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Langmuir 1989, 5 , 330-332
Infrared Studies of Valinomycin-Containing Langmuir-Blodgett Films V. A. Howarth and M. C. Petty* Molecular Electronics Research Group, School of Engineering and Applied Science, University of D u r h a m , Science Laboratories, S o u t h Road, Durham, D H 1 3LE, England
G. H. Davies and J. Yarwood Department of Chemistry, University of D u r h a m , Science Laboratories, S o u t h Road, Durham, DH1 3 L E , England Received August 22, 1988. I n Final Form: November 3, 1988 Valinomycin-containingLangmuir-Blodgett films have been prepared, and the structural changes occurring in these films when exposed to potassium solutions have been analyzed by using Fourier transform attenuated total reflection infrared spectroscopy. Pure valinomycin LB films are observed not to complex with potassium; the inclusion of a secondary component such as arachidic acid is necessary for complexation to occur. Furthermore, the ATR-IR spectra indicate major structural changes in the fatty acid matrix during complexation that have been interpreted as reflecting the migration of the valinomycin molecules toward the polar acid head groups.
Introduction Valinomycin is a membrane-active cyclodepsipeptide that can selectively complex with and transport potassium across both biological and synthetic membranes.lP2 As well as being important in the study of ion-selective transport through membranes, valinomycin can also be used as the basis for potassium ion sensing LangmuirBlodgett (LB) monomolecular films closely resemble biological membranes in their structure. It has already been shown that valinomycin may be deposited either as a pure material or in a mixture with a fatty acid, using the LB t e c h n i q ~ e . ~A? study ~ of the interaction between valinomycin-containing LB films and potassium solutions is expected to lead to a greater understanding of valinomycin in this artifical environment and be of relevance to potassium-sensing applications. Fourier transform attenuated total reflection (ATR) infrared spectroscopy' was chosen as the probe for this investigation because it has the extremely high sensitivity required to study monomolecular films8 and also because the valinomycin/potassium (VM/K+) complex has already been well characterized by IR techniques.+l2 We report here for the first time the observation that the VM/K+ complex is only formed in mixed LB films of valinomycin with a secondary component, such as arachidic acid. The inability of pure valinomycin LB films, which we have prepared, to complex with potassium must be related to the unusual conformations which the cyclodepsipeptide is forced to adopt in (1) Shemyakin, M. M.; Ovchinnikov, Yu. A.; Ivanov, V. T.; Antonov, V. K.; Vinogradova, E. I.; Shkrob, A. M.; Malenkov, G. G.; Evstratov, A. V.; Laine, I. A.; Melnik, E. I.; Ryabora, I. D. J. Membr. Biol. 1969, 1, 402-430. (2) Stark, G.; Ketterer, B.; Benz, R.; Lauger, P. Biophys. J . 1971,1I, 981-994. (3) Wen, C. C.; Lauks, I.; Zemel, J. N. Thin Solid Films 1980, 70, 333-340. (4)Sibbald, A. I E E Proc. I . 1983,130, 233-244. (5) Howarth, V. A.; Petty, M. C.; Davies, G. H.; Yarwood,J. Thin Solid Films 1988, 160, 483-489. (6) Peng, J. B.; Abraham, B. M.; Dutta, P. Ketterson, J. B. Langmuir
1987,3, 104-106. (7) Mirabella, F. M.; Appl. Spectrosc. Reo. 1985,21, 95-178. (8) Davies, G. H.: Yarwood, J. Spectrochim. Acta 1987, 43A, 161 . - -9-1 - - 62.1 - -- . (9) Asher, I. M.; Rothschild, K. J.; Anastassakis, E.; Stanley, H. E. J . Am. Chem. SOC.1977,99,2024-2032. (10) Asher, I. M.; Rothschild, K. J.; Stanley, H. E. J . Mol. Biol. 1974, 89,205-222. (11) Rothschild, K. J.; Asher, I. M.; Stanley, H. E. Anastassakis, E. J . Am. Chem. SOC.1977,99,2032-2039. (12) Grell, E.; Funck, T. Eur. J . Riochem. 1973,34, 415-424.
