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Langmuir 1994,10, 1354-1357
Stearic Acid Assisted Complexation of K+ by Valinomycin in Monolayers Vitaly Vodyanoy* Institute for Biological Detection Systems, Department of Physiology and Pharmacology, College of Veterinary Medicine, Auburn University, Auburn, Alabama 36849
Suram Pathirana and William C. Neely Department of Chemistry, Auburn University, Auburn, Alabama 36849 Received August 31, 1993. I n Final Form: March 7, 1994” Stearic acid/valinomycin mixed monolayers were transferred to solid substrates and were subjected to optical and electrochemicalinvestigations to determine the role of stearic acid in assisting valinomycin to complex potassium ions. Direct-view dark-field optical observations of multilayer/water interfaces revealed relatively uniform surfaces for mixed films with large and small concentrations of valinomycin (10 and 70 mol %), while films containing 40 mol 74 valinomycin showed nonuniform clustered surfaces. Fourier transform attenuated total reflection infrared spectra showedthe presence of complexed valinomycin molecules after samples were exposed to KC1 solutions. Langmuir-Blodgett multilayer systems of valinomycin/stearate mixtures were exposed to water solutions of KC1 and NaCl in a concentration range of 0.001-100 mM. The electriccurrent generated by these multilayer systems in contact with the electrolyte solutions was dependent on the K+ concentration. In contrast, these systems were not sensitive to Na+.
Introduction Valinomycin, a cyclododecadepsipeptideantibiotic, was initially isolated from the prokaryotic Streptomyces fulvissimus and is composed of a-amino and a-hydroxyl acids linked alternately in a 36-membered ring.l This molecule specificallycomplexes K+ and transports it across biological membranes. The nature of the valinomycin environment determines the conformation of the molecule and its ability to bind It has been recently demonstrated that valinomycin molecules were not able to complex K+ ions in pure valinomycin monolayers a t the aidwater interfacell or in monolayers deposited on solid substrates.6J2 In contrast, mixed monolayers of valinomycin and fatty acid (or phospholipid) were capable of complexing K+ i0ns.”7~9-~3 In our previous report13we have demonstrated that the sum of the partial molecular areas in the mixed valinomycin/stearic acid monolayers is significantly less than the calculated sum of the molecular areas of the pure components. This condensing effect was accompanied by two minima in the free energy of mixing, valinomycin *Abstract published in Advance ACS Abstracts, May 1, 1994. (1)Ovchinnikov, Yu. A. Membrane-active complexones, Elsevier: New York, 1974. (2)Lev, A. A. Modelling of ionic selectivity of cell membranes; Science: Leningrad, 1976. (3) Asher, I. M.; Rothschild, K. J.;Anastassakis, E.; Stanley, E. J. Am. Chem. SOC. 1977,99, 2024-2031. (4)Rothschild, K. J.; Asher, I. M.; Stanley, E.; Anastassakis, E. J.Am. Chem. SOC. 1977,99, 2032-2039. ( 5 ) Caspers, J.; Landuyt-Caufriez, M.; Ferreira, J.; Goormaghtigh, E.; Ruysschaert, J. M. J . Colloid Interface Sci. 1981, 81, 41C-418. (6)Howarth, V. A.; Petty, M. C.; Davies, G. H.; Yarwood, J. Langmuir 1989,5, 330-332. (7)Howarth, V. A.; Petty, M. C.; Ancelin, H.; Yarwood, J. Vib. Spectrosc. 1990, 1 , 29-23. (8)Ries, H. E.,Jr. Langmuir 1990, 6, 883-885. (9)Gabrielli, G.; Puggelli, M.; Gilardoni, A. B o g . Colloid Polym. Sci. 1992,89, 227-232. (10)Gilardoni, A.; Margheri, E.; Gabrielli, G. Colloids Surf. 1992,68, 235-242. (11)Sagawara, M.; Sazawa, H.; Umezawa, Y. Langmuir 1992,8,609-
fil2.
(12)Yang, X. L.; Dutta, P.; Wong, G. K.; Ketterson, J. B. Thin Solid Films 1992, 219, 210-214. (13)Pathirana, S.;Neely, W. C.; Myers, L. J.; Vodyanoy, V. Langmuir 1992,8, 1984-1987.
