Bilayer Separation Research Articles

Phosphatidylcholine-Cerebrosides Interactions: Influence of Cerebroside Acyl Chain Character

Abstract We have measured the swelling properties, and force decay with bilayer separation, for single phase multi-lamellar arrays made from dioleoylphosphatidylcholine, and cerebrosides with primarily non-hydroxl acyl chains. The phase characteristics and interactive forces are found to be similar to bilayers made from DOPC and cerebrosides with primarily hydroxyl acyl chains. In all cases, cerebrosides are found to have little effect on the hydration and electrostatic forces.

Forces between phospholipid bilayers

The measurement of forces between lipid bilayers is complicated by the coupling which exists between bilayer structure and interbilayer interaction. In fact, direct measurements are impracticable and methods must be sought which enable these two factors to be separated. A procedure is developed for calculating the bilayer stress, γ, and the interbilayer force, ƒ, from simultaneous X-ray measurements of the bilayer structure and water layer thickness and the activity of water. The method is applied to existing experimental measurements for the egg-lecithin–water system. The interbilayer ‘hydration force’ is found to be attractive, exponential with a decay length of 0.22 nm and to dominate completely the van der Waals dispersion force for bilayer separations well beyond the swelling limit. The spontaneous uptake of water by the lamellar phase is shown to be a consequence of the decrease in Gibbs energy associated with the lateral expansion of the bilayer. The elastic area compressibility modulus, ka, near to the swelling limits is found to be 140 mN m–1. The implications of these results for the phase behaviour of phospholipid–water mixtures and the stability of vesicle dispersions are discussed.

Anion influence on the binding of divalent cations to phosphatidylcholine

We have used the osmotic pressure technique of Rand, Parsegian and co-workers (Nature 259 (1976) 601–603) to investigate the effect of anion species on the binding of M 2+ to dipalmitoylphosphatidylcholine bilayers. Calcium and magnesium salts show a complex behavior which is consistent with both anion binding and screening. We observe virtually no change, within the accuracy of our experiment, in the decay of repulsive pressure with inter-bilayer separation for the acetate and nitrate salts of magnesium and calcium; however, the chloride salt does show a different pressure decay. At any given bilayer separation, 35 A ̊ < d w < 75 A ̊ , with calcium and magnesium salts present, the anions produce a decrease in the repulsive pressure in the order acetate − > Cl − > NO 3 −.

Effects of monovalent ion binding and screening on measured electrostatic forces between charged phospholipid bilayers

In an effort to determine the role that monovalent ions play in the modification of intermembrane forces, we have measured these forces between charged phospholipid bilayers in monovalent ionic solutions. The osmotic stress technique allowed the net electrostatic pressure between the bilayers to be measured while their separation was concurrently determined by x-ray diffraction. Taken together, these measurements yielded electrostatic pressure as a function of bilayer separation. We have related measured pressures to the bilayer surface charge density and surface potential through an exact solution of the full nonlinear Poisson-Boltzmann equation for this system. Quantitative differences in bilayer separation amongst monovalent alkali metal cations indicated differential binding of these to phosphatidylglycerol (PG), phosphatidylserine (PS), and phosphatidic acid (PA); binding affinity series were determined for Li+, Na+, K+, Cs+, and TMA+ ions to these lipids. The anions Cl-, Br-, I-, and CH3COO- were found to have no differential effect on the repulsive forces between PS bilayers. Debye lengths for the electric double layer estimated from the slopes of the experimental pressure curves were consistently longer than predicted on the basis of classic Gouy-Chapman theory. Estimates of the van der Waals Hamaker coefficient between bilayers of PS and PG in salt solution were found to be weaker than between phosphatidylcholine bilayers in pure water, a difference possibly due to electromagnetic retardation and ionic screening.

