Keywords: Fenvalerate, gramicidin, liposome, circular dichroism, pyranine, proton flux.

Transcription

1 Vol. 44, No. 6, May 1998 Pages SPECTROSCOPIC STUDIES OF THE INTERACTIONS OF THE PYRETHROID INSECTICIDE FENVALERATE WITH GRAMICIDIN J.K Ghosh, S. N. Sarkar and S.K. Sikdar" Molecular Biophysics Unit, Indian Institute of Science,Bangalore-5612, India Received November 19, 1997 Received after revision, January 21, 1998 SUMMARY: Fenvalerate is a pyrethroid insecticide which interacts with ionic channels. Using circular dichroism technique we have studied the interaction of fenvalerate with gral~cidin, a model channel peptide which transports ions. In most organic solvents, gramicidin exists as a double helix except in trifluoroethanot where it exists as a channel forming single stranded [36.3 helical monomer. In model lipid membranes, under certain experimental conditions, gramicidin exists as a channel forming single stranded [36.3 helical dimer. Our results show that fenvalerate interacts more with the single stranded 1363 helical monomer or dimer than with the double helical form of gramicidin. This was further confirmed by an increase in the rate of gramicidin mediated proton transport in liposomes by fenvalerate, using the ph sensitive fluorophore, pyranine. Keywords: Fenvalerate, gramicidin, liposome, circular dichroism, pyranine, proton flux. INTRODUCTION: Pyrethroids are a group of hydrophobic esters with structures based on the natural pyrethrins found in the flowers of Chrysanthemum sp. and are commercially used as insecticides. An ester linkage between an aromatic alcohol and an acid containing a gem- din~thyl group is associated with its high insecticidal activity (1). Like other pyrethroid insecticides, fenvalerate affects ionic transport by acting on membrane ionic channels (2,3). Fenvalerate also affects membrane fluidity (4). This investigation was aimed at studying the interaction of fenvalerate with gramicidin, a model channel helical peptide which transports ions when incorporated in membranes (5). The circular dichroism technique was used for this purpose. The effects Abbreviations: DMPC, di-myristoyl phosphatidyl choline; TFE, trifluoroethanol; FEN, fenvalerate; GRA, gramicidin-d; CD, circular dichroism; NMR, nuclear magnetic resonance; HPLC, high performance liquid chromatography. To whom correspondence should he addressed, sks@mbu.iisc.ernet.in; FAX: /98/ / Copyright by Academic Press Australia. 183 All rights of reproduction in any form reserved.

2 of fenvalerate on ion transport mediated by gramicidin incorporated in liposomes was studied using the fluorescence technique. The studies show that fenvalerate interacts more with the channel form of gramicidin helix compared to its interactions with other forms. MATERIALS AND METHODS: Gramicidin-D, Di-myristoyl phosphatidyl choline (DMPC) and Trifluoroethanol (TFE) were from Sigma (USA); 1-propanol and tetrahydrofuran were from E-Merck (India) and technical grade fenvalerate (FEN) was from Rallis (India). Gramicidin-D contains a mixture of three derivatives, with gramicidin-a consitituting 85% and is referred to as the channel forming peptide gramicidin-a. Gramieidin-D (GRA) was used as such without purification. The conformational states of gramicidin in lipid environments depends upon the method of sample preparation. Amongst different organic solvents, it is possible to directly incorporate gramicidin in channel state by co-dissolving gramicidin and lipid in TFE and DMSO (6). However, the channel conformation of gramicidin can also be induced after co-dissolving it with lipid in less polar solvents by prolonged heating or sonication (7). The channel conformation of gramicidin was prepared using both the methods. In the first method, GRA and DMPC were co-dissolved in chloroform in a round bottom flask. Chloroform was evaporated by a rot-evaporator to form a thin film on the wall of the flask. Traces of chloroform were removed by passing a stream &nitrogen gas, and keeping the flask in vacuum. The film was then hydrated by required volume of double distilled water to achieve lipid concentration of 4 mg/ml and lipid:peptide molar ratio of 5:1, followed by heating for 25 min. at about 55~ in a water bath. The sample was then sonicated by a Branson Sonifier with a microtip probe for about 1 rain, In the second method, TFE was used as the co-dissolving solvent. The liposomes were prepared by heating the hydrated dried film at 4~ for 15 rain in water bath followed by sonication. In order to form the gramicidin double helix in liposome the method of Bano et al., was followed using tetrahydrofuran as the eo-solubilising solvent (8). For ion permeation experiments, liposomes were prepared by co-dissolving GRA and DMPC (2 nag) in TFE The dried film was hydrated for sufficient time with 2 ml of aqueous soln. containing 5 mm sodium phosphate buffer (ph 7.5),.1 M NaC1 and.1 mm pyranine (9). Liposomes were prepared by sonicating the suspension for 15 rain. until the suspension became translucent. The liposome was then loaded on a sephadex G-5 column and eluted with the same buffer to remove external pyranine, The eluted vesicles were diluted to a final phospholipid concentration of 4. mm and gramicidin concentration of 2 nm. Gramieidin cohen, was determined by measuring the absorbance at 28 nm using an extinction co-efficient of 2,84 M "1 cm "l. The quality of liposome was routinely checked by observing the fluorescence quenclfing caused by the addition of HC1 on liposome not containing GRA The change in fluorescence quenching in a good liposome preparation was far less compared to liposomes containing GRA. To monitor proton flux through liposomal membrane, a proton gradient was created by adding 6 ml of 1N HC1 to 3 ml reaction mixture containing liposome (1). This lowered the ph of the external medium from 7.5 to 6.8. The proton transport was monitored by fluorescence quenching of entrapped pyranine (excitation wavelength-465 nm, emission wavelength-51 nm). 184