0743-7463/89/2405-0330$01.50/0
Table I. Band Assignments for Cast Films of Uncomplexed and Complexed Valinomycin" uncomplexed VM complexed VM ester, u(C=O) 1754 (29) 1744 (23) amide I, u(C=O) 1658 (35) 1650 (22)
amide 11, b(NH,) b(CH3) MCHJ
1536 (34) 1468 1448 sh 1390 1372
1537 (26) 1469 1454 sh 1393 1372
amide 111, u(CN) 1246 1248 ester, u(C-0-C) 1183 (29) 1191 (19) a Full widths at half-maxima (fwhm) are indicated in parentheses. such films. Furthermore, the ATR-IR spectra indicate major structural changes in the fatty acid matrix during complexation which we believe reflect changes in the molecular position of valinomycin in the LB film. The implied connection with the classical concept of carriermediated ion transport across biological membranes emphasizes the importance of structural studies of valinomycin-containing LB films.
Experimental Section Infrared spectra were obtained on a Mattson Sirius 100 Fourier transform spectrometer equipped with a liquid nitrogen cooled cadmium mercury telluride detector; all data were collected at a spectral resolution of 4 cm-'. A 1 x 20 X 10 mm silicon crystal was used to obtain the ATR-IR spectra. The crystal was prepared with a hydrophobic surface by refluxing in a 2% solution of dimethyldichlorosilane in l,l,l-trichloroethane. A cast film of valinomycin was obtained by depositing valinomycin in an Aristm chloroform solution onto the ATR silicon crystal surface and allowing the chloroform to evaporate. Similarly, a cast film of complexed valinomycin was prepared from a valinomycin-chloroform solution with a little potassium chloride added. Langmuir-Blodgett films were prepared by using standard equipment described e1~ewhere.l~Pure valinomycin and valinomycin/arachidic acid mixtures were spread from a chloroform solution onto either pure water (pH 5.8) or 0.05 M KC1 subphases (pH 5.8); the monolayers were subsequently transferred to the solid substrates at a surface pressure of 23 mN m-l. Before these films could be successfullydeposited it was necessary to first coat the clean, hydrophobic ATR crystal with four layers of cadmium stearate prepared on a 2 X M CdClz subphase at a pH of approximately 7.0 and a surface pressure of 30 mN m-l. (13) Roberts, G. G. Sens. Actuators 1983,4, 131-145.
0 1989 American Chemical Society
IR Study of Valinomycin LB films
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Figure 1. IR spectra of cast films of uncomplexed valinomycin (dashed line) and complexed valinomycin (solid line). For band assignments see Table I.
Results and Discussion The IR spectra of cast films of uncomplexed and complexed valinomycin are shown in Figure 1 (with band assignments shown in Table I). The important changes on complexation with potassium are summarized as follows. The ester carbonyl band at 1754 cm-' is simultaneously narrowed and shifted to give a more symmetric band at 1744 cm-l. The narrowing suggests that, in the complex, all six ester carbonyls experience a more similar chemical environment; possibly due to ion-dipole interactions with the K+ ion. In the uncomplexed valinomycin the presence of a "hydrogen-bonded" ester u(C=O) band is indicated by the shoulder a t 1739 cm-' in addition to "free" ester u(C=O) band at 1754 cm-l. A similar shift and narrowing transforms the amide I band (approximately v(C=O)) at 1658 cm-' into a narrower, more symmetric band at 1650 cm-'. In the complex, all six amide groups are involved in intramolecular hydrogen bonding. Again a more uniform environment for the amide groups is implied. The amide I1 band (approximately b(NH,)) at -1536 cm-' is also narrowed; the v(C0C) stretching band is both narrowed and shifted on complexation to high frequency. These characteristic changes are easily used to identify the valinomycin complex in the ATR-IR spectra of LB films of valinomycin and mixtures of valinomycin with arachidic acid. In the ATR spectra of up to 100 LB layers of pure valinomycin dipped on a 0.05 M KC1 subphase, no evidence for the VM/K+ complex was observed. Furthermore, LB films of pure valinomycin dipped on a pure water subphase and subsequently immersed in a saturated KCI solution exhibited no signs of complexation. The reason for these surprising results is a t present not fully understood but is probably related to the conformation of the valinomycin molecule in these films, which in turn depends on its local environment. In pure films of valinomycin the molecular environment is uniformly "valinomycin-like". It is possible that an intermolecular interaction between the polar carbonyl groups inhibits the formation of the complex by preventing coordination with the K+ ion. In mixed LB films of valinomycin and arachidic acid, the VM/K+ complex has been observed, and the evidence is presented in Figure 2. This is a difference spectrum between an 11.75:l (M) arachidic acid/valinomycin mixture dipped on a 0.05 M KC1 subphase and the same mixture dipped on a pure water subphase. Comparison of the band positions and half-widths for the spectrum in Figure 2 with those of complexed valinomycin in Table I
