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Langmuir 2006, 22, 2200-2204
Peptidolipid as Binding Site of Acetylcholinesterase: Molecular Recognition of Paraoxon in Langmuir Films Chengshan Wang, Changqing Li, Xiaojun Ji, Jhony Orbulescu, Jianmin Xu, and Roger M. Leblanc* Department of Chemistry, UniVersity of Miami, Coral Gables, Florida 33146 ReceiVed October 19, 2005. In Final Form: December 12, 2005 Peptidolipid C18H35O (stearoyl)-Phe-Trp-Ser-His-Glu (peptidolipid A) was synthesized and spread at the air-water interface to study the interaction with an organophosphorus compound. Paraoxon, sodium dihydrogen phosphate, or 4-nitrophenyl phosphate disodium was added to the subphase, but only paraoxon changed the surface pressure-area (π-A) isotherm of peptidolipid A. This indicated a specific interaction between paraoxon and peptidolipid A. To clarify which amino acid residue of peptidolipid A was responsible for the interaction, peptidolipid B, namely, C18H35O-Gly-His-Ser-Glu-Glu, was synthesized and studied as a Langmuir film. The difference between the π-A isotherms of peptidolipid B in the absence and presence of paraoxon in the subphase was minimal; consequently, the presence of amino acids phenylalanine (Phe) and tryptophan (Trp) in peptidolipid A may explain the interaction between peptidolipid A and paraoxon. The compression-decompression cycles and kinetic studies of peptidolipid A showed that the Langmuir film was stable. The in situ optical properties of the peptidolipid A Langmuir film such as UV-vis and fluorescence spectroscopies were examined to elucidate the interaction between peptidolipid A and paraoxon. UV-vis absorption of peptidolipid A was investigated in the presence and absence of paraoxon in the subphase. The emission maximum of fluorescence of Trp in peptidolipid A was observed at 351 nm on pure water, and the band intensity decreased when the concentration of paraoxon increased in the subphase. This suggested that the Trp was involved in the molecular recognition process. Epifluorescence micrographs showed domains of peptidolipid A on the pure water subphase. In the presence of paraoxon in the subphase, the Langmuir film of peptidolipid A showed a homogeneity, which was another indication of the recognition between paraoxon and peptidolipid A.
Introduction Organophosphorus (OP) compounds, such as paraoxon, are environmental pollutants in water and soil because they are widely used as pesticides.1 It is necessary to detect OP compounds and dispose them before their concentration reaches a dangerous level. To realize this aim, many analytical methods have been developed to detect the presence of OP compounds, e.g., gas chromatography,2,3 high-performance liquid chromatography (HPLC),4,5 and electrical methods of detection.6 A fast, efficient, and cheap method for the detection of organophosphorus compounds is the use of enzymes such as organophosphorus hydrolase (OPH) and acetylcholinesterase (AChE) as sensors.7-13 In the case of AChE, a nucleophilic attack of the phosphorus atom * To whom correspondence should be addressed. Fax: (305) 284-6367. Phone: (305) 284-2194. E-mail:
[email protected]. (1) Munnecke, D. J. Agric. Food Chem. 1980, 28, 105-111. (2) Mendoza, C. E. Thin-layer chromatography. In Pesticide Analysis; Dumas, K. G., Ed.; Marcel Dekker: New York, 1981; pp 1-44. (3) Das, K. G.; Kulkarni, P. S. Gas-liquid chromatography. In Pesticide Analysis; Dumas, K. G., Ed.; Marcel Dekker: New York, 1981. (4) Hanks, A. R.; Colvin, B. M. High-performance liquid chromatography. In Pesticide Analysis; Dumas, K. G., Ed.; Marcel Dekker: New York, 1981; pp 99-174. (5) Barcelo, D.; Lawrence, J. F. Residue analysis of organophosphorus pesticides. In Emerging Strategies for Pesticide Analysis; Charins, T., Sherma, J., Eds.; CRC Press: Boca Raton, FL, 1992; pp 127-150. (6) Palchetti, I.; Cagnini, A.; Del Carlo, M.; Coppi, C.; Mascini, M.; Turner, A. P. F. Anal. Chim. Acta 1997, 337, 315-321. (7) Dziri, L.; Boussaad, S.; Wang, S.; Leblanc, R. M. J. Phys. Chem. B 1997, 101, 6741-6748. (8) Dziri, L.; Boussaad, S.; Tao, N.; Leblanc, R. M. Langmuir 1998, 14, 48534859. (9) Constantine, C. A.; Mello, S. V.; Dupont, A.; Cao, X.; Santos, D., Jr.; Oliveira, O. N., Jr.; Strixino, F. T.; Pereira, E. C.; Rastogi, V.; Cheng, T.-C.; DeFrank, J. J.; Leblanc, R. M. J. Am. Chem. Soc. 2003, 125, 1805-1809. (10) Mello, S. V.; Mabrouki, M.; Cao, X.; Leblanc, R. M.; Cheng, T.-C.; DeFrank, J. J. Biomacromolecules 2003, 4, 968-973. (11) Constantine, C. A.; Gattas-Asfura, K. M.; Mello, S. V.; Crespo, G.; Rastogi, V.; Cheng, T.-C.; DeFrank, J. J.; Leblanc, R. M. J. Phys. Chem. B 2003, 107, 13762-13764.
from the paraoxon at the serine site which is part of the binding site of AChE and activated by the imidazole group of a histidine explained the affinity of the detection.14,15 Because of the high activity of serine at the binding site and the microenvironment in the pocket of AChE, this reaction is highly efficient. Despite the important role of enzymes in biology and chemistry, the source of enzymes is still highly limited and the extensive applications of enzymes are subsequently limited. This is the reason biomimicry of an enzyme by small molecules has attracted scientific interest. In our previous papers,8,13 we have used an AChE Langmuir film to detect paraoxon in the subphase. Here we synthesized two peptidolipids to mimic the binding site of AChE with the objective to recognize paraoxon. It has been shown that some peptidolipids composed of the amino acid residues of the binding site of an enzyme can form specific receptors for small target molecules.16,17 The rationale of the design of our peptidolipids was based on the structure of the AChE enzyme. As shown from single-crystal X-ray data,18 the binding site of AChE contains mainly Ser, His, and Glu. There is also an aromatic gorge formed by Trp and other aromatic residues, for example, Phe, to help paraoxon to reach the binding site.18 On the basis of these data, the first peptidolipid synthesized was C18H35O (stearoyl)-Phe-Trp-Ser-His-Glu (peptidolipid A), which contains amino acids from both the binding site and the aromatic gorge. The second peptidolipid was C18H35O (stearoyl)(12) Cao, X.; Mello, S. V.; Sui, G.; Mabrouki, M.; Leblanc, R. M.; Rastogi, V. K.; Cheng, T.-C.; DeFrank, J. J. Langmuir 2002, 18, 7616-7622. (13) Dziri, L.; Desbat, B.; Leblanc, R. M. J. Am. Chem. Soc. 1999, 121, 96189625. (14) Shen, T.; Tai, K.; Henchman, R.; McCammon, J. A. Acc. Chem. Res. 2002, 35, 332-340. (15) Nalivaeva, N. N.; Turner, A. J. Proteomics 2001, 1, 735-747. (16) Ariga, K.; Kunitake, T. Acc. Chem. Res. 1998, 31, 371-378. (17) Huo, Q.; Russell, K. C.; Leblanc, R. M. Langmuir 1998, 14, 2174-2186. (18) Sussman, J. L.; Harel, M.; Frolow, F.; Oefner, C.; Goldman, A.; Toker, L.; Silman, I. Science 1991, 253, 872-879.