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specific molecular area, and dipole moment, at low and high concentrations of valinomycin in mixed monolayers. Our data have indicated that the miscibility of the valinomycin and the stearic acid in mixed monolayers was a function of composition and that the system has lower miscibility in the range between 10 and 70 mol 7% valinomycin. The recent analysisll of surface area isotherms of mixed valinomycin/synthetic lipid monolayers at the air/water interface has found conditions when these two components are not miscible. The authors suggested that immiscibility was expressed by the coexistence of individual valinomycin and lipid domains (patches) and that valinomycin inside the valinomycin patches may be left uncomplexed with K+ions due to the absence of lipids needed for the complexation. In this work we have used high-resolution dark-field optical microscopy to directly observe surfaces of mixed stearic acid/valinomycin Langmuir-Blodgett films interfaced with water. We have found that surfaces of films made of pure components and mixed monolayers with large and small concentrations of valinomycin (10 and 70 mol 74 ) appear homogeneous while films containing 40 mol % valinomycin show distinctive patches. With electrochemicalmethods we have demonstrated that valinomycin/stearic acid systems are capable of specific complexation of K+. The ability of stearic acid to assist complexation of K+ by valinomycin in monolayers is higher for mixtures with high (70 mol % ) and low (10 mol %) concentrations of valinomycin. This information may be useful for understanding some fundamental properties of antibiotidcell membrane interactions.
Experimental Section Materials used and methods of sample preparation were completely described in the preceding paper.13 LangmuirBlodgett films were prepared using standard procedures.14J3 Mixed valinomycin/stearic acid monolayers were transferred at a surface pressure of 23 mN m-l onto 25 X 75 X 1 mm microscope slides for dark-field optical microscopy, or 50 X 10 X 3 mm, 45’ ~
(14)Gaines, G. L., Jr. Insoluble Monolayers at Liquid-Gas Interfaces; Interscience: New York, 1966.
0 1994 American Chemical Society
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Figure 1. Optical micrographs of monolayers deposited on the microscope slides by the Langmuir-Blodgett method (objective 100X/1.4, oil, dark-field illumination, mercury arc light): (A) 11monolayers of stearic acid, (B) 18 monolayers of valinomycin, (C-E) 18 mixed stearic acid/valinomycin monolayers containing 9,40, and 70 mol % valinomycin, respectively, (F) Fragment of a carbon grating replica calibrated to 0.46 pm/line. face angle, germanium parallelogram attenuated total reflection (ATR) plates (Wilmad), or silicon/silicon oxide plates (26 X 26 X .5 mm) for electrochemical measurements.13 The potassium/ sodium selectivity of the valinomycin/stearic acid multilayer was determined with the electrochemicalcell previously described.13 Current measurements were performed with the voltage clamped at 0 mV.l3 In this work optical images of multilayers were obtained with an Olympus BHS microscope fitted with a 100-W mercury lamp illumination source, a polarizer, a Naessens dark-field condenser
(COSE Corp., Canada), and a lOOX objective (oil, NA 1.4). The images were directed to a CCD color camera (WV-CL322, Panasonic), displayed on a video monitor (PVM-1343 MD, Sony Corp.), and hard copied with a video printer (UP 3000, Sony Corp.). After a valinomycin/stearate multilayer was deposited on a microscope slide a droplet of distilled water (5 pL) was delivered to the film surface and covered with a cover slide. The sample was then positioned onto a microscope stage for observation and photography. Small fragments of carbon grating replica (no. 10020, Ernest F. Fullam, Inc.) with 2160 parallel lines/"
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1356 Langmuir, Vol. 10, No. 5, 1994 were covered with distilled water and photographed for magnification calibrations in the same way as multilayers. Infrared spectra were recorded on a Fourier transform spectrophotometer (Sirrus, Mattson Instruments) equipped with a liquid nitrogen cooled cadmium mercury telluride detector. A 2F141-1Wilmad multipleinternal reflectionattachmentwas used for ATR measurements. The spectra were measured at a resolution of 4 cm-l. ATR plates were subjected to a cleaning in a PDS-3XG Harrick Plasma Cleaner before monolayer depositions. IR spectra were measured before and after valinomycin/stearicacid films were exposed to a 0.1 M KCl water solution for 5 min. Control samples were exposed to distilled water and to a 0.1 M NaCl water solution.