Langmuir, Vol. 5, No. 2, 1989 331 shows a close agreement, indicating that Figure 2 is, in fact, the spectrum of complexed valinomycin. In the full IR spectrum of the mixed LB film (not shown), the presence of both uncomplexed and complexed forms of valinomycin is inferred from the ester v(C=O) bands a t 1754 and 1744 cm-', respectively. From this we conclude that only a fraction of the valinomycin is complexed: approximately one-third for the 11.75:l mixture. It is worth noting the similarity between the arachidic acid/valinomycin system and the biological membrane. It is very encouraging that complexation occurs in our model system, but more fundamental is the observation that the presence of arachidic acid is necessary for complexation to take place in LB film structures. This is possibly related to the hydrophobic environment provided by the arachidic acid. The effect on immersing mixed arachidic acidlvalinomycin films dipped on a pure water subphase in KC1 solutions was also investigated. With the ATR technique it was possible to monitor the changes occurring in the LB film as a function of time. In Figure 3 the effects of a total of 10 min of immersion in a saturated KC1 solution on 14 layers of an 11.751 arachidic/valinomycin mixture are shown. These spectra were recorded in air after the corresponding immersion period in KCl had elapsed. A thousand scans were recorded for each spectrum, lasting a total of 10 min. After 10 s (spectrum not shown), the valinomycin is still largely in the uncomplexed state. After 1min, however, (Figure 3a), a significant amount of complexation has taken place, indicated by the ester v(C=O) and amide I bands at 1744 and 1653 cm-', respectively. Uncomplexed valinomycin is evident from the shoulder a t 1754 cm-' on the carbonyl band. A far more profound change in the spectrum after 1 min is the decrease in intensity of the cyclic acid dimer band at 1705 cm-'; at 10 min (Figure 3c) the intensity has reduced substantially. To interpret this change correctly, it is necessary to look a t related changes in the arachidic acid CH2 stretching bands shown in Figure 4. This is a difference spectrum showing the changes occurring in the structure after a 5-min immersion period in KC1 solution. Two positive features a t 2924 and 2856 cm-' appear, corresponding to the antisymmetric and symmetric CH2 stretching modes of the arachidic acid (there are no CH2 groups present in valinomycin). Since ATR is mainly sensitive to dipole changes parallel to the substrate, we conclude that the increase in v(CH2) band intensities (both of which have their transition dipoles parallel to the surface14 in an ordered film) is a result of some of the acid chains becoming more "ordered". The positive changes observed in this region also indicate that material is not disappearing from the LB film (unless there are major changes in extinction coefficients). It is therefore not possible to account for changes in the 1705-cm-' region in terms of loss of material or, indeed, in terms of conversion to salt, since no bands a t t r i b ~ t a b l e 'to ~ RCOO-K+ (expected a t 1555 cm-') are found. Instead, we propose that the changes in this band are due to dimers breaking apart as the hydrocarbon chains rearrange, thus leading to the formation of monomers or sideways dimers. Two pieces of evidence from Figure 3 suggest this. Firstly, a broadening of the ester carbonyl band is observed, and secondly, a relative increase in intensity with respect to the amide I band (cf. relative intensities in a cast film of complexed valinomycin) is noted. This implies the existence of another band in the region (14) Rabolt, J. F.; Burns, F. C.; Schlotter, N. E.; Swalen, J. D. J . Electron Spectrosc. Relat. Phenom. 1983, 30, 29-34. (15)Bellamy, L. J. Infrared Spectroscopy of Complex Molecules; Chapman and Hall: London, 1975.