3 All the CD spectra were recorded using a JASCO spectropotarimeter (model J- 5A), connected to a JASCO data processor (model 5iN) with a cuvette of 1 mm path length. For spectrophotometrie experiments, a JASCO uv/vis spectrophotometer model 785 was used. The fluorescence experiments were done with a SLM 8 C spectrofluorimeter (excitation and emission slits 8 nm each). RESULTS & DISCUSSION: Circular Dichroism studies: Gramicidin is a pentadecapeptide having alternating D and L amino acid residues (2). Molecular weight determination and different spectroscopic evidence showed that in most of the organic solvents, gramieidin exists as a mixture of dimeric forms in equilibrium with monomers (11,12). Extensive spectroscopic studies particularly multidimensional solution state NMR have shown that these dimerie forms are double helices (13). These helices are called 13-helices since they show the same extensive hydrogen bonding network similar to that found in dimeric 13-pleated sheets. The interaction of fenvalerate with gramicidin was studied by circular dichroism in different organic solvents. Fig. 1 shows the effect of fenvalerate on the CD spectrum of gramicidin in 1-propanol. Fig. 1A is a typical CD spectrum of gramieidin in 1-propanol having one negative peak at 228 nm with a shoulder at approximately 215 nm (11). Increasing concentrations of fenvalerate decreased both the negative peak and the shoulder (Fig. 1, B to G). Similar results were also obtained using dioxan (Figure not shown) as a solvent. fenvalerate interacts with gramieidin in its double helix conformation. The result suggests that The double helix conformation of gramicidin however, is not its channel conformation. NMR studies (14) and chemical modifications of the N-terminus (15) favours the N to N helical dimer model, resulting from the association of two helical monomers, one from each of the bilayer leaflets as the active channel form (16). study of interaction of fenvalerate with gramicidin is of interest under such solvent conditions where gramicidin exists as the helical dimer. The CD spectrum of gramicidin in TFE is different from that obtained in other organic solvents. Thin layer chromatography reveals only a single species and osmometry measurements suggest that gramicidin is a monomer in this solvent (11,17). The NMR and other spectroscopic data indicate that gramieidin in this solvent exists as monomer helix which is typical of the channel forming helix of gramicidin (18). Fig. 2 depicts the interaction of fenvalerate with gramicidin in TFE. Fig. 2A is the CD spectrum of 185

4 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL I i LO LO ci M -5.,.,.,.-4-1 J F -15 C I 1 _ ao Wave Length (nm) Fig. 1. CD spectrum of 55. BM gramicidin in l-propanol at different FEN:GRA molar ratios; A (.), B (.43), C (.86), D (1.72), E (3.5), F (4.36) and G (6.). gramicidin alone in TFE, with one sharp positive peak at 225 nm and a broad peak around 27 nm Increasing amounts of fenvalerate did not alter the broad peak, but the intensity of the sharp peak was progressively decreased with a slight red shift. The inset in Fig. 2 shows the plot of change in molar elliptieity (A) against the fenvalerate:gramicidin ratio. A plateau is reached near a ratio of 1:1, suggesting the stoichiometry of the interaction. The results suggest there may be a strong interaction of fenvalerate with the channel form of the gramicidin helix. The same plot in propanol or dioxan gives a much higher ratio (Figure not shown) indicating lesser interactions in these solvents. In order to investigate how fenvalerate interacts with the actual channel form i.e., N- N [36.3 helical dimer, the same interaction with gramicidin incorporated into liposome was studied. The conformation of gramicidin in phospholipid vesicles is dependent on the prior history of preparation, and depends on the co-solubilising solvent, incubation time, temperature during hydration and also the extent of sonication (8). Bano et al., using CD 186

