Langmuir 1992,8, 1984-1987
1984
Interaction of Valinomycin and Stearic Acid in Monolayers Suram Pathirana and William C. Neely Department of Chemistry, Auburn University, Auburn, Alabama 36849
Lawrence J. Myers and Vitaly Vodyanoy' Institute for Biological Detection Systems, Department of Physiology and Pharmacology, College of Veterinary Medicine, Auburn University, Auburn, Alabama 36849 Received March 13,1992. In Final Form: May 7, 1992 Surface pressure and surface potential as functions of surface area were measured for monolayers of valinomycin-stearic acid mixtures on the water-gas interface. Specific area per molecule of valinomycin, free energy of mixing, and dipole moment were calculated as a function of concentration of valinomycin. The s u m of the partial molecular areas in the mixture of valinomycin with stearic acid is significantly less than the calculated s u m of the molecular areas of the pure components. This condensing effect is accompanied by two minima in free energy of mixing, valinomycin specific molecular area, and dipole moment, at low and high concentrations of valinomycin in mixed monolayers. Our data indicate that the miscibility of the valinomycin and the stearic acid in mixed monolayers is a function of composition. Langmuir-Blodgett deposited multilayer5 of mixed valinomycin-stearate monolayers were exposed to water solutions of KC1and NaCl in a concentration range of 0.001-100mM. The electric current generated by the monolayer system in contact with electrolyte solutions was dependent on KC1 concentration. In contrast, the system was not sensitive to Na+. The valinomycin-stearate multilayer thus has potential application as a potassium ion sensor.
Introduction Valinomycin is a cyclododecadepsipeptide ionophore antibiotic comprised of 3 mol each of L-valine, D-whydroxyisovaleric acid, D-valine, and L-lactic acid linked alternately t o compose a 36-membered ring.l This remarkable molecular ring specifically complexes K+ and carries it across biological and artificial membranes. The valinomycin molecule is a good ionophore apparently because the K ions are embraced in the central polar cavity while the nonpolar exterior of the molecule is compatible with the hydrophobic interior of membrane5.l It has been recently demonstrated by infrared studies that pure valinomycin monolayers deposited on solid substrates were not able to complex K+.2 In contrast, mixed monolayers of valinomycin and fatty acid were capable of complexing K+. This leads to the important question of what role fatty acids play in assisting valinomycin in complexing K+. A study of the interaction of fatty acids with valinomycin can not only lead t o an answer t o the above question but may be of great importance to optimizing the conditions for a construction of a better potassium ion sensor. This information might also be useful for understanding of more fundamental properties of antibiotic/ cell membrane interactions. In this work, we have studied the effects of stearate molecules on the valinomycin in monolayers on water and have examined the K+/Na+ specificity of mixed stearic acid/valinomycin monolayers transferred onto silicon substrates.
