibroblast cells and extracted lipids were studied with fluorescent probes and were found to be very similar when grown on different choline analogues [309]. Thus, cells adjust their lipid head group composition by making compensatory changes in order to maintain similar interactive and repulsive properties, if forced to do so by changes in growth conditions. This is another form of the well-known homeoviscous adaptation of cells to changes in growth temperature or growth conditions by alteration of fatty acid and cholesterol content in order to maintain the majority of their lipids in the liquid-crystalline phase [310-312]. Regulation of the amounts of its major polar lipids occurs in Acholeplasma laidlawii in response to changes in the growth conditions or temperature [313-317]. This is an interesting example of regulation of the fipid content in order to maintain both the surface charge density and the tendency to form the hexagonal phase. The major polar lipids in this organism (which lacks a cell wall) are the glucosyl-containing M G D G , D G D G , glycerophosphoryl derivatives of M G D G and D G D G and PG [314]. The M G D G / D G D G ratio increases and the ratio of charged to neutral lipids decreases as the percent saturated fatty acid incorporated into the lipid increases [313-317]. Increased incorporation of cholesterol causes an increase in the unsaturated fatty acid content, a decrease in the M G D G / D G D G ratio and an increase in the amount of charged lipids [317]. A decrease in the growth temperature also causes an increase in the M G D G / D G D G ratio, a decrease in the amount of charged lipid and an increase in the unsaturated fatty acid content [316]. These changes, the ratio of M G D G / D G D G , ratio of charged to neutral lipids, degree of satura-

393 tion of the fatty acids, and the cholesterol content, can all be correlated with the tendency of MGDG to go into the HII phase and the effect of the other changes on the stability of the lamellar and hexagonal phases [318-320,353]. A high content of cis unsaturated fatty acids, high cholesterol content, low ratio of charged/neutral lipids, a high M G D G / D G D G ratio and an increase in temperature all favor the formation of non-lamellar phases, as occurs with PE (see subsection II-C3). Use of deuterium NMR and polarizing light microscopy showed that all mixtures of MGDG, DGDG and cholesterol which occur naturally under different conditions of fatty acid supplementation and growth temperature, form the lamellar phase, while unnatural mixtures with higher M G D G / D G D G ratios or high cholesterol content, form non-lamellar, isotropic inverted-micelle or reverse-cubic phases or the hexagonal phase [318-320]. Thus A. laidlawii appears to regulate its lipid composition in order to maintain the lamellar phase under normal conditions. Changes in the ratio of PG to neutral lipids which occur naturally also maintain the membrane surface potential at a constant level [321]. However, the presence of such a high concentration of an H H phase-forming lipid and the great sensitivity of the phase behavior to the composition suggest that the role of MGDG in this membrane is to undergo localized transitions to a non-lamellar phase in response to some stimulus from its environment. Thus the composition of Acholeplasma laidlawii must be regulated, not just in order to maintain the lamellar phase or a certain surface potential, but to carefully balance the stabilities of the lamellar and non-lamellar phases so that a non-lamellar phase can be readily induced with appropriate stimulation. One such stimulus could be divalent cation binding to the charged lipids. This would neutralize their charges, abolishing their inhibitory effect on HII phase formation, and/or cross-link two molecules of charged lipid making their structure compatible with the H H phase. Ca 2+ can induce a non-lamellar phase in P E / P G mixtures [138], even though it cannot phase separate out PG from this mixture, unlike PS. McElhaney [322] has coined the term 'homeophasic adaptation' to be used, rather than 'homeoviscous adaptation' for the maintenance of the liquid-crystalline-gel phase balance at the growth temperature of microorganisms by adaptive changes in lipid composition, to emphasize that this balance is more important than maintenance of a certain degree of fluidity. This term might be extended to also include adaptive changes which maintain the lamellar-hexagonal phase balance. An interesting example of regulation of lipid head group composition in order to maintain the cell permeability barrier is that of Staphylococcus aureus which regulates the ratio of its major lipids, PG and the positively charged lysylPG, in response to changes in the pH of the growth medium [187,323]. At pH 7.2, the major lipid is PG and the PG to lysylPG ratio is 6-19. At this ratio the cell should repel anions [324]. When the pH is lowered to 4.8, by either glucose fermentation or adjustment with acid, the PG/IysylPG ratio decreases to 0.6-1.3. At this ratio intermolecular hydrogen bonding between the two lipids will take place (see subsection II-E) stabilizing the membrane. In addition, if excess lysylPG is present it will repel cations and protons. This adjustment of the P G / lysylPG ratio allows S. aureus to protect its intracellular pH from the extracellular pH by causing the cell to repel protons at low pH and hydroxyl ions at high pH, as suggested by Houtsmuller and Van Deenen [323]. When lipids such as PE become very unsaturated they may be too widely separated for lateral intermolecular hydrogen bonding to occur in planar surfaces. One function of lipids with larger hydrogen bonding head groups which can extend laterally over a greater distance, like diglycosyldiacylglycerols and lysylPG, may be to maintain hydrogen bonding when highly unsaturated. This may be the role of an interesting lipid found so far only in two strains of Clostridium butyricum, a glycerol acetal of ethanolamine plasmalogen. An increase in growth temperature of the organism causes an increase in the amount of this lipid and of PE, and a decrease in PG [325]. When it is grown on oleic acid the amount of the glycerol acetal increases to 50% of the total phospholipid, double its amount when grown on elaidic acid. The transition temperature of the dielaidoyl form is similar to that of DEPE suggesting that it can also participate in intermolecular

