Alan McLaughlin

The adsorption of divalent cations to phosphatidylcholine bilayer membranes

Electrophoretic mobility and 31P NMR measurements were combined to test whether the combination of the Henry, Boltzmann and Grahame equations is capable of describing the adsorption of divalent cations of phosphatidylcholine membranes. Cobalt was chosen for this study because, of all the common divalent cations, its effects on the 31P NMR spectrum of phosphatidylcholine membranes are easiest to interpret. Both the 31P NMR data on the adsorption of cobalt and the zeta potential data calculated from the electrophoretic mobility in the presence of cobalt are well described by the combination of these three equations. Electrophoretic mobility measurements were also performed with a number of other divalent cations and the zeta potentials were, in all cases, well described by the combination of these three equations. The binding deduced from such measurements decreases in the sequence: Mn2+, Mg2+, Ca2+, Co2+, Ni2+, Sr2+, Ba2+. If we assume that a lipid molecule occupies an area of 60 A2 and that there is a 1 : 1 stoichiometry for the binding of the divalent ions to phosphatidylcholine, the dissociation constants are, respectively: 0.3, 1.0, 1.0, 1.2, 1.2, 2.8, 3.6 M.

The adsorption of divalent cations to phosphatidylglycerol bilayer membranes

The ability of Stern equation to describe the adsorption of divalent cations to phosphatidylglycerol membranes was tested by combining 31P-NMR and electrophoretic mobility measurements. In 0.1 M sodium chloride both the 31P-NMR and the zeta potential data are well described by the Stern equation. 31P-NMR and 13C-NMR results indicate that cobalt forms inner-sphere complexes only with the phosphate group of phosphatidylglycerol molecules and that a substantial fraction of the adsorbed cobalt ions form outer-sphere complexes. Evidence is presented that suggests the alkaline earth cations also bind to phospholipids mainly by forming outer sphere complexes. Electrophoretic mobility measurements were performed with several different divalent cations. In all cases the zeta potentials in 0.1 M sodium chloride were well described by the Stern equation. The intrinsic 1:1 association constants (M-1) for the phosphatidylglycerol complexes decreased in the sequence: Mn2+, 11.5; Ca2+, 8.5; Ni2+, 7.5; Co2+, 6.5; Mg2+, 6.0; Ba2+, 5.5 and Sr2+, 5.0.

Dimethonium, a divalent cation that exerts only a screening effect on the electrostatic potential adjacent to negatively charged phospholipid bilayer membranes.

SummaryCalcium and other alkaline earth cations change the electrostatic potential adjacent to negatively charged bilayer membranes both by accumulating in the aqueous diffuse double layer adjacent to the membrane and by adsorbing to the phospholipids. The effects of these cations on the electrostatic potential are described adequately by the Gouy-Chapman-Stern theory. We report the results of experiments with ethane-bis-trimethylammonium, a cation that has been termed “dimethonium” or “ethamethonium” in analogy with hexamethonium (hexane-1,6-bis-trimethylammonium) and decamethonium (decane-1,10-bis-trimethylammonium). We examined the effect of dimethonium on the zeta potential of multilamellar vesicles formed from the negative lipid phosphatidylserine (PS) and from 5 ∶ 1 phosphatidylcholine/phosphatidylserine mixtures in solutions containing 0.1, 0.01 and 0.001m sodium, cesium, or tetramethylammonium chloride. We also examined the effect of dimethonium on the conductance of planar PS bilayer membranes and the31P NMR signal from sonicated PS vesicles formed in 0.1m NaCl. We found no evidence that dimethonium adsorbs specifically to bilayer membranes. All the results, except for those obtained with vesicles of low charge density formed in a solution with a high salt concentration, are consistent with the predictions of the Gouy-Chapman theory. We conclude that dimethonium, which does not have the pharmacological effects of hexamethonium and decamethonium, is a useful divalent cation for physiologists interested in investigating electrostatic potentials adjacent to biological membranes.