10.1021/la052818+ CCC: $33.50 © 2006 American Chemical Society Published on Web 02/02/2006
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Figure 1. Chemical structures of peptidolipid A (C18H35O-Phe-Trp-Ser-His-Glu), peptidolipid B (C18H35O-Gly-His-Ser-Glu-Glu), paraoxon, 4-nitrophenyl phosphate disodium, and sodium dihydrogen phosphate.
Gly-His-Ser-Glu-Glu (peptidolipid B), which only contains the binding site amino acids. The sequence of peptidolipid B was chosen such that Ser was at the same position as that in peptidolipid A, and one Glu residue was added at the end of the peptide for solubility reasons. The His residue was isolated from the tail by placing a Gly residue near the stearoyl group. Structures of both peptidolipids are shown in Figure 1. Experimental Section Materials. Wang resin and amino acids used for peptidolipid synthesis were purchased from Advanced ChemTech (Louisville, KY). Other organic chemicals and solvents were of reagent grade and were obtained from VWR Co. (Westchester, PA). All the amino acids were in the l-configuration, except for glycine. The 1H NMR data were acquired with a Bruker 500 MHz spectrometer (Boston, MA). Low-resolution fast atom bombardment (FAB) of the sample was recorded on a VG-Trio 2000 mass spectrometer. High-resolution FAB was conducted on a 70-4F instrument and performed at the Mass Spectrometry Laboratory of the University of Illinois at UrbanaChampaign. Synthesis. Peptidolipids were synthesized via solid-phase (Fmoc) chemistry.19-21 Building blocks for the synthesis include FmocPhe-OH, Fmoc-Trp-(Boc)-OH, Fmoc-Ser-(But)-OH, Fmoc-His(19) Fields, G. B., Ed. Methods in Enzymology; Academic Press Inc.: New York, 1997; Vol. 289. (20) Kates, S. A.; Albericio, F. Solid-Phase Synthesis, a Practical Guide; Marcel Dekker: New York, 2000. (21) Fields, G. B.; Noble, R. L. Int. J. Pept. Protein Res. 1990, 35, 161-214.
(Trt)-OH, Fmoc-Glu-(OBut)-OH, Fmoc-Gly-OH, and stearic acid. Diisopropylcarbodiimide (DIC) and 1-hydroxylbenzotriazole (HOBT) were used for the coupling reactions. The Fmoc groups were deprotected with 20% piperidine solution in DMF (v/v) after an average coupling period of 1 h, and the extent of acylation was monitored via the standard ninhydrin test. After lyophilization the crude product was purified by semipreparative reversed-phase highperformance liquid chromatography (RP-HPLC) on Waters 2690 separation modules. The eluants were 0.1% trifluoroacetic acid in water (v/v; A) and 0.1% trifluoroacetic acid in 1-propanol/acetonitrile (50:50, v/v; B). The column conditions were as follow: Vydac 219TP1010 (diphenyl, 5 µm, 10 mm i.d. × 250 mm). The purity of the synthesized peptidolipids was verified by analytical RP-HPLC, 1H NMR, and mass spectrometry (MS). Analytical RP-HPLC was conducted on a small-scale column (Vydac 219TP54, diphenyl, 5 µm, 4.6 mm i.d. × 150 mm). The eluants were the same as in the aforementioned semipreparative RP-HPLC, and the elution gradient was 50-100% A in 45 min; the flow rate was 2 mL‚min-1. (1) Peptidolipid A: 1H NMR (500 MHz, DMSO) δ ) 8.28 (d, 1H), 8.20 (d, 2H), 8.08 (d, 1H), 7.95 (d, 1H), 7.55 (d, 1H), 7.30 (d, 2H), 7.23 (d, 1H), 7.18 (m, 5H), 7.05 (d, 2H), 6.91 (d, 1H), 4.56 (m, 1H), 4.48 (m, 1H), 4.41 (m, 1H), 4.28 (m, 1H), 4.19 (m, 1H), 3.53 (m, 2H), 3.20 (d, 2H), 3.15 (d, 2H), 2.90 (d, 2H), 2.35 (t, 2H), 2.05 (t, 2H), 1.90 (m, 2H), 1.70 (m, 2H), 1.30-1.12 (m, 28H), 0.91 (t, 3H); FAB-MS m/z 971.56 (MH+, calcd 971.56). (2) Peptidolipid B: 1H NMR (500 MHz, DMSO) δ ) 8.28 (d, 1H), 8.20 (d, 1H), 8.08 (d, 2H), 7.95 (d, 1H), 7.30 (s, 1H), 7.05 (s, 1H), 4.56 (m, 1H), 4.48 (m, 1H), 4.41 (m, 2H), 4.28 (m, 1H), 3.53 (m, 2H), 3.20 (d, 2H), 2.35