INTERACTIONS BETWEEN NEUTRAL PHOSPHOLIPID BILAYER MEMBRANES

We have obtained force vs. separation relations between bilayers in 10 different phospholipid preparations: dilauroyl-dimyristoyl-, dipalmitoyl-, distearoyl-, or dioleoylphosphatidylcholine (PC); egg phosphatidylethanolamine; cholesterol-containing bilayers of dipalmitoyl PC and of egg PC. The chemical potential of water in the multilamellar lattice is determined at all water contents and changes continuously with bilayer separation; no discrete classes of water are observed. The interbilayer van der Waals force is estimated from the balance of forces at the bilayer separation where the multilayer lattice is in equilibrium with pure water. Although quantitative differences are evident for different phospholipids, all force curves but one show a clear, exponentially decaying "hydration "repulsion" whose decay distance is 2-3 A . Estimates of forces between bilayer vesicles show great sensitivity to the identity of the phospholipid polar group and to the packing of the hydrocarbon acyl chains. On major implication of this is the likelihood of local structural changes and lipid segregation in the area of closest approach of interacting vesicles of mixed phospholipids.

Binding of divalent cations of dipalmitoylphosphatidylcholine bilayers and its effect on bilayer interaction.

We have confirmed that CaCl2 swells the multilayer lattice formed by dipalmitolyphosphatidylcholine (DPPC) in an aqueous solution. Specifically, at room temperature 1 mM CaCl2 causes these lipid bilayers to increase their separation, dw, from 19 A in pure water to greater than 90 A. CaCl2 concentrations greater than 4 mM cause less swelling. We have measured the net repulsive force between the bilayers in 30 mM CaCl2 at T = 25 degrees C (below the acyl chain freezing temperature). For interbilayer separations between 30 and 90 A, the dominant repulsion between bilayers is probably electrostatic; Ca2+ binds to DPPc lecithin bilayers, imparting a charge to them. The addition of NaCl to CaCl2 solutions decreases this repulsion. For dw less than 20 A, the bilayer repulsion appears to be dominated by the "hydration forces" observed previously between both neutral and charged phospholipids. From the electrostatic repulsive force, we estimate the extent of Ca2+ binding to the bilayer surface. The desorption and bound Ca2+, apparent when bilayers are pushed together, is more rapid than one would expect if an association constant governed Ca2+ binding. The association affinity does not appear to be a fixed quantity but rather a sensitive function of ionic strength and bilayer separation.

A comparison of surface forces and interfacial properties of mica in purified surfactant solutions

The results of direct measurements of forces between two mica surfaces in aqueous CTAB solutions at concentrations in the range 10 −6 M to 4 × 10 −3 M (c.m.c. ∼ 10 −3 M) are reported. These results are correlated with adsorption, wetting and adhesion properties, and together provide an almost complete description of a surfactant/solid interfacial system. Double-layer forces are altered by the effect of surfactant adsorption on the surface potential, as well as by the effect of the bulk surfactant concentration. At a CTAB concentration of about 1/20 c.m.c. a packed monolayer adsorbs on mica, exposing a hydrophobic surface to the solution; near the c.m.c. a second monalayer adsorbs, exposing a hydrophilic (bilayer) surface. Mica surfaces “coagulate” in the region of monolayer adsorption but are stable both at higher and lower surfactant concentrations. The force between mica surfaces in CTAB solutions at the c.m.c., where a bilayer adsorbs on each surface, exhibits a force maximum and shows no deviations from DLVO theory down to bilayer separations of about 1.5 nm, below which additional repulsive forces were measured down to final bilayer collapse. These may be due either to hydration effects or to simple compression of the bilayers, though both effects are likely to be occurring in this narrow distance regime. Large deviations from DLVO theory occur in the hydrophobic monolayer adsorption (coagulation) regime where the attractive force appears to be at least three times larger than the expected van der Waals force. Wetting studies of surfactant solutions on mica indicate that only advancing contact angles yield the correct values for the surface and interfacial energies, as calculated from the Young equation.

