Langmuir 2006, 22, 181-186
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Polymerization of a Cysteinyl Peptidolipid Langmuir Film Jianmin Xu,† Changqing Li,† Chengshan Wang,† Jinhai Wang,‡ Qun Huo,‡ and Roger M. Leblanc*,† Department of Chemistry, UniVersity of Miami, 1301 Memorial DriVe, Coral Gables, Florida 33124, and Nanoscience Technology Center and Department of Chemistry, UniVersity of Central Florida, 12424 Research Parkway, Suite 400, Orlando, Florida 32826 ReceiVed October 13, 2005. In Final Form: October 19, 2005 The surface pressure-area isotherm of a cysteinyl peptidolipid on a pure water subphase (pH 5.8) was compared with that on a water subphase saturated with oxygen and buffered with ammonium bicarbonate (pH 7.8). A reduction of the limiting molecular area was observed for the isotherm measured on the subphase saturated with oxygen. Hysteresis in the compression-decompression cycles of the Langmuir film was also observed. Taking into consideration the chemical structure of the peptidolipid, we rationalized that the free sulfhydryl groups of the peptidolipid were oxidized in the presence of oxygen in the alkaline subphase to form intermolecular disulfide bonds at the air-water interface. The surface topography of the peptidolipid Langmuir film was observed by epi-fluorescence microscopy and the Langmuir-Blodgett film by environmental scanning electron microscopy (ESEM). The micrographs showed evidence of the polymerization of the cysteinyl peptidolipid at the air-water interface. Furthermore, the XPS spectra of the Langmuir-Blodgett films also proved the existence of disulfide bonds. The control peptidolipid C18-SerGly-Ser-OH showed identical surface pressure-area isotherms in the presence or absence of an oxygen-saturated subphase.
Introduction A Langmuir film at the air-water interface represents a thin film technique that has broad applications in many important disciplines such as chemo- and biosensor developments,1,2 building blocks for nanomaterials,3 and a model system for mimicking biointerfaces and their functions.4 At air-solid interfaces, a Langmuir-Blodgett (LB) film and a self-assembled monolayer (SAM) are both thin film techniques that have been well studied.5-8 LB films are physically adsorbed onto the solid substrate surface, while self-assembled monolayers are covalently bonded to the substrate material.9 On the other hand, Langmuir film formation is a dynamic process in which the monolayer structure and property could be carefully controlled during the compression stage, while both the LB and SAM methods lack this useful feature. LB and SAM films are complementary tools in thin film-based research and may be used to target different areas of research. To overcome the limitation of the low stability of Langmuir films, cross-linking of the film could provide an attractive solution.10-12 Langmuir films could be polymerized through the photopolymerization of diacetylene groups13,14 or the sol-gel process at the air-water interface.15 The stability of these * To whom correspondence should be addressed. Telephone: (305) 2842194. Fax: (305) 284-6367. E-mail:
[email protected]. † University of Miami. ‡ University of Central Florida. (1) Kele, P.; Orbulescu, J.; Calhoun, T. L.; Gawley, R. E.; Leblanc, R. M. Langmuir 2002, 18, 8523. (2) Cheek, B. J.; Steel, A. B.; Miller, C. J. Langmuir 2000, 16, 10334. (3) Khomutov, G. B.; Gubin, S. P.; Khanin, V. V.; Koksharov, A. Y.; Obydenov, A. Y.; Shorokhov, V. V.; Soldatov, E. S.; Trifonov, A. S. Colloids Surf., A 2002, 198, 593. (4) deKruijff, B. Nature 1997, 386, 129. (5) Schwartz, D. K. Surf. Sci. Rep. 1997, 27, 245. (6) Ulman, A. An Introduction to Ultrathin Organic Films from LangmuirBlodgett to Self-Assembly; Academic Press: New York, 1991; p 278. (7) Schreiber, F. Prog. Surf. Sci. 2000, 65, 151. (8) Poirier, G. E. Chem. ReV. 1997, 97, 1117. (9) Gaines, G. L., Jr. Insoluble Monolayers at Liquid-Gas Interface; Interscience Publishers: New York, 1966; p 73.