2202 Langmuir, Vol. 22, No. 5, 2006
Figure 2. Surface pressure-area and surface potential-area isotherms of peptidolipid A at the air-water interface. (t, 4H), 2.05 (d, 2H), 1.90 (m, 4H), 1.70 (m, 2H), 1.30-1.12 (m, 28H), 0.91 (t, 3H); FAB-MS m/z 824.47 (MH+, calcd 824.47). General Methods for Surface Chemistry Study. All the isotherm measurements were conducted in a clean room (class1000) where constant conditions of temperature (20.0 ( 0.5 °C) and humidity (50 ( 1%) were maintained. The spreading solvent was chloroform/ methanol (5:1, v/v), and the solvents (HPLC grade) were purchased from VWR Co. The water utilized as the subphase (pH 5.5) for the monolayer study was obtained from a Modulab 2020 water purification system (Continental Water System Corp., San Antonio, TX) with a surface tension of 72.6 mN‚m-1 and a specific resistivity of 18 MΩ‚cm at 20.0 ( 0.5 °C. The spreading volume of the peptidolipid solutions was 20-30 µL. After spreading, a 15 min period was allowed for evaporation of the spreading solvent prior to compression. A Kibron µtrough (Kibron Inc., Helsinki, Finland) was utilized for surface pressure-area isotherm measurements. The compression rate was set at 5 Å2‚molecule-1‚min-1. Surface potential measurements were obtained on the Kibron trough using a Kelvin probe consisting of a capacitor-like system. The vibrating plate was set at approximately 1 mm above the surface of the monolayer, and a gold-plated trough was used as a counter electrode. The clean subphase was taken as the zero potential. The in situ UV-vis absorption spectra of the Langmuir monolayer were obtained with an HP spectrophotometer, model 8452 A, settled on a rail close to the KSV trough (KSV Instrument Ltd., Helsinki, Finland), suitable for approach toward the quartz window which is located at the center of the KSV trough. The fluorescence spectra of the Langmuir film were measured via an optical fiber (excitation and emission) positioned at the top of the KSV trough and connected to a Spex Fluorolog 1680 0.22 m double-beam fluorospectrometer (Horiba Jobin Yvon, Edison, NJ). The KSV minitrough (KSV Instrument Ltd.) has an area of 225 cm2 (7.5 × 30 cm). The trough is supplied with an electronic balance that uses a Wilhelmy plate as the pressure sensor with a sensitivity of 0.02 mN‚m-1. For monolayer compression, two symmetrically movable computer-controlled barriers were used. An epifluorescence microscope (Olympus IX-FLA) was used for acquiring the fluorescence micrographs of the Langmuir film at different surface pressures. A Kibron minitrough was used for the preparation of the monolayer. A thermoelectrically cooled Optronics Magnafire CCD camera detected the emission of a fluorescent probe, namely, 5-(octadecanoylamino)fluorescein (ODFL); this fluorescent probe was mixed with the peptidolipid prior to spreading, and the molar mixing ratio of ODFL to peptidolipid was 1:100. The fluorescence probe was excited at 480 nm, and the emission was recorded at 523 nm.