394

hydrogen bonding [326]. The glycerol on the C-1 carbon of the ether-linked chain may be able to hydrogen bond with the phosphate of a neighboring lipid. However, the transition temperature of the dioleoyl form is about 25 Cdeg higher than that of DOPE suggesting that its hydrogen bonding interactions persist in spite of the presence of two cis unsaturated chains, while those of DOPE may not, in the lamellar phase (see subsection II-C1). Thus, the increase in amount of this lipid for the organism grown on oleic acid may help to maintain the hydrogen bonding interactions of the lipids in the lamellar phase and stabilize the membrane. Curiously, however, the phase-transition temperature of the glycerol acetal lipid on cooling from the liquid-crystalline phase is 20 Cdeg less than on heating for both the dielaidoyl and dioleoyl forms, suggesting that its hydrogen bonding interactions are broken in the liquid-crystalline phase and the lipid must be supercooled in order for them to reform. Thus, it is not clear whether hydrogen bonding would be physiologically relevant for this lipid at temperatures above its transition temperature. This kind of hysteresis has not been observed for PE including the 1-saturated,2unsaturated natural species isolated from mammalian membranes [56]. The content of sphingolipids and their hydrogen bonding capacity in membranes is also related to the membrane structure and function. An increase in the sphingomyelin/PC ratio in red blood cells from some animal species decreases the lipid fluidity, permeability, osmotic fragility and susceptibility to damage by bile salts [327-329]. Karlsson [330,331] has demonstrated an intriguing correlation between the number of free hydroxyl groups on the sphingosine base and fatty acid of membrane sphingolipids and the degree of chemical and physical stress to which the membrane or organism is exposed. For example, the number of hydroxyl groups on the sphingosine of sphingomyelin is greater in the kidney medulla than in the cortex, which may be related to the increase in osmolarity from the cortex to the medulla. Sphingolipids of epithelial cells facing the lumen of the small intestine have more trihydroxy ceramide, while those of the underlying non-epithelial tissue have only monohydroxy ceramide [332]. These