5 QD r X 1 A 8 V B 6o ~ 4 I /\~ II II D E F G H I T T GO O O O r r O C~ ~J I._3_ ,5 2. FEN : GRA o 2 N -2 L I.~ I J Wave Length (nm) Fig. 2. CD spectrum of 28 I.tM gramicidin in TFE at different FEN:GRA molar ratios; A (.), B (.2), C (.61), D (.81), E (1.2), F (1.22), G (I.42) and H (1.83), The inset shows the plot of change in molar ellipticity of gramicidin CD spectrum at 225 nm vs. FEN:GRA molar ratio. and HPLC, showed that any CD spectrum of gramicidin incorporated in the phospholipid bilayer, can be deconvoluted as a linear combination of the reference subspectra of double stranded dimer and the helical monomer, regardless of sample preparation (19). Fig. 3 shows the change in CD spectrum of gramicidin in DMPC liposome in the presence of increasing fenvalerate concentrations. Gramicidin exhibits two positive peaks, one at 217 nm, and the other at 235 nm The spectrum in Fig. 3A is characteristic of a single stranded fight handed channel conformation of gramicidin (2,21). With addition of fenvalerate, the molar elliptieity of the gramieidin CD spectrum at 217 nm decreased appreciably. This suggests there is a strong interaction of fenvalerate with the channel 187

6 25 A ' ' ' ' 2 o 15 d X i ~ 5,,---i o --5 L I I I I I --I Wave Length (nm) Fig. 3. CD spectra of 13. I.tM gramieidin incorporated into DMPC liposome (molar ratio oflipid/peptide = 5.) at different FEN:GRA molar ratios: A (.), B (.73), C (1,46), D (22), E (2.9), F (4.3) and G (5.8). form of gramicidin. The same interaction in liposome was studied, under conditions where gramicidin exists as a double helix of dimeric species, with the spectrum exhibiting one prominent negative peak at 229 nm (8). The addition of fenvalerate at a FEN:GRA ratio of 5., failed to produce any change in the spectral characteristic (Figure not shown, but data are presented in Fig. 4A), although at an almost similar FEN:GRA ratio, a profound change in the CD spectrum typical of single stranded fight handed conformation was observed. This suggests, that fenvalerate may interact preferentially with the single stranded channel forming helix than with the double helix. In an attempt to understand whieh conformation of gramicidin fenvalerate favours, the percentage change in the molar ellipticity of gramicidin at the characteristic wavelength (the wavelengths at which molar ellipticity of gramicidin is affected by fenvalerate) is plotted, against different FEN:GRA ratios in different environments. Fig. 4 shows four such plots. Figures 4A and 4B axe plots of percentage change in molar 188

7 I - - I I I I I I Bo ~ D N 6 ~ c ~ 4 o x: B 2O J 1 1 J i Fenvalerate : Gramicidin Fig. 4, Plot of percentage change in molar ellipticity of gramicidin CD spectrum vs. FEN:GRA molar ratio at different characteristic wavelengths and environments; (A)- at 229 nm in DMPC tiposome with gramicidin in its double helix conformation (see Materials and Method and text), (B)- at 228 nm in 1-propanol (data from Fig.l), (C) - at 225 nm in TFE (data from Fig. 2), (D) -at 217 nm in DMPC liposome with gramicidin in its single stranded channel conformation (data from Fig. 3). ellipticity against FEN:GRA molar ratio in liposome and 1-propanol respectively, where gramicidin exists as a double helix. Figures 4C and 4D are similar plots in TFE and liposome, where gramicidin exists as a channel forming monomer and dimer respectively, While the percentage change in molar ellipticity of gramicidin maximizes at FEN:GRA molar ratios of 2 and 4 in Figs. 4C and 4D, this does not seem to be the case even at high FEN:GRA molar ratios of 5 and 6 in Figs. 4A and 4B respectively. Further experiments with higher FEN:GRA molar ratios were not attempted since such high ratios make any studies of receptor-ligand interactions irrelevant. The simplest way to detect a change in affinity of fenvalerate for different forms of gramicidin is to took for changes in FEN:GRA molar ratios that provide a half maximal effect. The half maximal ratios for Figs. 4C and 4D are approximately 1 and 2 respectively. Although determination of half maximal ratios from Figs. 4A and B were not 189

