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J. Phys. Chem. B 2008, 112, 5250-5256
Infrared Reflection-Absorption Spectroscopy and Polarization-Modulated Infrared Reflection-Absorption Spectroscopy Studies of the Organophosphorus Acid Anhydrolase Langmuir Monolayer Chengshan Wang,† Jiayin Zheng,† Liang Zhao,† Vipin K. Rastogi,‡ Saumil S. Shah,‡ Joseph J. DeFrank,‡ and Roger M. Leblanc*,† Department of Chemistry, UniVersity of Miami, Coral Gables, Florida 33146, and U.S. Army Edgewood Chemical and Biological Center, Aberdeen ProVing Ground, Maryland 21010 ReceiVed: October 1, 2007; In Final Form: January 8, 2008
The secondary structure of the organophosphorus acid anhydrolase (OPAA) Langmuir monolayer in the absence and presence of diisopropylfluorophosphate (DFP) in the subphase was studied by infrared reflectionabsorption spectroscopy (IRRAS) and polarization-modulated IRRAS (PM-IRRAS). The results of both the IRRAS and the PM-IRRAS indicated that the R-helix and the β-sheet conformations in OPAA were parallel to the air-water interface at a surface pressure of 0 mN‚m-1 in the absence of DFP in the subphase. When the surface pressure increased, the R-helix and the β-sheet conformations became tilted. When DFP was added to the subphase at a concentration of 1.1 × 10-5 M, the R-helix conformation of OPAA was still parallel to the air-water interface, whereas the β-sheet conformation was perpendicular at 0 mN‚m-1. The orientations of both the R-helix and the β-sheet conformations did not change with the increase of surface pressure. The shape of OPAA molecules is supposed to be elliptic, and the long axis of OPAA was parallel to the air-water interface in the absence of DFP in the subphase, whereas the long axis became perpendicular in the presence of DFP. This result explains the decrease of the limiting molecular area of the OPAA Langmuir monolayer when DFP was dissolved in the subphase.
1. Introduction Organophosphorus (OP) compounds are widely used in pesticides, insecticides, and chemical warfare nerve agents.1-3 The development of methods to detect these compounds is not only a requirement to protect the environment but also necessary to warn of sudden OP compound leaks or chemical warfare attacks. Consequently, several methods have been developed to detect OP compounds, for example, gas chromatography,4,5 high-performance liquid chromatography,6,7 and capillary electrophoresis.8 A fast, efficient, and specific method to detect OP compounds is to use enzymes in UV-vis or fluorescence sensors.9-14 Organophosphorus acid anhydrolase (OPAA; E.C.3.1.8.2), with a molecular weight of 58 500 Da, is one of the enzymes which can be used in the fabrication of sensors to detect OP compounds, because OPAA has a high ability to cleave the P-F bond in OP compounds, for example, diisopropylfluorophosphate (DFP), which has a similar structure to sarin (Scheme 1).15,16 Langmuir monolayer and Langmuir-Blodgett (LB) film techniques are widely used methods to build up ultrathin film devices and sensors.10,17-19 The surface chemistry properties of OPAA have been examined in detail to optimize the conditions to make OP sensors by the LB film technique.15,16 The limiting molecular area of the surface pressure-area isotherm of the OPAA Langmuir monolayer in the absence of DFP was 3430 Å2‚molecule-1, whereas the limiting molecular area decreased to 2800 Å2‚molecule-1 when the concentration * Corresponding author. Fax: +1-305-284-6367. Tel.: +1-305-2842194. E-mail:
[email protected]. † University of Miami. ‡ U.S. Army Edgewood Chemical and Biological Center.