polymerized films was greatly improved due to the robustness of the polymer backbone. Huo et al.16 started a novel research direction by using peptidolipids to simulate specific artificial protein structure. During the compression of the peptidolipid Langmuir film, the peptide moieties organize into supramolecular assemblies with proteinlike structures. These artificial protein structures may be used to develop biomimetic sensors and model systems for studying interactions at biointerfaces. Since the proteinlike supramolecular assemblies are formed through noncovalent bonding, the stability of these assemblies needs to be improved to broaden their application. Unfortunately, the two abovementioned polymerization approaches are not suitable for the cross-linking of a peptidolipid Langmuir film: diacetylene groups on the hydrocarbon chains, via photopolymerization, may interact with the aromatic amino acids such as tryptophan, tyrosine, and phenylalanine. The sol-gel process proceeds through proton catalysis in acidic media; under that condition, a peptidolipid Langmuir film could have an effect on the supramolecular arrangement through hydrogen bonding interactions between the peptidolipids. Considering the fact that most protein tertiary structures are stabilized with disulfide bonds between cysteine residues,17,18 we rationalize that disulfide bond formation may be an appropriate way to cross-link the peptidolipid Langmuir film, and therefore to increase the stability of the film. Disulfide bonds could be formed from cysteine residues in many different ways.19,20 S-Protected cysteines could be oxidized to form disulfide bonds by I2 in various solvents such as acetic (10) Kim, K.; Kim, C.; Byun, Y. Abstracts of Papers of the American Chemical Society; American Chemical Society: washington, DC, 2002; Vol. 223, p 258. (11) Aoki, A.; Miyashita, T. Polymer 2001, 42, 7307. (12) Heger, R.; Goedel, W. A. Supramol. Sci. 1997, 4, 301. (13) Tomioka, Y.; Imazeki, S. J. Phys. Chem. 1991, 95, 7007. (14) Meyer, S.; Smith, P.; Wittmann, J. C. J. Appl. Phys. 1995, 77, 5655. (15) Oswald, M.; Hessel, V.; Riedel, R. Thin Solid Films 1999, 339, 284. (16) Huo, Q.; Sui, G. D.; Kele, P.; Leblanc, R. M. Angew. Chem., Int. Ed. 2000, 39, 1854. (17) Thornton, J. M. J. Mol. Biol. 1981, 151, 261. (18) Betz, S. F. Protein Sci. 1993, 2, 1551. (19) Kellenberger, C.; Hietter, H.; Luu, B. Pept. Res. 1995, 8, 321.
10.1021/la0527700 CCC: $33.50 © 2006 American Chemical Society Published on Web 11/24/2005
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Figure 1. Chemical structures of the cysteinyl peptidolipid C18-Cys-Gly-Cys-NH2 and control peptidolipid C18-Ser-GlySer-OH.