332 Langmuir, Vol. 5, No. 2, 1989 I
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Howarth et al.
1600
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1
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,
,
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1200
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Figure 2. Difference spectrum between an 11.75:l (M) arachidic acid/vdinomycin mixture dipped on a 0.05 M KC1 subphase and the same mixture dipped on a pure water subphase. The spectrum shows complexed valinomycin.
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3000 2950 2900 2050 Wavonumber
Figure 4. Difference spectrum showing the changes occurring in the CH2 stretching region for 14 layers of an 11.75:l (M) arachidic acid/valinomycin mixture after a 5-min immersion period in saturated KCl.
tures in pure water suggest that the valinomycin molecules remain in the complexed state. This may be related to the hydrophobic surface of our LB assembly-in the biological membrane, the potassium is released at an acid head groupsolution interface, but for the conventional “Y-type” deposition the alkyl chains of the final layer will be outermost.
-0.010
1760 1680 1600 K2O 1440 Wawnumbar
Figure 3. Effects of immersion in saturated KC1 solution on 14 layers of an 11.75:l (M) arachidic acid/valinomycin mixture: (a) after 1 min; (b) after 5 min; (c) after 10 min.
1740-1750 cm-l, possibly associated with acid sideways dimers. The structural changes detected in the arachidic acid matrix probably reflect changes in the molecular position of valinomycin molecules within the matrix. Recent NMR work16 has shown conclusively that, at least for sonicated phospholipid vesicle-based systems, the valinomycin molecule occupies an average location in a relatively hydrophobic region (in our case, amongst the hydrocarbon chains) and that it has a tendency to move to a more polar region (in our case nearer to the acid head groups) on complexation. This is thought16J7to be due to competition for hydrogen bonds on the valinomycin and the presence of a charged (K+) center. Our results are entirely consistent with this picture in that both alkyl chain and acid head group vibrational “markers” indicate a movement from hydrophobic to (more) hydrophilic regions of the surfactant structure. The results of experiments concerned with immersing complexed LB films of arachidic acid/valinomycin mix(16) Meers, P.; Feigenson, G. W. Biochem. Biophys. Acta 1988, 938, 469-482. (17)Dum, W. L.; Hauptman, H.; Weeks, C. M.; Norton, D. A. Science 1972, 176, 911-914.
Conclusions In conclusion, we have prepared valinomycin-containing Langmuir-Blodgett films and analyzed the structural changes occurring in these films when they are exposed to KC1 solutions by using Fourier transform infrared ATR spectroscopy. A detailed examination has revealed that, in order to complex with K+, valinomycin must be mixed with arachidic acid. Significant complexation occurred after 1 min, but more interesting and profound structural changes in the arachidic acid matrix have been inferred from the IR spectra. A marked decrease in intensity of the acid dimer v(C=O) band and enhancement of v(CH,) bands suggest structural changes of the arachidic acid hydrocarbon chains and acid dimer head groups, possibly reflecting the migration of valinomycin molecules toward the polar acid head groups on complexation. The connection with K+ transport is obvious and demonstrates the close resemblance of the mixed LB film system with the biological membrane. However, an important distinction is that dissociation of the VM/K+ complex does not appear to occur in the LB films reported in this work. We have demonstrated that the novel combination of the LB technique to fabricate thin (less than 20 nm) K+-sensitive films and ATR spectroscopy to probe these layers reveals valuable information concerning complexation and the associated structural changes in the films. The LB technique is an elegant method of engineering the molecular environment, and it is hoped with further work that this technique can elucidate the molecular interactions responsible for the behavior of pure valinomycin LB films. The importance of these studies is clearly their relevance to analogous processes in the biological environment and the use of such films in sensor applications. Acknowledgment. We thank the Science and Engineering Research Council for financial support. Registry No. VM, 2001-95-8; K, 7440-09-7; arachidic acid, 506-30-9.