SCHEME 1: Hydrolysis Reaction of DFP Catalyzed by OPAA
of DFP was 1.1 × 10-5 M in the subphase (shown in Figure 1a).15 As shown in Scheme 1, OPAA hydrolyzes DFP, and consequently, DFP in the subphase can change either the percentage of the secondary structure (R-helix, β-sheet, turns, and unordered structures) or the orientation of the secondary structure of the OPAA Langmuir monolayer, one of which or both might be responsible for the 20% decrease of the limiting molecular area. The circular dichroism (CD) technique is widely used to analyze the percentage of secondary structures of enzymes and has consequently been utilized to analyze the OPAA aqueous solution in the absence and presence of DFP (shown in Figure 1b) to explain the decrease of the limiting molecular area. However, the difference between the two spectra in Figure 1b was minor and the same conclusion was drawn when the two spectra were analyzed by the software of CDPro, which showed that the percentage of the R-helix conformation decreased from 38.9% to 35.5% and the β-sheet increased from 14.9% to 16.2% when DFP was added to the subphase at a concentration of 1.1 × 10-5 M.15 The CD data do not clearly show that the conformation of the OPAA is involved in the interaction between OPAA and DFP in aqueous solutions and, consequently, could not explain the large decrease of the limiting molecular area when DFP interacts with the OPAA Langmuir monolayer. The second hypothesis could be the change of the
10.1021/jp709591e CCC: $40.75 © 2008 American Chemical Society Published on Web 03/29/2008
IRRAS and PM-IRRAS of the OPAA Langmuir Monolayer
Figure 1. (a) Surface pressure-area isotherm of the OPAA Langmuir monolayer and (b) CD spectra of the OPAA aqueous solution (0.08 mg‚mL-1) using 0.1 cm optical path length. The labels are for the absence (s) and presence (O) of DFP (1.1 × 10-5 M) either in the subphase (a) or in the aqueous solution (b). (From ref 15.)
orientation of the secondary structures of the OPAA Langmuir monolayer to explain the limiting molecular area result, and this is why we decided to use IR spectroscopy. Infrared reflection-absorption spectroscopy (IRRAS)20-25 and polarization-modulated IRRAS (PM-IRRAS)26-30 have been widely used to analyze the structure of Langmuir monolayers. The IRRAS technique can detect not only the orientation change of functional groups, such as alkyl chains20-22 and nitro groups,25 but also the interaction between lipids in the Langmuir monolayer and the protein and peptides in the subphase.23,24 Because of the strong water vapor bands in the range of 1800-1400 cm-1, the IRRAS spectrum of proteins has to be measured under careful control of water vapor. Because the PM-IRRAS technique can technically waive the water vapor bands without any careful control of water vapor,26-30 PM-IRRAS has been widely used to analyze the percentage and the orientation change of the secondary structure of an enzyme’s Langmuir monolayer caused by the addition of substrate molecules in the subphase. Consequently, in this paper, both IRRAS and PM-IRRAS techniques were utilized to confirm the hypothesis of the orientation change of the secondary structure of the OPAA Langmuir monolayer in the presence of DFP in the subphase. 2. Experimental Section 2.1. Materials. Purified OPAA (E.C.3.1.8.2) was obtained from the U.S. Army Laboratory (Edgewood Chemical and Biological Center, APG, MD) with a purity of 85-90%. OPAA aqueous stock solution (2 mg‚mL-1) was kept in the refrigerator at 4 °C. Bis-tris-propane (Bis-Tris), NaOH, MnCl2, KCl, KH2PO4, dithiothreitol, and DFP were purchased from VWR Co. (Westchester, PA). BTP buffer (pH ) 6.8) was prepared with 10 mM Bis-Tris, 0.05 mM MnCl2, and 0.1 mM dithiothreitol. The OPAA concentration was diluted to 0.5 mg‚mL-1 by BTP buffer, then used for Langmuir monolayer preparation. The water used as subphase was purified by a Modulab 2020
J. Phys. Chem. B, Vol. 112, No. 16, 2008 5251 water purification system (Continental Water Systems Corp., San Antonio, TX). The pure water has a specific resistance of 18 MΩ‚cm and a surface tension of 72.6 mN‚m-1 at 20 ( 1 °C. The subphase for all the experiments was phosphate buffer (pH ) 7.2) which was prepared with 0.1 M KH2PO4 and 0.1 M NaOH. Potassium chloride was added into the subphase solution as an electrolyte, and the concentration was 0.5 M. 2.2. General Methods for Surface Chemistry Study. The surface chemistry experiments were done in a clean room (class 1000), with a constant temperature of 20.0 ( 0.5 °C and a relative humidity of 50% ( 1%. A Kibron µ-trough (Kibron, Inc., Helsinki, Finland) with an area of 124.5 cm2 (5.9 cm × 21.1 cm) was utilized for Langmuir monolayer preparation. A total of 40 µL of OPAA aqueous solution (0.5 mg‚mL-1) was spread dropwise on the surface of the subphase. After spreading the enzyme, 15 min was allowed for film equilibration. Molecular interaction between the OPAA and DFP was studied at the air-water interface by dissolving the DFP in the subphase at a concentration of 1.1 × 10-5 M and then spreading the enzyme at the air-water interface. 