ceramide hydroxyl groups may contribute to a hydrogen bonding network which stabilizes the membrane and decreases its permeability. The number of ceramide hydroxyl groups of sphingolipids in the brain and myelin of adults varies from one to two. The ratio of h y d r o x y / nonhydroxy fatty acids increases in myelin sphingolipids during development in the human and rat [333-336]. This ratio tends to increase with the complexity of the nervous system in different species [337]. However, myelin from some species of Urodela completely lacks the hydroxy fatty acid form of cerebroside and cerebroside sulfate [338]. Interestingly, the velocity of nerve conduction is significantly reduced in this species relative to that in species which possess the hydroxy fatty acid forms, suggesting a possible involvement of the membrane stabilizing effects of ceramide hydroxyl groups in nerve conduction. An increase in the ratio of hydroxy/nonhydroxy fatty acids of ceramide lipids of N. crassa and of ciliary membranes of Tetrahymena pyriformis NT-1 cells occurs with increasing growth temperature [339,340]. This change in the ciliary membranes is accompanied by decreased fluidity of the membranes and lipid extracts as detected with a fluorescent probe [341]. However, other changes in lipid composition occur as well, so that the change in fluidity cannot be attributed solely to the change in hydroxylation. Tetrahymena, grown in the presence of the branched fatty acid isovalerate, increases its ratio of hydroxy/nonhydroxy fatty acids [342], possibly to compensate for the fluidizing effect of the branched fatty acid. Thus, changes in ceramide hydroxylation, as well as changes in fatty acid saturation and head group composition, can occur in response to changes in growth conditions. Kaya et al. [340] provided evidence that hydroxylation can occur through direct hydroxylation of the ceramide bound fatty acid rather than free fatty acid, and thus could constitute a rapid response to some change in the cell's environment. Invertebrates are rich in plasmalogens and an increase in the growth temperature of fish causes an increase in their amounts of plasmalogens as well as less unsaturated fatty acids [343]. Tetrahymena grown at a higher temperature also increase their percentage of ether-linked lipids [344]. The

395 increased head group hydrogen bonding caused by the ether linkage in ethanolamine plasmalogens may help maintain the normal lipid fluidity and the balance between the gel and liquid-crystalline phases, and the lamellar and hexagonal phases. Extremely halophilic bacteria contain exclusively diether-type lipids [345], which may help decrease their permeability and protect them against the high NaC1 concentration in their environment [139]. Ethanolamine plasmalogens may be synthesized in membranes also because of a need for their increased tendency to go into the hexagonal phase. The ethanolamine plasmalogen of myelin goes into the hexagonal phase at 18C suggesting that the high content of this lipid in myelin may have a purpose in causing fusion or other functions mediated by the hexagonal phase, under some conditions or during myelin biogenesis [56]. The percentage of the total ethanolamine plasmalogen in myelin increases during development in the human and rat [127,144]. IV-B2. Response to signals Rapid enzymatic modification of lipid head groups can also occur in response to various stimuli and serve as a mechanism of transmission of signals to other constituents of the membrane or to the inside or outside of the cell. These modifications may often change the hydrogen bonding properties of the lipids; this may partially constitute their mechanism of action. One example of this is the rapid turnover of PI and its phosphorylated forms [346]. The inositol triphosphate released and diacylglycerol transiently produced act as second messengers by triggering other responses [347]. Diacylglycerol can hydrogen bond with repulsively charged lipids as demonstrated with dimyristin and DMPM [4] (see subsection II-E). Unsaturated forms of diacylglycerol can induce the hexagonal phase in PE, PC and PS [223,272]. This could allow it to induce fusion. Long chain alkanes can also induce the hexagonal phase in PE but a higher concentration is required [223]. Although the small polar head group of diacylglycerol may be partly responsible for its ability to induce the hexagonal phase, as suggested by the authors of these studies, its ability to hydrogen bond with these lipids is probably involved also. Less widely recognized as a potential second messenger during PI turnover is the PA which is also transiently produced [186,250,305]. This could significantly affect the hydrogen bonding interactions in localized domains of the membrane. The change in structure and hydrogen bonding properties of PI which occurs upon inositol phosphorylation may also constitute a message in itself. This may affect the Ca z+ binding of PI as suggested by Brockerhoff [286], as well as its lipid-lipid and lipid-protein interactions in the membrane. Another example of the involvement of enzymatic modification of lipid head groups in transduction of biochemical signals is that of methylation of PE, which occurs to a significant extent in liver [180] and to a lesser degree in other organs, where it is of interest, however, because it occurs in response to neurotransmitters, chemotactic peptides, antigen-antibody interactions, etc., demonstrated by Hirata and Axelrod [198]. Methylation is mediated by two different methyltransferases located asymmetrically in the membrane, resulting in translocation of the methylated PE to the outer surface. The different hydrogen bonding properties of PE and its methylated forms may be involved in this translocation. The monomethylPE formed in non-hepatic tissue is present only transiently and appears to be sequestered within the bilayer, since it cannot be hydrolyzed by phospholipase C added to either side of the membrane [198]. Hirata and Axelrod [210] have suggested that Mg 2 complexes PE to the enzyme bringing the polar head group into the bilayer where it is then methylated by the first transferase located within the bilayer. It may then be methylated again by the second transferase on the other side of the bilayer. An alternative explanation is that PE may be methylated within the bilayer while in the form of inverted micelles. Since monomethylPE can form inverted micelles almost as well as PE, it can remain within the micelles where it then becomes methylated again by the second transferase on the other side of the bilayer. Since dimethylPE behaves more like PC and has a lesser tendency to form inverted micelles, it then goes back into the lamellar phase but on the extracellular side this time, where it is methylated further to PC. Since monomethylPE behaves similarly to PE