Experimental Section Valinomycin of the highest purity available was obtained from Sigma and waa used without further purification. Stearic acid from Sigma was recrystallized 5 times. Anhydrous hexane obtained from Aldrich waa used as the solvent in preparing the spreading solutions, which were prepared in an atmosphere of dry nitrogen. The pure solutions of stearicacid and valinomycin (1) Ovchinnikov,Iu. U. Membrane-actiue complexones, Elsevier: New York. 1974. (2j Howarth, V. A.; Petty, M. C.; Daviee, G. H.;Yarwood, J.Langmuir 1989,5,330-332. ~
0743-746319212408-1984$03.00/0
had concentrationsof 0.4and 0.17mg/mL, respectively. A series of mixed stearic acid/valinomycin spreading solutions, ranging from 0 to 1mole fraction of valinomycin, was spread on the subphase solution of 0.03 mM CdCl2 and 0.4mM NaHCOS (pH 8.4) made with deionized doubly distilled water (Millipore, Mill-Rowater system). Each monolayer was allowed to equilibrate and to stabilize for 5 min before data collection. Surface pressuresurface area (*-A) isotherms were measured at 15 i 0.1 OC. By use of the minimum dispersion of mean molecular area as a criterion of the optimal rate of compression: a rate of 0.5 cm2 s-l was chosen for these experiments. Control experimentswith spreadingsolvents gave no measurablechange in surface pressure after evap~ration.~ Each isotherm was replicated 3-4 times. Measurements of surface pressure were performed on a KSV 2200LB Langmuir-Blodgett film balance.6 The system contains a Wilhelmy type surface balance (0-100mN/m; sensitivity,0.05 mN/m) and a Teflon trough (45X 15 cm2). The temperature of the subphasewas controlled by circulating water through a quartz coil located on the bottom of the trough. Temperature was measured by a thermistor located just below the water surface. Surface potential measurements were done with a 2*oPo air electrode located 2 mm above the water surface, connected to an electrometer,and referenced to an Ag-AgC1 electrode immersed in the s u b p h a ~ e . ~ ~ ~ Langmuir-Blodgett films were prepared using standard procedure.' Mixed monolayers were transferredat a surface pressure of 23 mN m-1 onto silicon/silicon oxide (Si/SiOz)plates (26 X 26 mm2). The potassium/sodium selectivity of the valinomycin/ stearic acid multilayer was determined with the electrochemical cell, composed of the Si/SiOz plate, covered with valinomycin/ stearic acid monolayers as a K+sensor (Figure 1). The reference electrode, made of Si/SiO2, was immersed into a small pool (300 rL)of KC1 (or NaC1) water solutions isolated by an O-ring. The cell was assembled in a Teflon holder (not shown) and was contained in a grounded metal box on an isolation table (GS-34, (3) Ito, H.; Morton, T. H.; Vodyanoy, V. Thin Solid Films 1989,180, 1-13. (4) Amett, E. M.; Gold, J. M. J. Am. Chem. SOC.1982,104,636-839. (5) Vodyanoy, V.; Bluestone, G. L.; Longmuir, K. J. Biochim. Biophys. Acta 1990,1047, 284-289. (6) Pathirana, S.; Neely, W. C.; Myers, L. J.; Vodyanoy, V. J. Am. Chem. SOC.1992,4, 1404-1405. (7) Gaines, G . L., Jr. Insoluble Monolayers at Liquid-Gas Interfaces; Interscience: New York, 1966.
0 1992 American Chemical Society
Interaction of Valinomycin and Stearic Acid in Monolayers
Langmuir, Vol. 8, No. 8,1992 1985 AGME= f ( A s v - NsAs - NvAv) dII AGM1= RTNs In N ,
+ RTNv In Nv
(2) (3)
AGM = AGME-k RTNs In N s + RTNv In Nv
(4) where A G Mis~the excess free energy of mixing, AGMis the free energy of mixing, and AGMIis the free energy of mixing for an ideal system, Asv, As, and AVare the areas of the mixed and pure monolayers, II is the surface pressure, R is the universal gas constant, and T is the absolute temperature. The values of dipole moment ( p ) were calculated from the equation
Figure 1. Diagrammatic representation of an electrochemical
cell: 1,valinomycin/stearic acid multilayer; 2, silicon oxide layer,
3, silicon plate; 4, O-ring;5, water pool containing K+or Na+;6,
reference electrode made of oxide-passivated silicon plate; 7, voltage clamp and recording system.a