2.3. IRRAS and PM-IRRAS Measurements. IRRAS measurements at the air-water interface in the presence and absence of DFP were performed by the EQUINOX 55 Fourier transform infrared (FTIR) spectrometer (Bruker Optics, Billerica, MA) equipped with an XA-511 external reflection accessory suitable for the air-water interface experiments. The IR beam was conducted out of the spectrometer and focused onto the airwater interface of the Kibron µ-trough. The reflected IR beam went to a HgCdTe (MCT) detector cooled by liquid nitrogen. The spectra were acquired with a resolution of 8 cm-1 by coaddition of 1200 scans. PM-IRRAS spectra were measured by the same system as IRRAS, and 400 scans were collected for each spectrum with a resolution of 8 cm-1. The incident IR beam was modulated by a photoelastic modulator (PEM, Hinds Instruments, Inc., Hillsboro, OR) between parallel (p) and perpendicular (s) polarization to the plane of incidence. The optimal value of the angle of incidence for the detection was 70° relative to the optical axis normal to the interface. The twochannel processing of the detected IR signal gives the PMIRRAS signal S ) ∆R/R ) (Rp - Rs)/(Rp + Rs) where Rp and Rs were the polarized reflectance. The normalized PM-IRRAS spectrum is displayed by ∆S ) (S - Sb)/Sb, where Sb is the signal of the bare surface of the subphase and S is the signal of the surface with the sample Langmuir monolayer.25-29 The FTIR spectra of the OPAA aqueous solution (1 mg‚mL-1) were measured by the EQUINOX 55 spectrometer with a bioATRcell II unit accessory on the A729/Q baseplate with a resolution of 4 cm-1 and coaddition of 300 scans. 3. Results and Discussion 3.1. FTIR Spectra of the OPAA Aqueous Solutions in the Absence and Presence of DFP. Before the measurement of IRRAS and PM-IRRAS of the OPAA Langmuir monolayer, the FTIR spectra of OPAA in aqueous solutions were measured first as a control, and the results are shown in Figure 2. In the absence of DFP, two dominant peaks were observed in the spectrum, namely, 1650 and 1547 cm-1, which were assigned totheR-helixconformationbasedonexperimentalobservations31-33 and theoretical calculations.34-37 This result correlated with the data of the CD spectroscopy study,15 which showed that the R-helix was the dominant secondary structure in OPAA. Two minor peaks at 1450 and 1400 cm-1 were assigned to the methyl and isopropyl residue groups of the amino acids in OPAA.38,39
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Figure 2. FTIR spectra of OPAA (1 mg‚mL-1) in aqueous solution in the absence and presence of DFP at a concentration of 1.1 × 10-5 M.
There were two shoulder peaks in the amide I band, namely, 1685 and 1640 cm-1, which were assigned to the β-sheet conformation and the unordered structures, respectively. The peak of the β-sheet at 1630 cm-1 was not observed because it was covered by the peaks of the R-helix due to the low percentage of β-sheet in OPAA secondary structure. After DFP was added to the solution at a concentration of 1.1 × 10-5 M, the position of the peaks of OPAA did not change. This result correlated with the CD data15 which showed that the percentage change of secondary structures was about 3%, a change which is difficult to detect by FTIR. The peak intensities at 1450 and 1400 cm-1 increased. This was due to the isopropyl group of DFP. On the whole, little difference was detected between the spectra in the absence and presence of DFP in the aqueous solutions. 3.2. IRRAS Spectra of the OPAA Langmuir Monolayer at a Surface Pressure of 15 mN‚m-1 with Different Incident Angles. Before the discussion of the IRRAS results, it is necessary to present the different selectivity rules of the IRRAS of s and p polarization. For the IRRAS of s polarization, only the vibration parallel to the air-water interface can be detected. The bands of the IRRAS are always negative, and the intensity of the bands decreases with the increase of the incidence angle. As for the IRRAS of p polarization, the bands are more sensitive to the orientation of the vibration and consequently have been used to calculate the tilt angle of alkyl chains.20-22 If the vibration is parallel to the air-water interface, the band is initially negative and the band intensities increase with the increasing incidence angle until the Brewster angle is reached. Above the Brewster angle the band becomes positive, and the intensity decreases with the further increase of the incident angle. For the vibrations perpendicular to the air-water interface, the bands are positive first and then become negative beyond the Brewster angle. If the vibration is tilted to the air-water interface, the intensity of the band is weak and even zero. The IRRAS spectra of p polarization of the OPAA Langmuir monolayer are shown in Figure 3a. When the incident angle was below the Brewster angle (54.5° for the 2920 cm-1 IR light),21 all of the peaks were negative. The peaks became positive when the incident angle was above the Brewster angle, which indicated that the vibrations were parallel to the airwater interface. The peaks at 1685, 1672, 1650, and 1630 cm-1 were assigned to the amide I band, whereas the peaks at 1547 and 1530 cm-1 were assigned to the amide II band. The peaks at 1650 and 1547 cm-1 were assigned to the R-helix conformation,16 whereas the peak at 1672 cm-1 was assigned to turns conformation. The peaks at 1685, 1630, and 1530 cm-1 were assigned to the β-sheet conformation. The peaks of β-sheet were much stronger compared with the ones in the aqueous solutions.