acid with methanol, acetic acid with water, methanol with water, or dimethylformamide (DMF), chloroform, and dichloromethane. Deprotected cysteines may be oxidized into disulfide bonds in a DMSO/aqueous hydrochloric acid system21 or directly in the presence of atmospheric oxygen at high dilution under slightly alkaline conditions. This work investigates the possibility of disulfide bond formation at the air-water interface to improve the rigidity of the Langmuir film. In this study, a lipid tripeptide with two unprotected cysteine residues was designed, and the amino acid sequence of the peptidolipid is presented in Figure 1a. This peptidolipid contains free sulfhydryl groups; therefore, it is plausible to perform the easiest oxidizing procedure, oxygen oxidation in basic media. Hence, we regulated the subphase by flushing oxygen into it and adding ammonium bicarbonate buffer (pH 7.8) to make the subphase slightly basic. It is expected that compressing the peptidolipid molecules on this regulated subphase would cause disulfide bond formation, and subsequently, intermolecular disulfide bond formation could lead to a polymerized network. The surface pressure and surface potential measurements were measured to characterize and analyze the Langmuir film that formed, while the topography of the Langmuir film and LB film was examined by epi-fluorescence and environmental scanning electron microscopy (ESEM), respectively. A control peptidolipid C18-Ser-Gly-Ser-OH (Figure 1b) without a cysteine residue was also designed so it could be compared with the cysteinyl peptidolipid. Experimental Section Material. The Wang resin, Rink resin, and amino acids used for peptidolipid synthesis were purchased from Advanced ChemTech (Louisville, KY). All the amino acids were of the l-configuration, except for glycine. Other organic chemicals and solvents were of either HPLC grade or reagent grade and were obtained from Aldrich (St. Louis, MO). The 1H NMR data were acquired with a Bruker 500 MHz spectrometer. Low-resolution FAB spectra were 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 (Urbana, IL). Synthesis. Peptidolipids were prepared by a solid-phase methodology22-24 on Rink resin (0.6 mmol/g) and Wang resin (1.3 mmol/g). Briefly, coupling reactions were carried out with a 3-fold excess of Fmoc amino acids with DIC or DIC/HOBT as the activating agent in DMF. Deprotection of the FMOC group was achieved with 20% (v/v) piperidine in DMF for 30 min. After an average coupling period of 1 h, the extent of acylation was monitored via the standard (20) Kamber, B.; Hartmann, A.; Eisler, K.; Riniker, B.; Rink, H.; Sieber, P.; Rittel, W. HelV. Chim. Acta 1980, 63, 899. (21) Tamamura, H.; Matsumoto, F.; Sakano, K.; Otaka, A.; Ibuka, T.; Fujii, N. Chem. Commun. 1998, 1, 151. (22) Fields, G. B., Ed. Methods in Enzymology; Academic Press: New York, 1997; Vol. 289. (23) Kates, S. A.; Albericio, F. Solid-Phase Synthesis, a Practical Guide; Marcel Dekker: New York, 2000. (24) Fields, G. B.; Noble, R. L. Int. J. Pept. Protein Res. 1990, 35, 161.
Xu et al. ninhydrin test. In the event of incomplete coupling, the coupling procedure was repeated. After the final deprotection, cleavage of the cysteinyl peptidolipid from the Rink resin was conducted with the following cleavage cocktail: trifluoroacetic acid (TFA), phenol, water, thioanisole, and 1,2-ethanedithiol (82.5:5:5:5:2.5, v/v); the cleavage of control peptidolipid C18-Ser-Gly-Ser-OH from Wang resin was performed with TFA. The cleavage time was 2 h. After removal of the TFA under a stream of nitrogen and in a vacuum, the free peptidolipid was precipitated with diethyl ether, filtered off, washed with water and diethyl ether, extracted with 50% (v/v) aqueous acetic acid, and lyophilized. The synthesized peptidolipids were purified on Water 2690 separation modules. The following eluants were used: 0.1% TFA in water (v/v; A) and 0.1% TFA in n-propanol and acetonitrile (50:50, v/v; B). A Vydac 219TP1010 column (diphenyl, 10 µm, 10 mm inside diameter × 250 mm) was used. The purity of the synthesized peptidolipids (95%) was verified by analytical