Myelin swelling and measurement of forces between myelin membranes

MYELIN has been observed by many workers to swell to a limited extent in water1,2; a swelling thought to result from electrostatic repulsion between the myelin membranes1. The extracellular space between membrane pairs increases by approximately 80 A on immersion in water, whereas the intracellular space changes by only a few angstroms1,2. On the other hand, in phospholipid bilayer systems, very low surface charge densities (approximately 2 mol per cent charged phospholipid or 1 charge per 3,500 A2) cause otherwise neutral phospholipid bilayers, which normally swell to a very limited extent, to separate indefinitely in water as a result of electrostatic repulsion3,4. At even lower surface charge densities, intermediate bilayer separations between that of neutral bilayers (about 27 A) and charged bilayers (indefinite swelling) have rarely, if ever, been observed. Why, then, does nerve myelin swell to only a limited extent in water? We describe here our attempts to answer this question, and report that myelin is prevented from swelling indefinitely by mechanical forces. We have also succeeded in measuring the net repulsive force between myelin membrane pairs.

X-ray diffraction study of myelin structure in immature and mutant mice

X-ray diffraction patterns were obtained from freshly dissected central and peripheral nerves of quaking, myelin synthesis deficiency ( msd), and trembler mutants, as well as immature and adult normal mice. The patterns were compared with respect to strength of myelin diffraction, background scatter level, repeat period, and intensity and linewidth of Bragg reflections. The deficiency of myelin in optic nerves was found to be (in decreasing severity): quaking > immature > trembler ⋍ normal adult; and in sciatic nerves: trembler > immature > quaking msd ⋍ normal adult. Repeat periods about 3 Å less than that for normal adult sciatic myelin were detected in corresponding nerves from immature, quaking, and trembler mice. In some trembler sciatic nerves a second phase having a 190–200 Å period and accounting for about 60% of the total ordered myelin was also evident. Comparison of electron density profiles of membrane units calculated from the repeat periods and diffracted intensities for sciatic myelins indicate structural differences at the molecular level. The main findings are: (1) quaking myelin shows a significant elevation of density in the external protein-water layer between membrane bilayers; (2) the membrane bilayer of immature myelin is ≈ 2 Å thinner than that for normal adult; (3) the membrane bilayer of the more compact phase in trembler myelin is ≈ 5 Å thinner than for normal; and (4) the difference in repeat periods for the two phases present in some of the trembler nerves can be accounted for predominantly by distinct membrane bilayer separations at the external boundary.

Apparent modification of forces between lecithin bilayers.

Small sugar solutes effect variation in the equilibrium separation of lecithin bilayers in aqueous solution. Since sugars have negligible influence on bilayer structure, they probably act by modifying interbilayer forces. The observed widening and narrowing of the bilayer separation is correlated with the predicted weakening and strengthening of the attractive van der Waals forces between lipid bilayers that occurs with increasing sugar concentrations.

An interacting spin label study of lateral expansion in dipalmitoyllecithin-cholesterol bilayers

The lateral expansion in dipalmitoyllecithin-cholesterol multibilayer films has been investigated using interacting pairs of cholestane spin labels. The abrupt lateral expansion occurring in dipalmitoyllecithin at the phase transitio has been directly demonstrated, as also has the extremely high lateral expansivity occurring in the region of lateral phase separation of dipalmitoyllecithin-cholesterol bilayers. A higher lateral expansivity is found above the dipalmitoyllecithin phase transition than below it. The measured increase in lateral molecular separation at the phase transition correlates well with the change in X-ray short spacing, and also with other, indirect, dimensional measurements on the change in area per molecule in the bilayer. Cholesterol is found to have opposite effects on the lateral molecular packing in dipalmitoyllecithin bilayers above and below the phase transition. These opposing effects are also manifest in a reduction, with increasing cholesterol composition, of the total lateral expansion over the transition region. The measured lateral separations are used to calculate the decrease by cholesterol of the intermolecular contribution to the latent heat absorbed over the transition region. The calculations are consistent with the calorimetric measurements of the total latent heat. These results suggest that the observed decrease in the calorimetric onset temperatures for lateral phase separations could be attributed to the decreased lateral expansion in the bilayers containing cholesterol. The broadening of the transition region could also be correlated with a decrease in the cooperativity of intermolecular interactions resulting from the increased intermolecular spacing induced by cholesterol below the transition.