Figure 3. IRRAS spectra of the OPAA Langmuir monolayer at a surface pressure of 15 mN‚m-1 with different incident angles: (a) p polarization; (b) s polarization.
This result was due to the following reasons: First, the carbonyl groups in the β-sheet were parallel to the air-water interface, although the percentage of β-sheet was low in the OPAA secondary structures. Second, the carbonyl groups in the R-helix were not as parallel to the air-water interface as the ones in the β-sheet, because the R-helix had a high percentage in the OPAA secondary structure and it is difficult for all of the R-helices to be strictly parallel to the air-water interface. As for the IRRAS spectra of s polarization which are shown in Figure 3b, the strongest peak was at 1650 cm-1 which was assigned to the R-helix, and the peak at 1672 cm-1 was assigned to the turns conformation. For the amide II band, the peaks at 1560 and 1547 cm-1 were assigned to the R-helix and the peak at 1530 cm-1 was assigned to the β-sheet. The shoulder peak at 1640 cm-1 was assigned to the unordered structures. Although the parallel orientation of the carbonyl groups in the β-sheet has been proved by the IRRAS of p polarization, the peak at 1630 cm-1 was weak because of the low percentage of β-sheet conformation in OPAA. For the peak at 1672 cm-1, the peak intensity decreased with the increase of the incident angle, which was possibly due to a characteristic of the IRRAS of s polarization on a dielectric surface. 3.3. IRRAS Spectra of the OPAA Langmuir Monolayer at Various Surface Pressures. The incident angle at 60° for the IRRAS of p polarization was selected because of the good signal-to-noise ratio under this incident angle. For the same reason, the incident angle at 35° was selected for the IRRAS of s polarization. The p-polarized spectra of OPAA Langmuir monolayer on phosphate buffer (pH 7.2) are shown in Figure 4a. The peaks’ positions changed little with the increase of surface pressure, whereas the peaks’ intensities increased with the surface pressure which was due to the increased number of amide groups per unit area. The relative intensity between the amide I and amide II bands at 20 mN‚m-1 was smaller than the one at 10 mN‚m-1, which indicated that the carbonyl groups
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Figure 5. PM-IRRAS spectra of the OPAA Langmuir monolayer with an incident angle at 70°.
Figure 4. IRRAS spectra of the OPAA Langmuir monolayer at different surface pressures: (a) IRRAS of p polarization with an incident angle at 60°; (b) IRRAS of s polarization with an incident angle at 35°.
in OPAA became more tilted at the air-water interface. The results of the s-polarized spectra are shown in Figure 4b. The peaks’ positions under different surface pressures were the same as the ones under 15 mN‚m-1. Although the orientation of the carbonyl groups became more tilted when the surface pressure increased, the relative intensity of the peaks did not change in the s-polarized spectra. This was due to the fact that IRRAS of s polarization is not sensitive enough to detect the orientation change of the vibration modes. On the basis of the results shown in Figures 3 and 4, it can be concluded that OPAA was not denatured at the air-water interface because of the appearance of the major peak at 1650 cm-1 which was assigned to the R-helix conformation. Although the minor difference between the spectra of the OPAA Langmuir monolayer at 0, 5, 10, and 15 mN‚m-1 was detected (Figure 4a), a strong evidence which can clearly prove the change of the orientation of secondary structures of the OPAA Langmuir monolayer was still absent. Although the IRRAS technique has been used to calculate the tilted angle of alkyl chains at the air-water interface successfully,20-22 it was difficult for the IRRAS technique to calculate the tilted angle of amide groups because the refractive index of water fluctuates seriously for the light with wavenumbers between 1400 and 1800 cm-1.40 On the other hand, the PM-IRRAS technique can technically waive the water vapor effect, not by the careful control of water humidity during the experiment. Consequently, the PM-IRRAS technique has been widely used to detect the orientation change of the secondary structures of proteins.26,27,30 Consequently, PMIRRAS was utilized to analyze the orientation change of the secondary structure in the following. 3.4. PM-IRRAS Spectra of OPAA Langmuir Monolayer in the Absence of DFP. The PM-IRRAS spectra are shown in Figure 5. The peaks at 1653, 1560, and 1540 cm-1 were assigned to the R-helix conformation. The peaks at 1695 and 1525 and shoulders at 1625 cm-1 were assigned to the β-sheet conforma-
