2 Measurement of the Adsorption of Ca and 2+
Mg
2+
to Phosphatidyl Choline Bilayers
L. J. LIS and R. P. R A N D Department of Biological Sciences, Brock University, St. Catharines, Ontario L2S 3A1 Canada V. A . P A R S E G I A N National Institutes of Health, Bethesda, M D 20014 We have used osmotic stress (4) to examine the effect of Mg and Ca on the interactions between dioleoylphosphatidylcholine or dipalmitoyl phosphatidyl choline bilayers. From the net repulsive forces between bilayers we are able to infer electrostatic potentials and charge densities at the site of ion binding; these quantities are sensitive to bilayer separation. We find that at any particular bilayer separation dioleoyl phosphatidyl choline bilayers (melted hydrocarbon chains) adsorb less charge than dipalmitoyl phosphatidyl choline bilayers (frozen hydrocarbon chains) and that the binding of Ca is greater than that of Mg for both kinds of bilayers. 2+
2+
2+
2+
T T 7 e have previously measured the net repulsive force between dipal* * mitoyl phosphatidyl choline ( D P P C ) bilayers in a multilayer lattice charged by the adsorption of C a ( I ) . F r o m these forces we were able to infer electrostatic potentials and charge densities at the site of ion binding by integrating a nonlinear Poisson-Boltzmann equation. This initial study on D P P C showed that as bilayers are pushed together, bound C a desorbs. W h e n N a C l is added to the C a C l solution, membrane repulsion decreases even though more cationic charge appears to adsorb to the bilayer ( I ) . I n this chapter we report the net repulsive force and estimate the associated electrostatic potentials and surface charge densities as a function of bilayer separation between bilayers of D P P C or of 2+
2+
2
0-8412-0473-X/80/33-188-041$05.00/l © 1980 American Chemical Society
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BIOELECTROCHEMISTRY: IONS, SURFACES, M E M B R A N E S
dioleoyl phosphatidyl choline ( D O P C ) i n 3 0 m M C a C l or M g C l solutions. The calculated surface potentials and charge densities, at least for D P P C bilayers i n 3 0 m M C a C l and lOOmM N a C l , are higher than those inferred by L a u et al. (2) from electrophoresis and N M R spectroscopy on multilamellar vesicles. O u r present force measurements as well as the electrophoretic studies (2) show that D O P C bilayers bind less divalent charge than do those of D P P C . W e find a preference for C a over M g * for both phospholipids while electrophoresis (2) suggests ion preference by saturated chain PCs only. 2
2
2
2+
2
Experimental The phosphatidyl cholines PCs used in this study were obtained from the Sigma Chemical C o . The D P P C used here showed swelling behavior in C a C l solutions similar to that of D P P C previously obtained from other sources ( I ) . A l l lipids showed less than 1% impurity by thin-layer chromatography ( T L C ) . Water was doubly distilled and salts were of reagent grade. Dextran was obtained from Pharmacia Chemicals and mixed i n known proportions with salt solutions prior to contact with lipid. Samples were made either by adding lipid to known amounts of salt solution or by adding lipid to dextran-plus-salt solutions. Mixtures were allowed to equilibrate for 48 hr and then were mounted between mica windows. Mixing and waiting at 50°C for up to 48 hr provided no advantage in reaching equilibria nor d i d the addition of a C a ionophore, A23187. A l l measurements were at room temperature ( 2 5 ° C ) . The x-ray diffraction and force-measuring techniques have been described elsewhere ( 3 , 4 ) . Lamellar phases produce a series of x-ray reflections whose spacings are integral multiples of the bilayer repeat spacing, d. The lipid bilayer thickness, d and bilayer separation, d , can be determined for each value of d. W h e n bilayers are forced apart by electrostatic repulsion ( 1 , 5 ) , they maintain the same thickness they have i n excess pure water (44 A for D P P C and 30.5 A for D O P C at 2 5 ° C ) . The bilayer separation differs from the multilayer repeat spacing, d, by this constant lipid bilayer thickness. The net repulsive force between bilayers is given by the experimentally measured osmotic pressure of the dextran solutions ( 6 ) . The difference between the osmotic pressure exerted by dextran i n salt solutions and dextran i n pure water (6) is no greater than the error i n measuring the dextran concentration ( 7 ) . 2
2+
h
w
Simultaneous measurements of d and osmotic pressure provide a relation between the separation of bilayers and their mutual repulsive pressure. Measurement of the electrostatic repulsion is, i n fact, a determination of the electrostatic potential midway between bilayers relative to the zero of potential i n the dextran reservoir. The full nonlinear Poisson-Boltzmann differential equation governing this potential has been integrated ( I ) from the midpoint to the bilayer surface to let us infer the surface potential. The slope of this potential at the surface gives a measure of the charge bound.
2.
LIS E T A L .
Adsorption of Ca * and Mg
43
2
Results DPPC. D r y D P P C was mixed with solutions of C a C l or M g C l to make samples of approximately 30 w t % lipid. T h e lamellar repeat distances d for these mixtures are shown i n Figure 1 as a function of salt concentration. In 0.7mM C a C l or 9 m M M g C l solutions, D P P C multilayers suddenly swell to the maximum allowed by the amount of water in the mixture. I n C a C l solutions, they remain maximally swollen at concentrations up to 3 0 m M C a C l ; but i n M g C l the swelling decreases steadily with increasing salt concentration. Figure 2 shows the relationship between net repulsive force between bilayers and the bilayer separation for D P P C bilayers (frozen chains) either i n 3 0 m M C a C l or i n 3 0 m M M g C l . I n all cases where the D P P C bilayers are separated by more than 20 A of salt solution, the force to maintain a particular bilayer separation is greater in C a C l than i n M g C l . As described above, we calculate surface potential, and area per Me *, S, from the data i n Figure 2. In Figures 3 and 4, the variations i n ^ and S respectively are shown for both C a and M g data sets, respectively. More C a is bound than M g . 2
2
2
2
2
2
2
2
2
2
2
2
2+
g
2+
2 +
2+
IOOO [MeCI ]
(mM)
Figure 1. Repeat distance, d, of the lamellar phase formed by 30 wt % DPPC in various concentrations of CaCl or MgCl . T = 25°C: (%), CaCl and (A), MgCl . 2
2
2
g
44
BIOELECTROCHEMISTRY:
A A
•••
0 OA
2
(dynes/tm )
•
•
0
A A
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IONS, SURFACES, M E M B R A N E S