396

and dimethylPE behaves similarly to PC and can even substitute for them in auxotrophic mutants [307,308], methylation of PE could be a mechanism for changing the hydrogen bonding properties of a small domain of lipid and causing transitions between the inverted micelle and lamellar phases. This could help regulate fusion, endocytosis and other functions putatively mediated by inverted micelles [10]. Gagne et al. [77] have shown that monomethylPE can support fusion of vesicles better than the dimethyl form.

V. Summary
The great variety of different lipids in membranes, with modifications to the hydrocarbon chains, polar groups and backbone structure suggests that many of these lipids may have unique roles in membrane structure and function. Acidic groups on lipids are clearly important, since they allow interaction with basic groups on proteins and with divalent cations. Another important property of certain lipids is their ability to interact intermolecularly with other lipids via hydrogen bonds. This interaction occurs through acidic and basic moieties in the polar head groups of phospholipids, and the amide moiety and hydroxyl groups on the acyl chain, sphingosine base and sugar groups of sphingo- and glycolipids. The putative ability of different classes of lipids to interact by intermolecular hydrogen bonding, the molecular groups which may participate and the effect of these interactions on some of their physical properties are summarized in Table IX. It is frequently questioned whether intermolecular hydrogen bonding could occur between lipids in the presence of water. Correlations of their properties with their molecular structures, however, suggest that it can. Participation in intermolecular hydrogen bonding increases the lipid phase transition temperature by approx. 8-16 Cdeg relative to the electrostatically shielded state and by 20-30 Cdeg relative to the repulsively charged state, while having variable effects on the enthalpy. It increases the packing density in monolayers, possibly also in the liquid-crystalline phase in bilayers, and decreases the lipid hydration. These effects can probably be accounted for by transient, fluctuating hydrogen bonds involving

only a small percentage of the lipid at any one time. Thus, rotational and lateral diffusion of the lipids may take place but at a slower rate, and the lateral expansion is limited. Intermolecular hydrogen bonding between lipids in bilayers may be significantly stabilized, despite the presence of water, by the fact that the lipids are already intermolecularly associated as a result of the hydrophobic effect and the Van der Waals' interactions between their chains. The tendency of certain lipids to self-associate, their asymmetric distribution in SUVs, their preferential association with cholesterol in noncocrystallizing mixtures, their temperature-induced transitions to the hexagonal phase and their inhibitory effect on penetration of hydrophobic residues of proteins partway into the bilayer can all be explained by their participation in intermolecular hydrogen bonding interactions. Such interactions at the polar head group region as in phospho- and glycolipids help result in the 'wedge shape' invoked by many investigators to explain their results. The occurrence of many of these effects above the gel to liquid-crystalline phase transition temperature indicates that intermolecular hydrogen bonding persists in the liquid-crystalline phase, particularly if the lipids can form a more highly curved bilayer. Thus, the presence of 'interactive' or 'repulsively charged' groups on lipids may help organize membrane lipids into domains of different composition, help maintain the asymmetric distribution of lipids in membranes, regulate the stability of lamellar and non-lamellar phases, such as inverted micelles, which may be involved in a number of membrane functions, and regulate the conformation and activity of extrinsic membrane proteins and enzymes. Many of these intermolecular interactions depend on ionizable groups whose pK values are sensitive to ionic strength, divalent cations and the surface charge density of the bilayer. Changes in pH, the concentration of mono- and divalent ions, near-neighbor lipids and proteins can alter the state of ionization and hydrogen bonding properties of the lipids. Thus lipids could act as receptors for intra- or extracellular signals, transmit or amplify these signals to other membrane components and take part in dynamic membrane func-