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Results Figure 2 represents the pressurearea isothermsof mixed valinomycin/stearic acid monolayerson the buffered water solution. The pure stearic acid isotherm replicates published d a h 7 When valinomycin is added to stearic acid monolayer a very distinctive shoulder appears in the pressure-area isotherm. The extrapolation of the linear portion of this shoulder to the "zero pressure" area (Figure 2) determines the mean molecular area (A,) of the mixed monolayer, and the value of A, is increased when concentration of valinomycin is increased. For pure valinomycin monolayers this extrapolated area reaches 300.8 f 1.1 (SD) A2/molecule. The value of the specific molecular area of the valinomycin derived from the pressure-area isotherms appeared to be a function of the valinomycin concentration. Figure 3A shows that this function has two minima, at 0.1 and 0.7 mol fraction of valinomycin. Positions of the two minima are reproduced in the valinomycin concentration dependence of the free energy of mixing (A&) shown in the Figure 3B. All experimental values of AGM show a significant departure from that corresponding to an ideal system. The departure from the ideality can be also illustrated with valinomycin concentration dependences of the mean molecular area and the surface dipole moment of mixed monolayers (Figure 3C,D). The straight line in the Figure 3C represents the ideal concentration dependence of molecular area calculated by the equation, Aided = N,A, + NJ,, where A, and A, are specific molecular areas determined by "zero pressure" extrapolations of pressure area isothermsfor pure valinomycin and stearic acid monolayers, respectively (A, = 20.57 f 0.11 and A, = 300.8 f 1.1 A2/molecule;*SD). The straight line in Figure 3D shows the ideal concentration dependence according to the equation, Pideal = PVNV+ p a s , where PV and PQ are maximum values of the vertical components of surface dipole moments for pure valinomycin and stearic acid monolayers, respectively (pS= 98.0 f 1.5 and PV = 2097.7 f 110.1 mD; fSD).
(i2)
Figure 2. Surface pressure-area isotherms of mixed valino-
mycin/stearic acid monolayers on buffered water solutions at the indicated concentration of valinomycin at 15' C. Each isotherm was obtained by averagingthree or four runs. Standard deviations of the surface pressure and area were determined at 11equally spaced points along the curves at each mole fraction of valinomycin. Mean standard deviations at 0.0, 0.55, 0.105, 0.150,0.191, and 0.230 mol fraction of valinomycin are as follows: 0.079,0.078,0.148,0.065,0.084,0.075mN/m;0.081,0.161,0.098, 0.096, 0.141, 0.322 AZ/molecule. Newport). Current measurements were performed at the voltage clamped at 0 mVa8 The specificmolecular area of valinomycin (Av)in valinomycin/ stearic acid mixed monolayers was estimated by the equationg A, = (A, - A>J/NV
p = AAV/12* (5) where A (Azmolecule-') is the molecular area, AVis the surface potential in millivolts, and p is in millidebye units.7
(1)
where A, and A, are extrapolated "zero-pressure" areas7for the mixed monolayer and the stearic acid monolayer, respectively, and N,and N,are mole fractions of stearic acid and valinomycin, respectively(Figure 2). Thermodynamic properties derived from the isothermal compression data for monolayers of stearic acid, valinomycin and its mixtures, were calculated by using the following equations1*13-3-5 (8) Vodyanoy, V.; Halverson, P.; Murphy, R. B. J. Colloid Interface Sci. 1982,&3,247-257. (9)Barnes, G. T.J. Colloid Interface Sci. 1991,144,299-300. (10)Vilallonga, F.;Altachul, R.; Fernandez, M. S.Biochim. Biophys. Acta 1967,135,406-415. (11) Vilallonge, F.Biochim. Biophys. Acta 1968,163,290-300. (12) Gershfeld, N.L.; Pagano, R. E. J. Phys. Chem. 1972,1231-1237.
Discussions Surfacepressure-area isotherms for mixed valinomycin/ stearic acid monolayers (Figure 2) are in good agreement with those measured by RieB and Swift." Our results for pure stearic acid and pure valinomycin monolayers are in accord with published data.7J5J6 (13)Rakshit, A. K.;Zograf, G.; Jalal, I. M.; Gunstone, F. D. J. Colloid Interface Sci. 1981,466-473. (14)Ries, H.E.,Jr.; Swift, H.S . J . Colloid Interface Sci. 1978,64, 111-119. (15)Kemp, G.; Wenner, C. E. Biochim. Biophys. Acta 1972,282,l-7.
Pathiram et al.
1986 Langmuir, Vol. 8, No. 8,1992 .
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