RP-HPLC, 1H NMR, and MS. Analytical HPLC was conducted on a small-scale column (Vydac 219TP54, diphenyl, 5 µm, 4.6 mm inside diameter × 150 mm). The eluants were the same as those used in the aforementioned semipreparative RP-HPLC, and the elution gradient was from 95 to 80% eluant B over 40 min for both peptidolipids; the flow rate was 0.7 mL/min. (a) C18-CysGly-Cys-NH2: 1H NMR (500 MHz, DMSO) δ 8.10 (m, 2H), 5.67 (m, 2H), 4.41-4.30 (m, 2H), 3.71-3.67 (m, 2H), 3.50-3.32 (m, 2H), 2.30-2.19 (m, 4H), 1.54 (t, 2H), 1.35-1.28 (m, 28H), 0.88 (t, 3H); FAB-MS m/z 547.348 (MH+, calcd 547.327). (b) C18-SerGly-Ser-OH: 1H NMR (500 MHz, DMSO) δ 8.10 (m, 2H), 4.334.28 (m, 2H), 3.82-3.46 (m, 8H), 2.19-2.17 (t, 2H), 1.52 (t, 2H), 1.35-1.28 (m, 28H), 0.92 (t, 3H); FAB-MS m/z 517.424 (MH+, calcd 517.415). General Methods for Surface Chemistry. All the surface chemistry studies were conducted in a clean room (class 1000) where constant conditions of temperature (20.0 ( 0.5 °C) and humidity (50 ( 1%) were maintained. The spreading solvent was chloroform and methanol (5:1, v/v), and the solvents were purchased from Fisher Scientific Co. (Pittsburgh, PA). The water utilized as a subphase for the monolayer study was purified with a Modulab 2020 water purification system (Continental Water System Corp., San Antonio, TX) with a specific resistance of 18 MΩ‚cm and a surface tension of 72.6 mN m-1 at 20.0 ( 0.5 °C. For the oxidation of the cysteinyl peptidolipid, the water subphase was regulated with ammonium bicarbonate (NH4HCO3) to make its pH to 7.8, followed by a flush with oxygen for 45 min, at a bubbling rate of 1.0 mL/min. The spreading volume of the peptidolipid solution was 30-70 µL for the surface pressure-area and the surface potential-area measurements. After the solution was spread, a 15 min period was allowed for the complete evaporation of the spreading solvent prior to compression. The compression rate for Langmuir troughs was set up at 5 Å2 molecule-1 min-1. A KSV minitrough (KSV Instrument Ltd., Helsinki, Finland) was utilized for surface pressure-area isotherm measurements. Computer-controlled symmetrically movable barriers were used to regulate the surface area. The trough dimensions were 0.6 cm × 7.5 cm × 30.0 cm. The surface pressure was measured by the Wilhelmy method with a sensitivity of (0.01 mN m-1. Surface potential measurements were recorded using a homemade trough. Two symmetrically movable barriers controlled by the computer were used to regulate the surface area. The dimensions of the trough were 0.6 cm × 12.0 cm × 100.0 cm. The surface potential was measured using the ionizing electrode method. A reference platinum electrode was immersed in the reference trough compartment, and an americium electrode (Am241) was placed ∼1-2 mm above the Langmuir film under study. All the surface pressure- and surface potential-area isotherms were measured at 20.0 ( 0.5 °C. Topographical Studies. An epi-fluorescence microscope (Olympus IX-FLA) was used for acquiring the fluorescence micrographs of the Langmuir film at the air-water interface. A Kibron minitrough (area available for spreading solution of 5.9 cm × 19.5 cm, Kibron Inc., Helsinki, Finland) was used for the preparation of the monolayer. A thermoelectrically cooled Optronics MagnafireTM CCD camera detected the emission of a fluorescent probe, namely, (5octadecanoylamino)fluorescein (ODFL); this fluorescent probe was
Cysteinyl Peptidolipid Langmuir Film
Figure 2. Surface pressure- and surface potential-area isotherms of C18-Cys-Gly-Cys-NH2. mixed with the peptidolipid prior to spreading, and the ODFL: peptidolipid molar mixing ratio was equal to 1:200. For the purpose of LB film topological study, we used a Philips/ Electroscan environmental scanning electron microscope (XL 30-FEG) equipped with a field emission electron gun to study the Langmuir film deposited on the surface of a mica plate. All imaging was performed in a “wet mode”, using a water vapor pressure in the range of 0.9-1.1 mbar as the imaging gas and protective atmosphere. Images presented herein were gaseous secondary electron images (GSEs). A wide