397 TABLE IX SUMMARY OF ABILITY OF VARIOUS LIPIDS TO PARTICIPATE IN INTERMOLECULAR HYDROGEN BONDING AND EFFECT ON PHASE BEHAVIOR Lipid Ionization state a (- + ) (-+) (- ) (- +) (- - +) (- - ) ( - ) (- ) (- - ) (0 or - ) (- ) ( - ~2) (-) (0) (0) (- + ) (- ) (0) Participation in intermolecular hydrogen bonding b ++ + + + + + + + +
-

Potential hydrogen donating and accepting groups PO-, C--O e PO-, NH~ PO-, NH 2 PO-, N H ~ , COOH PO-, COO-, NH~ PO -, COO-, NH 2 PO-, POH PO-, POH POPOH, COH, P=O PO-, COH PO-, POH
PO-

Effect on Tm c 0 ++ 0 + + + + + + + 0 + +
0

Effect on hydration c + n.d. + n.d. n.d. n.d. n.d. n.d. + n.d.

+

Formation of hexagonal phase a + + + n.d. n.d.

-

PC PE PS

PA

PG PM
MGDG

DGDG Sphingomyelin Cerebroside sulfate Cerebroside

+ + + + +

+ + f g + +

COH COH PO-, HNC=O, COH SO-, HNC--O, COH COH, HNC=O

+ + + + + + + + +

+ - g

+ -

a As in footnote a of Table I. b With itself. All lipids indicated can hydrogen bond heteromolecularly with other suitable lipids. Number of ( + ) indicates degree of interaction, ( - ) indicates absence of interaction. c Relative to PC. ( + ) indicates greater than or equal to PC, ( - ) indicates less than PC. Number of ( + ) or ( - ) indicates degree of difference from PC. (0) indicates similar to PC. d ( + ) means yes under appropriate conditions, ( - ) means no. e May be possible for all lipids with ester-linked chains. f Possibly (see text). Depends on ionic strength. n.d. = not reported

tions by undergoing phase transitions or causing protein conformational changes. Changes in the hydrogen bonding and other properties of the lipids could also occur through e n z y m a t i c a l t e r a t i o n o f t h e lipid. T h u s , t h i s c o u l d be a mechanism by which the effects of other chemical signals are mediated. Enzymatic modification could occur transiently for an immediate a n d r a p i d r e s p o n s e as i n P I t u r n o v e r a n d m e t h y l ation of PE, or more permanently in response to changes in growth conditions or during development and differentiation. Further studies of the properties of lipids, individually and in combinat i o n , will h e l p t o u n d e r s t a n d t h e r o l e s o f s p e c i f i c lipids in membranes.

Acknowledgements

The author gratefully acknowledges a Scientist Award from the Medical Research Council of C a n a d a . A p p r e c i a t i o n is e x p r e s s e d t o D r s . H . E i b l , T. H a i n e s , D . M a r s h , I. P a s c h e r , G . S h i p l e y a n d their collaborators for permission to reproduce f i g u r e s f r o m t h e i r m a n u s c r i p t s . D r s . T. H a i n e s , I. P a s c h e r a n d D. V a u g h a n a r e s i n c e r e l y t h a n k e d f o r a l l o w i n g m e t o see a n d c i t e w o r k p r i o r t o its publication. Drs. K.M. Keough and J.R. Silvius are also thanked for reading this manuscript and for their helpful suggestions.

398

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