Results and Discussion Surface Pressure-Area and Surface Potential-Area Isotherms of Peptidolipid A. The surface pressure-area (πA) and surface potential-area (∆V-A) isotherms of peptidolipid A on a pure water subphase are shown in Figure 2. The π-A
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Figure 3. Surface pressure-area isotherms of peptidolipid A at the air-water interface when the subphase was (0) pure water, (O) aqueous NaH2PO4 (1.5 × 10-3 M), (4) aqueous 4-nitrophenyl phosphate disodium (1.5 × 10-3 M), and (3) aqueous paraoxon (1.5 × 10-3 M).
isotherm of peptidolipid A showed a condensed phase behavior with a lift-off at 94 Å2‚molecule-1 and a collapse surface pressure at 62 mN‚m-1. The limiting molecular area obtained by extrapolating the low compressibility region of the isotherm at nil surface pressure was 69.4 Å2‚molecule-1. This experimental limiting molecular area is close to the theoretical one calculated by the space-filling CPK (Corey-Pauling-Koltum) model, i.e., 68.0 Å2‚molecule-1. The ∆V-A isotherm measures the changes in the perpendicular dipole moment during compression of the Langmuir film. This measurement gives a good indication of the interaction between the polar moieties of the amphiphilic molecules at the air-water interface. When the subphase was pure water, the surface potential of peptidolipid A fluctuate around zero when the molecular area was larger than 120 Å2‚molecule-1. The fluctuation ((50 mV) was due to the formation of domains in the Langmuir film. When the monolayer was further compressed, the surface potential stabilized and continuously increased up to a surface potential of 120 mV at a molecular area of 67.2 Å2‚molecule-1, a condition under which the monolayer is in the solidlike phase. π-A Isotherms of Peptidolipid A with Small Molecules in the Subphase. When paraoxon was added to the subphase at a concentration of 1.5 × 10-3 M, the π-A isotherm still showed a condensed behavior with a lift-off at 105 Å2‚molecule-1, but the collapse surface pressure and the limiting molecular area decreased to 50 mN‚m-1 and 62.5 Å2‚molecule-1, respectively. The lower limiting molecular area in the presence of paraoxon indicates an interaction between paraoxon and peptidolipid A. To verify which moiety of paraoxon could interact with peptidolipid A, sodium dihydrogen phosphate or 4-nitrophenyl phosphate disodium was also dissolved in the subphase (see the structure of the derivatives in Figure 1). Figure 3 shows the π-A isotherms of peptidolipid A in the presence of one of the substrates in the subphase. When sodium dihydrogen phosphate or 4-nitrophenyl phosphate disodium was solubilized in the subphase at a concentration of 1.5 × 10-3 M, the π-A isotherm was the same as the π-A isotherm of peptidolipid A on the pure water subphase. Only paraoxon in the subphase causes significant change of the π-A isotherm, which indicates the specificity of the interaction between peptidolipid A and paraoxon. The question is which amino acid residue is responsible for this interaction. To answer this question, peptidolipid B was synthesized. Surface Pressure-Area Isotherm of Peptidolipid B. The π-A isotherms of peptidolipid B in the absence and presence
Peptidolipid as Binding Site of AChE
Figure 4. Surface pressure-area isotherms of peptidolipid B at the air-water interface in the absence (0) and presence (b) of paraoxon in the subphase (1.5 × 10-3 M).
Figure 5. Compression-decompression cycles of peptidolipid A up to 20 mN‚m-1 on pure water. Inset: Stability isotherm of peptidolipid A at the air-water interface.