range gaseous secondary electron detector was used without an external aperture. It has been previously demonstrated that a low-vacuum field emission gun ESEM could be successfully applied to LB film studies.25 X-ray Photoelectron Spectroscopy (XPS). The XPS spectra were obtained in a Phi 5400 XPS system equipped with an Mg KR source (1253.6 eV, 300 W) with a base pressure of 8 × 10-9 Torr. For XPS measurements, the pass energy of the analyzer was fixed to 35.75 eV. The binding energy scale of the spectrometer was calibrated by using Au 4f7/2 ) 84.0 eV and Cu 2p3/2 ) 932.67 eV. The charging of sample was corrected by Si 2p3/2 ) 103.1 eV and O 1s ) 532.3 eV from the quartz substrate. Peak fitting was carried out by employing a symmetric Gauss-Lorentz sum function with a full width at half-maximum (fwhm) of 1.14 eV. The spin-orbit splittings were set at 1.2 eV for S 2p signals while keeping the respective intensity ratio of 2:1. Before peak fitting, a Shirley background was subtracted.
Results and Discussion Surface Pressure-Area and Surface Potential-Area Isotherms of C18-Cys-Gly-Cys-NH2. The surface pressure (π)- and surface potential (∆V)-area (A) isotherms of the amphiphilic C18-Cys-Gly-Cys-NH2 at the air-water interface are shown in Figure 2. Compression of the peptidolipid results in a π-A isotherm that shows a nil surface pressure higher than 52 Å2/molecule, followed by the lift-off at 52 Å2 and a knee in the region of 52-45 Å2/molecule, indicating a transition from a liquid expanded phase to a liquid condensed phase. Further compression causes an abrupt increase in the surface pressure; that is to say, the surface pressure increases from 3 to 54 mN m-1 which corresponds to a compression from 45 to 17 Å2/molecule. The Langmuir film is observed to collapse at 54 mN m-1. The limiting molecular area value, extrapolated from the linear portion of the π-A curve to nil surface pressure, which is characteristic of the smallest molecular area occupied by the peptidolipid before reaching the collapse of the monolayer, is 39 Å2/molecule. This measured limiting molecular area is close to the value of 35 Å2/molecule predicted with the space-filling CPK (Corey-Pauling-Koltum) model.
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Figure 3. Surface pressure of C18-Cys-Gly-Cys-NH2 on the pure water subphase (b) (pH 5.8) and the oxygen-saturated subphase (O) (ammonium bicarbonate buffer at pH 7.8).
Figure 4. Compression-decompression cycles of C18-Cys-GlyCys-NH2 at surface pressures of up to 20 mN m-1 on an oxygensaturated subphase (ammonium bicarbonate buffer at pH 7.8).
The surface potential-area isotherm of the peptidolipid at the air-water interface is also shown in Figure 2. The surface potential is observed to be nil up to 67 Å2/molecule. Further compression of the Langmuir film causes an increase in the region of 67-59 Å2/molecule, followed by a sharp increase (59-47 Å2/molecule) in the surface potential. Afterward, a steady increase is observed (47-32 Å2/molecule), indicating that further compression caused moderate changes in the orientation of the molecular dipoles. It has to be noted that the lift-off of the surface pressure at ∼47.5 Å2/molecule corresponds to the slope change of the ∆V-A isotherm. Below this value, the van der Waals interaction plays a major role compared to the electrostatic interaction, as shown by a large increase in the surface pressure compared to the surface potential. Surface Pressure-Area Isotherm and in Situ Epi-Fluorescence Microscopy in the Presence of Oxygen in the Subphase. We then regulated the subphase with ammonium bicarbonate to make it slightly basic (final concentration of the subphase is 0.1 mg/mL, at pH 7.8), followed by flushing of the subphase with oxygen for 45 min (oxygen bubbling rate of 1.0 mL/min) to ensure oxygen saturation. The surface pressure-area isotherm was run on this subphase under continuous flushing of oxygen. Figure 3 shows two isotherms. We observed that (1) the isotherm for the oxygen saturated subphase lift-off at a smaller (25) Sui, G. D.; Micic, M.; Huo, Q.; Leblanc, R. M. Langmuir 2000, 16, 7847.