tion. The peak positions of the R-helix and the β-sheet were different to the ones in the IRRAS spectra of the OPAA Langmuir monolayer under the same experimental conditions, because the peak positions in PM-IRRAS were also related to the orientation of the secondary structure.41 When the surface pressure was 0 mN‚m-1, the amide II band of OPAA disappeared, which was similar to the results reported by Cornut et al.41 This result is a clear evidence which showed that the carbonyl groups in both the R-helix and the β-sheet conformations were parallel to the air-water interface. The amide II band was detected at 5 mN‚m-1, and the intensity of the amide II band increased with the increase of surface pressure. This clearly showed that the R-helix and the β-sheet conformations became tilted when the surface pressure increased, which correlated with the conclusion from p-polarized data shown in Figure 4a. On the basis of the results of IRRAS and PM-IRRAS shown in Figures 3-5, it can be concluded that, at 0 mN‚m-1, the carbonyl groups in the β-sheet conformation were parallel to the air-water interface, whereas the R-helix was mostly parallel to the air-water interface, because the percentage of the R-helix conformation was high in OPAA and it is difficult for all the carbonyl groups in the R-helix to be strictly parallel to the airwater interface. Consequently, the peak of the β-sheet at 1630 cm-1 was as strong as the peak of the R-helix at 1650 cm-1 in p-polarized spectra (Figures 3a and 4a) and in the PM-IRRAS spectra (Figure 5), the amide II band disappeared under 0 mN‚m-1. With the compression of the OPAA Langmuir monolayer, the OPAA molecules were more packed, and consequently, the orientation of the carbonyl groups became tilted. This explains why the relative intensity of the amide I band and amide II bands decreased in p-polarized spectra (Figure 4a), whereas in PM-IRRAS spectra, the amide II bands appeared at 5 mN‚m-1 and became stronger and stronger with the increase of surface pressure (Figure 5). The orientation of the secondary structure of the OPAA Langmuir monolayer in the absence of DFP has been clarified. In order to make clear the reason why the limiting molecular area of the OPAA Langmuir monolayer decreased by 20% in the presence of DFP in the subphase, IRRAS and PM-IRRAS were further used to analyze the orientation of secondary structures of the OPAA Langmuir monolayer in the presence of DFP. 3.5. IRRAS Spectra of the OPAA Langmuir Monolayer in the Presence of DFP in the Subphase (1.1 × 10-5 M) at a Surface Pressure of 15 mN‚m-1. The IRRAS of the p-polarization results are shown in Figure 6a. All of the bands were negative at first and then became positive after the incident angle was above the Brewster angle. The peak of the R-helix at 1650 cm-1 and the peaks of the β-sheet at 1685, 1630, and
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Figure 6. IRRAS spectra of the OPAA Langmuir monolayer at a surface pressure of 15 mN‚m-1 in the presence of DFP in the subphase (1.1 × 10-5 M) with different incident angles: (a) p polarization; (b) s polarization.
1530 cm-1 were observed as shown in Figure 6a. A new peak at 1610 cm-1 was detected, which was assigned to the intermolecular β-sheet in the OPAA Langmuir monolayer.42 This indicated that OPAA aggregated in the Langmuir monolayer. The s-polarized results are shown in Figure 6b; the peaks at 1650, 1560, and 1547 cm-1 were assigned to the R-helix, whereas the shoulder peaks at 1685, 1630, and 1530 cm-1 were assigned to the β-sheet conformation. The peaks of the turns conformation at 1672 cm-1 disappeared in Figure 6b. According to the previous CD results of OPAA, the percentage of the turns conformation increased a little when DFP was added to the subphase at a concentration of 1.1 × 10-5 M.15 Consequently, the decrease of the peaks of the turns conformation in the s-polarized spectra was due to the tilted orientation of the turns conformation at the air-water interface. The peak at 1640 cm-1 was assigned to unordered structures. The peak at 1610 cm-1 was not observed in the s-polarized spectra, which indicated that the percentage of the intermolecular β-sheets was low, although the orientation of the carbonyl groups in the intermolecular β-sheets was parallel to the air-water interface. 3.6. IRRAS Spectra of the OPAA Langmuir Monolayer in the Presence of DFP in the Subphase (1.1 × 10-5 M) at Various Surface Pressures. The incident angle at 60° was selected for the IRRAS of p polarization and 35° was selected for s polarization. The p-polarized spectra of the OPAA Langmuir monolayer are shown in Figure 7a, and the s-polarized results are shown in Figure 7b. In both parts a and b of Figure 7, the intensity of all the peaks increased with the surface pressure and the relative intensity of the peaks changed little. This indicated that the orientation of the secondary structure of the OPAA Langmuir monolayer did not change during the compression of the Langmuir monolayer and the number of the enzyme molecules per unit area increased with the increase of surface pressure. In order to confirm this conclusion, PM-IRRAS spectra of the OPAA Langmuir monolayer were measured in the presence of DFP, and the results are in the following.