[16] Measuring electrostatic potentials adjacent to membranes

Publisher Summary This chapter presents the method for measuring electrostatic potentials adjacent to membranes. It is suggested that one can measure the potential at the hydrodynamic plane of shear, at the surface of the membrane, and within the bilayer. Four different techniques are described for measurement, all of which utilize ions as probes. (l) 31 P nuclear magnetic resonance (NMR) can be used for measurements using a paramagnetic cation, such as Mn, as a probe. The NMR measurements sense the potential at a well-defined site, the phosphodiester group at the membrane surface, but the technique requires small sonicated vesicles and relatively large amounts of them. (2) Fluorescence measurements can be made using 2-( p toluidinyl)naphthalene 6-sulfonate (TNS). (3) Electron paramagnetic resonance measurements using spin labels as probes are applicable. The anion TNS and the charged spin labels are amphipathic molecules that adsorb hydrophobically to the membrane-solution interface. TNS and spin labels can be used to probe the surface potential of biological membranes, but they permeate some membranes and are sensitive to many parameters other than the electrostatic potential. Fluorescent probes, for example, respond to the dielectric constant. (4) The conductance of planar membranes can be measured using nonactin K or carbonyl-cyanide-p-trifluoro-methoxyphenylhydrazone as cationic or anionic probes.

The interaction of cobalt with glycerophosphoryl choline and phosphatidyl choline bilayer membranes

Abstract The interaction of cobalt with glycerophosphoryl choline (a model for the polar headgroup region of phosphatidyl choline membranes) has been investigated. Because of the long T2M the effects of cobalt on the 31P NMR spectrum are predominantly due to the hyperfine interaction, which considerably simplifies the analysis. The frequency of the bound species, Δωm is calculated to be −1442 ppm (at 70°C), while τM, the lifetime of the complex, is calculated to be 2.95 × 10−7 seconds (at 70°C) with an activation energy of 10.0 kcal/mole. The observed effects of the 31P NMR spectrum are used to calculate the amount of bound cobalt-glycerophosphoryl choline complex, and a binding constant for the complex (5.1 M). It is shown that 31P NMR is very useful for studying the binding of cobalt (as a model divalent cation) to phospholipid bilayer membranes. In particular, the technique can distinguish direct binding from electrostatic screening.

Nuclear Magnetic Resonance Studies of the Adsorption of Divalent Cations to Phospholipid Bilayer Membranes

Thermodynamic aspects of the adsorption of divalent cations to phospholipid bilayer membranes can be reasonably well described by the Gouy-Chapman-Stern theory (S. McLaughlin, this volume). To proceed beyond this thermodynamic description requires molecular information about the structure of the divalent cation-phospholipid complexes. Because most experimental techniques, i.e., equilibrium dialysis (Portis et al., 1979) or ion-sensitive electrodes (McLaughlin et al., 1981), measure only the loss of divalent cations from the aqueous medium, they give no information on the bound complexes. For example, they cannot be used to determine which groups in the phospholipid molecule provide ligands for the divalent cation, or to distinguish between inner-sphere complexes, where the ligand is inserted into the first coordination sphere of the divalent cation, and outer-sphere complexes, where the ligand and the fully hydrated cation form an “ion pair” (Basolo and Pearson, 1967; Hewkin and Prince, 1970; Ahland, 1972; Beck, 1968). This review briefly discusses how nuclear magnetic resonance (NMR) can provide this type of molecular information and can also be used to quantitatively test one of the major assumptions of the Gouy-Chapman-Stern theory.

The Interaction of cobalt with glycerophosphoryl glycerol and phosphatidyl glycerol bilayer membranes

Abstract The frequency shift, Δω M , of the 31 P NMR signal from the inner-sphere complex between cobalt and the phosphodiester group of glycerophosphoryl glycerol is calculated to be 3980 ppm at 70°C. This value of Δω M is used to calculate the lifetime, τ M , of the inner-sphere complex between cobalt and the phosphodiester group of phosphatidyl glycerol, from an analysis of the temperature dependence of the effects of cobalt on the 31 P NMR signal from phosphatidyl glycerol membranes. The value of τ M (1.5 μsec at 20°C) is used in a companion study [A. Lau, A. C. McLaughlin, and S. McLaughlin, Biochim. Biophys. Acta 645 , 279 (1981)] to investigate the importance of outer-sphere complexes, i.e., weak ion pairs between the charged phosphodiester group and the completely hydrated divalent cation, in the interaction of cobalt with phosphatidyl glycerol membranes.