of paraoxon are shown in Figure 4. When the subphase was pure water, the lift-off was at 104.5 Å2‚molecule-1, whereas the collapse surface pressure and the limiting molecular area were decreased to 50 mN‚m-1 and 56.1 Å2‚molecule-1, respectively. The experimental limiting molecular area, 56.1 Å2‚molecule-1, is close to the value calculated using the space-filling CPK model, i.e., 55.0 Å2‚molecule-1. When paraoxon was added to the subphase, practically no change of the π-A isotherm of peptidolipid B was observed. Both peptidolipids A and B contain Ser, His, and Glu; therefore, the change in the π-A isotherm of peptidolipid A is due to the presence of Trp and Phe residues. Compression-Decompression Cycles and Kinetic Study of Peptidolipid A. The compression-decompression cycles and long-term stability of peptidolipid A were studied up to a surface pressure of 20 mN‚m-1, and the results are shown in Figure 5. For the compression-decompression cycles, the decompression cycles overlapped the compression cycles, which indicated that the Langmuir film of peptidolipid A was stable even after six cycles (Figure 5). The molecular area of peptidolipid A did not change much (3 Å2‚molecule-1) after the surface pressure was held at 20 mN‚m-1 for 1 h (inset in Figure 5). This result indicated that peptidolipid A formed a stable Langmuir film at the airwater interface. In Situ UV-Vis Spectroscopy of Peptidolipid A at the AirWater Interface. Three aromatic amino acids of peptidolipid A have absorption maxima between 200 and 300 nm. The UV-vis absorption spectrum in aqueous solution (pH 5.5) was studied for each aromatic amino acid. Tryptophan has a higher energy
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Figure 6. In situ UV-vis spectra of peptidolipid A at the air-water interface (pH 5.5). Inset: Absorbance at 271 nm against surface pressure.
absorption maximum at 220 nm and another one at 279 nm with two shoulders at 271 and 287 nm. The molar absorptivity at λmax ) 271 and 279 nm was 5.7 × 103 and 6.7 × 103 M-1‚cm-1, respectively. Phenylalanine absorption is similar to that of benzene, with maxima at 251, 258 (258 ) 198 M-1‚cm-1), and 262 nm, whereas histidine has a maximum only at 210 nm (210 ) 5.2 × 104 M-1‚cm-1), which corresponds to peptide bonding (results not shown). Our results correspond to those given by Santos et al.22 for the aqueous solution work. Because of the low numbers of molecules per unit area in a monolayer at the airwater interface, we may expect to detect only the tryptophan residue on the basis of the relatively high extinction coefficient at around 270 nm. In situ UV-vis absorption spectra of peptidolipid A at the air-water interface were recorded at five different surface pressures as shown in Figure 6. All the aromatic amino acids absorb at 210 nm; consequently, a high absorbance was observed at this wavelength for peptidolipid A. Due to the formation of domains, increasing the surface pressure will increase the compactness of the monolayer, which results in scatter of the incident light at the air-water interface. The spectra between 300 and 500 nm showed that the baseline increases with an increase of surface pressure, which is due to the effect of scattering light. At 341 nm, there is no assignment of the band because we did not observe any linearity of A341 against surface pressure, and no band appears at this wavelength for the three aromatic amino acids in aqueous solution. However, the shoulder at 270 nm is assigned to the tryptophan residue because the band position corresponds to that of tryptophan in aqueous solution, and A270 vs surface pressure showed a linear dependency as expected (see Figure 6, inset). The presence of paraoxon in the subphase at a concentration of 1.5 × 10-3 M does not change the fingerprint of the UV-vis spectra at the surface pressures indicated in Figure 6. To observe any interaction between peptidolipid A and paraoxon, it was necessary to use tryptophan as an intrinsic fluorescence probe to detect any interaction. In Situ Fluorescence of Peptidolipid A at the Air-Water Interface. The in situ fluorescence spectra of Langmuir films were measured via an optical fiber which brings the excitation light and gathers the emission light by reflection at the airwater interface. A fluorescence spectrum was measured for peptidolipid A on the pure water subphase (pH 5.5), and an emission band was recorded at 351 nm as shown in Figure 7 (see the inset). The emission band was assigned to the tryptophan (22) Santos, N. C.; Castanho, M. Trends Appl. Spectrosc. 2002, 4, 113-125.