Wang et al.
Figure 7. IRRAS spectra of the OPAA Langmuir monolayer in the presence of DFP in the subphase (1.1 × 10-5 M) at different surface pressures: (a) IRRAS of p polarization with an incident angle at 60°; (b) IRRAS of s polarization with an incident angle at 35°.
Figure 8. PM-IRRAS spectra of the OPAA Langmuir monolayer in the presence of DFP in the subphase (1.1 × 10-5 M) with an incident angle at 70°.
3.7. PM-IRRAS Spectra of the OPAA Langmuir Monolayer in the Presence of DFP in the Subphase (1.1 × 10-5 M). The PM-IRRAS spectra are shown in Figure 8. The amide II band disappeared, and only two peaks at 1653 and 1635 cm-1 were shown in the spectrum at a surface pressure of 0 mN‚m-1 in the PM-IRRAS (Figure 8). The peak at 1635 cm-1 was possibly a combination peak of the unordered structures and the β-sheet. The peak at 1635 cm-1 was stronger than the one of the R-helix at 1653 cm-1, which was possibly due to the following two reasons: (i) the percentage of the β-sheet increased in the presence of DFP and (ii) the carbonyl groups in β-sheet became more parallel to the air-water interface. The amide II band did not appear when the surface pressure increased, which indicated that the carbonyl groups in both the R-helix and the β-sheet conformations kept parallel to the airwater interface with the increase of surface pressure. Consequently, the PM-IRRAS data in Figure 8 correlated with the conclusion from the IRRAS data (Figure 7, parts a and b). On the basis of the results shown in Figures 6-8, the carbonyl groups in both the R-helix and the β-sheet of the OPAA
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SCHEME 2: Model of the Interaction between the OPAA Langmuir Monolayer and DFP Dissolved in the Subphase
Langmuir monolayer are parallel to the air-water interface. This parallel orientation of carbonyl groups did not change with the increase of surface pressure. Consequently, it is possible to present a model of the orientation of the secondary structure of the OPAA Langmuir monolayer in the absence and presence of DFP, which is helpful to clarify the reason for the decrease of the limiting molecular area of the OPAA Langmuir monolayer in the presence of DFP in the subphase. 3.8. Model of the Orientation of Secondary Structure of the OPAA Langmuir Monolayer in the Absence and Presence of DFP in the Subphase. Because the single-crystal structure of OPAA is still unknown, it is difficult to present a detailed model of the interaction between the OPAA Langmuir monolayer and DFP dissolved in the subphase. However, a simple model can still be presented according to the results shown above. We hypothesized that the molecular shape of OPAA is elliptic as shown in Scheme 2a. When DFP is absent in the subphase, the orientation of the binding site of OPAA is parallel to the air-water interface at 0 mN‚m-1. Under this orientation, the β-sheet conformation is parallel and the R-helix is almost parallel to the air-water interface as explained in the second section of the Results and Discussion. Consequently, the amide II band, namely, the peaks at 1560, 1540, and 1525 cm-1, disappeared at 0 mN‚m-1 in the PM-IRRAS spectrum shown in Figure 5. With the compression of the OPAA Langmuir monolayer, the film becomes more compact and the orientation of the binding site becomes tilted (Scheme 2a). This explains why the intensity ratio of the amide I band (1650 and 1630 cm-1) and the amide II band (1547 and 1530 cm-1) at 20 mN‚m-1 was smaller than the one at 10 mN‚m-1 in Figure 4a and the intensity of the amide II band (1560, 1540, and 1525 cm-1) increased with the increase of surface