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Figure 5. Epi-fluorescence micrographs of C18-Cys-Gly-Cys-NH2 (a) on a pure water subphase and (b) on an oxygen-saturated subphase (ammonium bicarbonate buffer at pH 7.8). Both are measured at a surface pressure of 30 mN m-1 (image size, 448 µm × 357 µm).
Figure 6. Environmental scanning electron microscopic images of LB films (before deposition, the Langmuir film was run on an oxygensaturated alkaline subphase) deposited on mica at (A) 10 and (B) 30 mN m-1. Imaging conditions: wet mode, 0.9 mbar/1.1 mbar; magnification, 400× at 10.0 keV.
surface area per molecule, i.e., 38 Å2/molecule, and (2) the extrapolation of the linear portion of the isotherm to zero surface pressure presents a limiting molecular area of 33 Å2/molecule compared to 39 Å2/molecule for the pure water subphase. Meanwhile, the π-A isotherm of the peptidolipid in the slightly basic subphase with argon flushing the subphase at the same bubbling rate and flushing time as oxygen is the same as the isotherm of the pure water subphase. It indicates that the decrease in the limiting molecular area might be due to disulfide bond formation, not to the ionization of the functional group. The compression-decompression cycle (three cycles) of the peptidolipid up to 20 mN m-1 on the oxygen-flushed subphase was also assessed and is shown in Figure 4. It is found that in each compression-decompression cycle, when the barrier returns to a wider area, the decompression curve does not overlap with the compression curve. This irreversibility of the isotherm at a surface pressure of up to 20 mN m-1 is an indication of the formation of domains at the air-water interface during compression. On the basis of the observation given above, we have experimental evidence that the compression of the cysteinyl peptidolipid molecules on the oxygen-flushed subphase could result in the formation of intermolecular disulfide bonds by the cysteine residues. Upon spreading, the peptidolipid molecules are far from one another at the interface, while compression of the monolayer brings the molecules close to each other. At the oxygen-rich interface, the SH groups of the peptidolipid are oxidized to form disulfide bonds. The formation of covalent disulfide bonds reduces the space between the peptidolipid molecules; this is consistent with the observation that the cross-
sectional area per molecule measured from the π-A isotherm on the modified subphase is smaller than that on the pure water subphase. To further verify this reasoning, in situ epi-fluorescence microscopy was employed to observe the topography of the Langmuir film during compression. In Figure 5, the epi-fluorescence micrographs of the Langmuir film at 30 mN m-1 on both the regular subphase and the oxygen-saturated subphase are shown. It has been observed that at the same surface pressure, the Langmuir film of the former (Figure 5a) is quite homogeneous, while in the latter case (Figure 5b), the film appears to be heterogeneous and the domains with irregular sizes could be clearly seen. The results of epi-fluorescence measurements revealed the formation of disulfide bonds between cysteinyl peptidolipid molecules at the air-water interface. Furthermore, to study the effects of the ending group, the π-A isotherm and epi-fluorescence of C18-Cys-Gly-Cys-OH were also studied in both oxygen- and argon-saturated subphases (pH 7.8) and showed no difference with those of the NH2 ending group peptidolipid (data not shown here). Topographic Study by ESEM. The ESEM microscopic technique was used to study the topological feature of the cysteinyl peptidolipid in LB films. The Langmuir film formed in the oxygen-saturated subphase was successfully transferred onto mica by Y-type deposition with a transfer ratio of 1 at 10 and 30 mN m-1. It is found that the LB film of peptidolipid molecules deposited onto mica is resistant to electron beam irradiation at intensities of up to 10 keV. Figure 6 shows the ESEM images of the deposited LB films at two different surface pressures, i.e., 10 and 30 mN m-1. It can be seen that at a low surface pressure
Cysteinyl Peptidolipid Langmuir Film
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Figure 7. S 2p XPS spectra of a C18-Cys-Gly-Cys-NH2 LB film deposited on a quartz substrate at 30 mN m-1: (A) one layer and (B) five layers.