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Figure 7. In situ fluorescence quenching of peptidolipid A at the air-water interface in the presence of paraoxon (0, 1 × 10-5, 5 × 10-5, 1 × 10-4, and 1.5 × 10-3 M) measured at 20 mN‚m-1. Inset: Fluorescence spectrum of peptidolipid A at 20 mN‚m-1.
residue as observed at 351 nm in aqueous solution22 (results not shown). The presence of paraoxon at different concentrations quenched the fluorescence of tryptophan as shown in Figure 7. At a concentration of paraoxon of 1.5 × 10-3 M, the intensity of fluorescence completely vanished. This experimental observation supports the hypothesis of an interaction between peptidolipid A and paraoxon. Epifluorescence Micrographs of Peptidolipid A in the Absence and Presence of Paraoxon in the Subphase. Figure 8 shows the epifluorescence micrographs of peptidolipid A at different surface pressures in the absence (Figure 8A) and presence (Figure 8B) of paraoxon at 1.5 × 10-3 M in the subphase. Peptidolipid A at the air-water interface showed domain formation at all surface pressures, although at surface pressures higher than 30 mN‚m-1, the domains were compressed and showed a more uniform topography (Figure 8A). In the presence of paraoxon in the subphase, the epifluorescence micrographs showed a homogeneous film at all the surface pressures (Figure 8B). How do we explain the fact that a homogeneous film was formed in the presence of paraoxon? First, peptidolipid A has a hydrophobic tail of 17 carbon atoms (stearoyl group) and a hydrophilic moiety of 5 amino acids. The comparison between surface pressure-area isotherms of stearic acid and peptidolipid A shows that the limiting molecular area of the stearic acid is 22.5 Å2‚molecule-1 compared to 69.4 Å2‚molecule-1 for peptidolipid A. This experimental result is interpreted mainly by the interaction between polar moieties because if it were due to van der Waals interaction or hydrophobic-hydrophobic interaction, the limiting molecular area would be observed at about 22 Å2‚molecule-1 as for the stearic acid monolayer, and this is not the case. The long-range interaction between polar moieties certainly plays a role in the formation of domains observed by epifluorescence microscopy (see Figure 8A). Formation of domains will show an empty space at the air-water interface that means “dark spots” in the epifluorescence micrographs (Figure 8A). Because of the large dipole moment of paraoxon, this molecule will certainly interact with the polar moiety of peptidolipid A, then breaking the domains as shown in Figure 8B. Parts A and B of Figure 8 support the evidence of an interaction between peptidolipid A and paraoxon.
Figure 8. Epifluorescence micrographs of peptidolipid A in the absence (A) and presence (B) of paraoxon in the subphase (1.5 × 10-3 M).
Conclusion Surface pressure-area and surface potential-area isotherms of peptidolipid A were studied on a pure water subphase. Paraoxon and two other small molecules, namely, sodium dihydrogen phosphate and 4-nitrophenyl phosphate disodium, which have a moiety of the structure of paraoxon, were solubilized in the subphase, but only paraoxon causes changes in the π-A isotherm of peptidolipid A. This indicated that there was a specific interaction between peptidolipid A and paraoxon. Phe and Trp were responsible for the interaction because peptidolipid B, which has no aromatic amino acid, does not interact with paraoxon. The compression-decompression cycles and long-term stability of the peptidolipid A Langmuir film showed that the peptidolipid A monolayer was stable. UV-vis spectra of peptidolipid A in the absence and presence of paraoxon were recorded. Although there was no change in the fingerprint of the spectrum in the presence of paraoxon, this result does not mean that there was no interaction. We used the tryptophan residue as an intrinsic probe of peptidolipid A to observe an interaction between the peptidolipid and paraoxon. We observed a decrease of the intensity of fluorescence at 351 nm when the concentration of paraoxon was increased. This result supports our hypothesis on the interaction between peptidolipid A and paraoxon. Epifluorescence micrographs of peptidolipid A mixed with a fluorescein derivative (molar ratio 100:1) in the absence and presence of paraoxon also support this hypothesis. Acknowledgment. This research was supported by the National Science Foundation (Grant CHE-0091390) and the U.S. Army Research Office (Grant DAAD19-03-1-0131). LA052818+