Results Figure 1 shows dark-field optical micrographs of valinomycin/stearate Langmuir-Blodgett films interfacing distilled water. Parts A and B represent pure stearate and pure valinomycin films, respectively, while parts C-E show, respectively, 9, 40, and 70 mol % mixtures of valinomycin and stearic acid. A small fragment of carbon grating replica with 2160 parallel lines/" is shown in Figure 1F. The surface of a pure stearate film (Figure 1A) reveals a dark fine granulated two-dimensional structure, while the surface of a pure valinomycin multilayer (Figure 1B) appears much brighter with a range of small and large white granules. Mixed valinomycin/stearate films of 9 and 70 mol % valinomycin show relatively uniform distributions of white granules (Figure lC,E). Films with 9 mol % valinomycin show low-density domains of relatively small granules, as compared to the greater density of granules in samples with 70 mol % valinomycin. In contrast, mixed films containing 40 mol % valinomycin show a nonuniform distribution of white granules assembled in clusters (Figure 1D). At higher magnification, a representative structure of these white clusters is seen in Figure 2A and the replica carbon grating in Figure 2B. The conformation of valinomycin determines its ability to bind and to carry ions.14 We have found that the electric current generated by the mixed stearic acid/valinomycin system containing 70 mol % valinomycin in contact with electrolyte solutions appeared to be determined by [K+] when the concentration of KC1 was increased from lo4 to 10-l M. In contrast, the sample was not sensitive to [Na+] in the same concentration range (Figure 4). Similar experiments were carried out for mixed multilayers containing 9 and 40 mol % valinomycin. From these data, values of the formation constants (kf)for the K+/ valinomycin complex were estimated. The values of Kf for the 10% and 70% mixtures were about 5 X 105 M-l while the Kffor the 40% mixture was 4 times less than this value. Figure 3 shows the effect of immersing mixed valinomycin/stearic acid films containing 9 mol % valinomycin in a 0.1 M KC1 solution for 5 min. The difference IR spectrum (Figure 3) shows complexation of valinomycin as verified by positions of optical bands specific for complexed valin~mycin.~,~,~ These characteristic bands were not observed in control films exposed to a distilled water or a NaCl solution. Band assignments for uncomplexed and complexed valinomycin obtained in this work, together with those found in the literature, are given in Table 1.
respectively.)
Discussion A dark-field ultramicroscopictechnique was introduced first for observationsof small colloidalparticles15and later was adapted for monolayers on the liquid-gas interface.l6
The technique was subsequently modified1' and used for microscopic examinations of monolayers on aqueous17J8 and solid substrate^.^^^^^ In the present work we have applied dark-field microscopy of high resolution to observe
(15) Seidentopf,H.; Zsigmondy, R. Ann. Phys. (N.Y.) 1903,10,1-39. (16) Zocher, H.; Stiebel, F. 2. Phys. Chem. 1930,147,401-417.
(17) Adam, N. K. Trans. Faraday SOC.1993,29,90-106. (18) Bruun, H. H. Ark. Kemi 1955,8,411-422.
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Figure 3. Difference spectrum between 10 mixed stearic acid/ valinomycin monolayers containing 9 mol % valinomycin before the sample was immersed in a 0.1 M KC1 solution for 5 min. 120 0
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Figure 4. Electric current response (relative to doubly distilled deionized water) of a mixed stearic acid/valinomycin film made of 12 monolayers containing 70 mol % valinomycin to K+ and Na+ solutions of different concentrations. Ion solutions were applied sequentially. When the ion current was stabilized after the solution application, the current was recorded and the system was washed with distilled water and then the next solution was applied. The experiment was replicated three times. The data points represent the mean values of the stabilized current, while the curve is a second-order polynomial interpolation. Table 1. Band Assignments for Complexed and Uncomplexed Valinomycin ununcomplexeda complexeda complexedb complexedb primary vibration (cm-l) (cm-1) (cm-9 (cm-I) 1754 1744 1738 ester C=O stretch 1755 1650 1651 1658 amide C=O stretch 1665 1536 1537 1543 amide NH2 bending 1539 1191 1196 1181 1186 ester COC stretch a Data obtained in this work from the difference spectrum between 10 mixed stearic acid/valinomycin monolayers containing 9 mol 5% valinomycin after and before the sample was immersed in a 0.1 M KCl solution for 5 min. * Data published by Howarth et aE.* for cast mixed stearic acid/valinomycin films.