pressure in Figure 5. After DFP is added to the subphase, there is an interaction between DFP and OPAA in the Langmuir monolayer. This interaction induces the orientation of the binding site of OPAA perpendicular to the air-water interface (Scheme 2b). Under this orientation, the R-helix conformation in OPAA is still parallel to the air-water interface (the R-helix is oriented perpendicular to the plane of the paper), whereas the β-sheet conformation becomes perpendicular to the air-water interface. Because the carbonyl groups in the R-helix are parallel to the elongation direction of the R-helix and the orientation of the carbonyl groups in the β-sheet conformation is perpendicular to the elongation direction of the β-sheet, the carbonyl groups in both the R-helix and the β-sheet of OPAA are still parallel to the air-water interface in the presence of DFP in the subphase. Consequently, the amide II bands (1560, 1540, and
1525 cm-1) disappeared in Figure 8. Because the perpendicular orientation of the β-sheet makes it easier for OPAA to aggregate to form more hydrogen bonds, the peak of the intermolecular β-sheet at 1610 cm-1 appeared in the p-polarized spectra shown in Figures 6a and 7a. Because the OPAA still interacts with DFP when the surface pressure increases, the orientation of the binding site remains unchanged with the increase of surface pressure (Scheme 2b). This explains the reason why the intensity ratio of the amide I band (1650, 1630, and 1610 cm-1) and amide II band (1547 and 1530 cm-1) in Figure 7a remained unchanged and the amide II band (1560, 1540, and 1525 cm-1) was not detected in Figure 8 when the surface pressure was increased. Because the long axis of the elliptic OPAA is perpendicular to the air-water interface, the molecular area is smaller than the value without DFP in the subphase. 4. Conclusion OPAA possibly has an elliptic shape, and the long axis was parallel to the air-water interface at 0 mN‚m-1 in the absence of DFP. Under this orientation, both the R-helix and the β-sheet conformations in OPAA were parallel to the air-water interface. When the surface pressure increased, the R-helix and the β-sheet conformations became tilted. When DFP was added to the subphase at a concentration of 1.1 × 10-5 M, the long axis became perpendicular to the air-water interface. The orientations of the R-helix and the β-sheet were parallel and perpendicular to the air-water interface, respectively. The orientations of the two conformations did not change with the increase of surface pressure, because of interaction between OPAA in the Langmuir monolayer and DFP in the subphase. This change of orientation of the long axis was responsible for the decrease of the limiting molecular area of the OPAA Langmuir monolayer. Acknowledgment. This research was supported by the U.S. Army Research Office (DAAD 19-03-1-0131). References and Notes (1) Munnecke, D. J. Agric. Food Chem. 1980, 28, 105-111. (2) Karalliedde, L.; Wheeler, H.; Maclehose, R.; Murray, V. Public Health 2000, 114, 238-248. (3) Marrs, T. C.; Maynard, T. J.; Sidell, F. R. Chemical Warfare Agents: Toxicology and Treatment; John Wiley and Sons: Chichester, U.K., 1996. (4) Das, K. G.; Kulkarni, P. S. Gas-liquid chromatography. In Pesticide Analysis; Dumas, K. G., Ed.; Marcel Dekker: New York, 1981. (5) Mendoza, C. E. Thin layer chromatography. In Pesticide Analysis; Dumas, K. G., Ed.; Marcel Dekker: New York, 1981; pp 1-44. (6) 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.