8 possible, by extrapolating the curves, the half maximal ratios appear to be greater than 3. These results suggest that fenvalerate has more affinity for gramicidin when it exists in single stranded helix form as a monomer or a dimer (Figs, 4C and 4D respectively) than the double helix form. M1 the interactions mentioned above have also been studied by spectrophotometric techniques. In all cases, the absorption spectra of gramicidin plus fenvalerate is almost the algebraic sum of gramicidin and fenvalerate spectra alone. Since the absorption spectrum is concerned with the electronic energy level of a molecule, the additive nature of the absorption spectrum ofgramicidin plus fenvalerate suggests that the binding of fenvalerate to gramicidin does not alter the electronic energy levels. But, this binding may be associated with the change in orientation of the chromophores within the gramicidin molecule i.e., conformational change of gramicidin following interaction with fenvalerate. Thus the CD spectrum of gramicidin, being sensitive to its conformation, changes with the addition of fenvalerate. Functional studies: Pyranine is a suitable ph sensitive fluorophore (9) which has been used to monitor the proton flux mediated by gramicidin (1). The effect of fenvalerate on the kinetics of gramicidin mediated proton transport was examined by monitoring the fluorescence quenching of pyranine entrapped in liposomes following the external addition of HCt (Fig. 5), Fig. 5A shows a typical biphasie decrease in the control, consisting of an initial fast phase of proton influx mediated by gramicidin and a slow gramicidin insensitive phase attributed to a charge compensating counter-ion redistribution (1). Fig. 5B shows the change in fluorescence quenching of another aliquot of pyranine entrapped liposomes incubated with fenvalerate. There is an increase in the rate of fluorescence quenching of the initial fast phase, which suggests that the kinetics of the gramicidin mediated proton influx is faster in the presence of fenvalerate. Extending the observations to gramicidin channels, it is likely that fenvalerate interacts with gramicidin to stabilize the functional open configuration of the gramicidin channel. Although the structural features of the gramicidin channel cannot be compared with the more complex membrane ion channel proteins, it is likely that the action of fenvalerate on gramicidin may be similar to those reported for voltage gated Na channels for instance where fenvalerate has been shown to stabilize the Na channel in the open state (22). i9

9 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL p-- L HCI ~z 4 -- Ld I.-- z UJ U Z -- W k) W...J 2oo I 3 sec I Fig. 5. Effect of fenvalerate on the gramicidin mediated proton transport in unilamellar DMPC vesicles. Plot of change in fluorescence intensity of pryranine entrapped in vesicles containing 2 nm gramicidin alone (trace A) and vesicles containing the same amount of gramicidin incubated with 1.5 p,m fenvalerate (trace B), following a rapid decrease in external ph. Unilameltar vesicles were prepared as described in the Materials and Method section. Downward arrow indicates the addition of 6 p,1 1 N HC1 to 3 ml reaction mixture containing vesicles in the phosphate buffer, which lowered the ph of the external medium from 7.5 to 6,8. From the circular dichroism studies mentioned above, it may be concluded that fenvalerate interacts with the channel form of gramicidin and probably disturbs its conformation. This conclusion is supported by functional studies which indicated an increase in the rate of gramicidin mediated proton transport by fenvalerate. Acknowledgements: J.K.G. was supported by a Post Doctoral fellowship from DBT, India, and S.N.S. was supported by a Senior Research fellowship from CSIR~ India. We thankfully acknowledge the help of Dr. N.V. Joshi (NIMHANS, Bangalore) and his research students fur the use of their spectrofluorimeter. References: 1. Elliott, M. and James, N.Y. (1978) Chefn. Soc. Rev. 7, Beeman, R.W. Recent Advances in Mode of Action of Insecticide. (1982). Ann. Rev.Entomol. 27, Gammon, D. and Caside, J.E. (t983) Neurosci. Lett., 4,

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