surfaces of mixed stearic acidlvalinomycinmultilayers and to examine our suggestion13about the dependence of the physical state of the mixed monolayer on the valinomycin concentration. In the range between 10 and 70 mol 5% valinomycin,valinomycin molecules in mixed monolayers may be assembled in microscopic domains.llJ3 In view of the fact that pure valinomycin monolayers were observed (19)Gaines, G.L.,Jr.; Ward, W. J., 111. J. ColloidZnterface Sci. 1977, 60,210-213. (20)Asher, S. A.; Pershan, P. Biophys. J. 1979,27, 393-422.
not to complex with potassium ion,6it appears that, in the interior of these valinomycin domains, complexation is not complete. Thus, the monolayer composition and the physical state of the valinomycin and stearic acid in mixed monolayers13 appear to be important conditions for the ability of the valinomycin molecules to complex K+ ions. By the nature of the dark-field illumination, particles or aggregates in monolayers scatter light and appear as more illuminated than a monolayer, which appears dark.18 The surfaces of pure stearate multilayers interfacing water (Figure 1A) appear to form a very dense two-dimensional system of points and spots. This image agrees very well with those obtained by the dark-field optical ultramicroscopy for monolayers of behenic acid on a water surface18 and by differential interference contrast microscopy for transferred multilayers of stearic acid,21 as well as with photographs of stearic acid monolayers, transferred onto supporting films of carbon, obtained with dark-field electron microscopy.22 The pure valinomycin multilayer (Figure 1B) appears much brighter than a pure stearate multilayer. White granules of different sizes cover the entire surface of the valinomycin sample. When valinomycin and stearic acid are mixed in monolayers, the appearance of the surface depends very strongly on the concentration of valinomycin. Films containing 9 and 70 mol % valinomycin show relatively uniform surfaces (Figure lC,E). In contrast, multilayers containing 40 mol % valinomycin show a nonuniform distribution of white granules assembled in large clusters (Figure 1D). These results are in accord with our thermodynamic analysis of mixed stearic acidlvalinomycin monolayers which show that the system has lower miscibility in the range of 10-70 mol % valinomycin.ls According to our electrochemical data, the ability of stearic acid to assist complexation of K+ by valinomycin is larger for mixtures with 10 and 70 mol % valinomycin. The specificity of complexation of valinomycin with K+ was demonstrated by the dependence of the electric current generated by valinomycinlstearic acid films's on the K+ concentration, but not on the Na+ concentration (Figure 4). The specific complexation of valinomycin with K+ in mixed multilayers was further confirmed by infrared spectroscopic data. FTIR-ATR data (Figure 3, Table 1) obtained for mixed stearic acidlvalinomycin multilayers are in good agreement with published data.'^^*^ IR spectra of the mixed multilayer containing 9 mol % valinomycin reveal the presence of complexed valinomycin molecules after the sample was exposed to a KC1 solution. The complexation was not observed in control films exposed to distilled water or to a NaCl solution. The method of dark-field ultramicroscopy applied in this work for observations of multilayerlwater interfaces presents certain features which make it very useful in examining surfaces in biological work. It provides real time direct-viewoptical images of high resolution. Samples need no freezing, dehydration, staining, shadowing,marking, or any other manipulations, so that they can be investigated in the natural water environment. Acknowledgments. We t h a n k Dr. Gaston Naessens for expert technical assistance. This work was supported by U S . Army Grant DAAL03-90-GO009 and Federal Aviation Administration Grant 93-G-058(V.V.) and Office of Naval Research Grant N00014-90-5-1515 (W.C.N.). (21)Braun, H.G.; Fuchs, H.; Schrepp, W. Thin Solid Films 1988,159, 4. . -m- -- ~- -i . (22)Matsumoto, M.; Uyeda, N.; Fujiyoshi, Y.; Aoyama, K. Thin Solid Films 1993,223, 358-367.