5256 J. Phys. Chem. B, Vol. 112, No. 16, 2008 (7) 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. (8) Clement, R. E.; Eiceman, G. A.; Koester, C. J. Anal. Chem. 1995, 67, 221R-255R. (9) Orbulescu, J.; Constantine, C. A.; Rastogi, V. K.; Shah, S. S.; DeFrank, J. J.; Leblanc, R. M. Anal. Chem. 2006, 78, 7016-7021. (10) Wang, C.; Li, C.; Ji, X.; Orbulescu, J.; Xu, J.; Leblanc, R. M. Langmuir 2006, 22, 2200-2204. (11) Ji, X.; Zheng, J.; Xu, J.; Rastogi, V. K.; Cheng, T. C.; DeFrank, J. J.; Leblanc, R. M. J. Phys. Chem. B 2005, 109, 3793-3799. (12) Cao, X.; Mello, S. V.; Leblanc, R. M.; Rastogi, V. K.; Cheng, T. C.; DeFrank, J. J. Colloids Surf., A 2004, 250, 349-356. (13) Constantine, C. A.; Gatta´s-Asfura, K. M.; Mello, S. V.; Crespo, G.; Rastogi, V.; Cheng, T. C.; DeFrank, J. J.; Leblanc, R. M. Langmuir 2003, 19, 9863-9867. (14) Constantine, C. A.; Mello, S. V.; Dupont, A.; Cao, X.; Santos, D.; Oliveira, O. N., Jr.; Strixino, F. T., Jr.; Pereira, E. C.; Cheng, T. C.; DeFrank, J. J.; Leblanc, R. M. J. Am. Chem. Soc. 2003, 125, 1805-1809. (15) Zheng, J.; Constantine, C. A.; Zhao, L.; Rastogi, V. K.; Cheng, T. C.; DeFrank, J. J.; Leblanc, R. M. Biomacromolecules 2005, 6, 15551560. (16) Mello, S. V.; Mabrouki, M.; Cao, X.; Leblanc, R. M.; Cheng, T. C.; DeFrank, J. J. Biomacromolecules 2003, 4, 968-973. (17) Orbulescu, J.; Kele, P.; Kotschy, A.; Leblanc, R. M. J. Mater. Chem. 2005, 15, 3084-3088. (18) Zheng, Y.; Orbulescu, J.; Ji, X.; Andreopoulos, F. M.; Pham, S. M.; Leblanc, R. M. J. Am. Chem. Soc. 2003, 125, 2680-2686. (19) Kele, P.; Orbulescu, J.; Calhoun, T. L.; Gawley, R. E.; Leblanc, R. M. Langmuir 2002, 18, 8523-8526. (20) Wang, Y.; Du, X.; Guo, L.; Liu, H. J. Chem. Phys. 2006, 124, 134706. (21) Wang, Y.; Du, X.; Miao, W.; Liang, Y. J. Phys. Chem. B 2006, 110, 4914-4923. (22) Du, X.; Miao, W.; Liang, Y. J. Phys. Chem. B 2005, 109, 74287434. (23) Cai, P.; Flach, C. R.; Mendelsohn, R. Biochemistry 2003, 42, 94469452.
Wang et al. (24) Dieudonne, D.; Gericke, A.; Flach, C. R.; Jiang, X.; Farid, R. S.; Mendelsohn, R. J. Am. Chem. Soc. 1998, 120, 792-799. (25) Wang, C.; Zheng, J.; Oliveira, O. N., Jr.; Leblanc, R. M. J. Phys. Chem. C 2007, 111, 7826-7833. (26) Huo, Q.; Dziri, L.; Desbat, B.; Russell, K. C.; Leblanc, R. M. J. Phys. Chem. B 1999, 103, 2929-2934. (27) Dziri, L.; Desbat, B.; Leblanc, R. M. J. Am. Chem. Soc. 1999, 121, 9618-9625. (28) Calvez, E. L.; Blaudez, D.; Buffeteau, T.; Desbat, B. Langmuir 2001, 17, 670-674. (29) Wang, X.; Zheng, S.; He, Q.; Brezesinski, G.; Mohwald, H.; Li, J. Langmuir, 2005, 21, 1051-1054. (30) Zheng, J.; Desbat, B.; Rastogi, V. K.; Shah, S. S.; DeFrank, J. J.; Leblanc, R. M. Biomacromolecules 2005, 7, 2806-2810. (31) Shafer, P. T. Biochim. Biophys. Acta 1974, 373, 425-435. (32) Susi, H.; Timasheff, S. N.; Stevens, L. J. Biol. Chem. 1967, 242, 5460-5466. (33) Susi, H; Byler, D. M. Biochem. Biophys. Res. Commun. 1983, 115, 391-397. (34) Pauling, L.; Corey, R. B. Proc. Natl. Acad. Sci. U.S.A. 1951, 37, 729-740. (35) Krimm, S. J. Mol. Biol. 1962, 4, 528. (36) Miyazawa, T. J. Chem. Phys. 1960, 32, 1647-1652. (37) Miyazawa, T.; Blout, E. R. J. Am. Chem. Soc. 1960, 83, 712-719. (38) Ronzon, F.; Desbat, B.; Buffeteau, T.; Mingotaud, C.; Chauvet, J.; Roux, B. J. Phys. Chem. B 2002, 106, 3307-3315. (39) Mendelsohn, R.; Mantsch, H. H. In Fourier Transform Infrared Studies of Lipid-Protein Interaction. Progress in Protein-Lipid Interactions; Watts A., de Pont, J. J. H. M., Eds.; Elsevier: Amsterdam, 1986; Vol. 2, pp 103-146. (40) Bertie, J. E.; Ahmed, M. K.; Eysel, H. H. J. Phys. Chem. 1989, 93, 2210-2218. (41) Cornut, I.; Desbat, B.; Turlet, J. M.; Dufourcq, J. Biophys. J. 1996, 70, 305-312. (42) Kerth, A.; Erbe, A.; Dathe, M.; Blume, A. Biophys. J. 2004, 86, 3750-3758.