Figure 8. Surface pressure-area isotherms of C18-Ser-Gly-SerOH on argon-saturated (b) and oxygen-saturated (9) subphases (ammonium bicarbonate buffer at pH 7.8).
(10 mN m-1) the peptidolipid molecules are dispersed and not tightly packed, and a few domains could be identified with sizes ranging from 2 to 16 µm. At a high surface pressure (30 mN m-1), the pattern of domains is different: all the irregular domains disappear, and regular-sized fibrillar-like domains are formed. Those fibrils are elongated with an almost uniform diameter of 2 µm. This observation could serve as visual evidence of the formation of domains at the air-water interface due to the crosslinking of the cysteinyl groups. XPS Measurements. It is well-known that the carboxyl group is easier to ionize when pH values increase, so the XPS spectra of C18-Cys-Gly-Cys-OH LB films were also investigated. In full-range XPS spectra (not shown here), both the one-layer C18-Cys-Gly-Cys-OH LB film sample and the five-layer C18-Cys-Gly-Cys-OH LB film sample show signals of carbon, nitrogen, and sulfur as well as signals of silicon and oxygen from a quartz substrate; no other elements were detected. Figure 7a shows the S 2p XPS spectrum obtained for the one-layer C18-Cys-Gly-Cys-OH LB film. The S 2p signal is fitted with two sum functions, which are centered at 162.8 and 163.8 eV. According to previously published data, the peak at ∼163.8 eV can be assigned to the sulfur of free thiol groups (SH).26-28 The feature at ∼162.8 eV is assigned to the sulfur in a SS group, (26) Bourg, M. C.; Badia, A.; Lennox, R. B. J. Phys. Chem. B 2000, 104, 6562. (27) Castner, D. G.; Hinds, K.; Grainger, D. W. Langmuir 1996, 12, 5083.
Figure 9. Compression-decompression cycles of C18-Ser-GlySer-OH at surface pressures of up to 30 mN m-1 on an oxygensaturated subphase (ammonium bicarbonate buffer at pH 7.8).
according to the result obtained from (S,S-dioxo)-L-cystine.29 The spectrum obtained from the five-layer C18-Cys-Gly-CysOH LB film is shown in Figure 7b. Similarly, two XPS features were observed at 163.8 eV for the SH group and at 162.9 eV for the SS group. In both spectra, the existence of the SS group is clearly shown by XPS. Therefore, the results are also proof that the decreasing molecular area in the oxygen-saturated subphase is due to the formation of SS structure, not to the ionization of the functional groups. Surface Pressure-Area Isotherms and Epi-Fluorescence Micrographs of the Control C18-Ser-Gly-Ser-OH Peptidolipid. Because C18-Ser-Gly-Ser-OH has structure and hydrophilic properties similar to those of C18-Cys-Gly-Cys-OH, it is used as a control peptidolipid in our study. Figure 8 displays the surface pressure-area isotherms of C18-Ser-Gly-Ser-OH on alkaline (pH 7.8) argon and the oxygen-saturated subphase. The results show that two π-A isotherms are almost identical. The lift-off of the π-A isotherms appears at 35 Å2/molecule, and the transition from the liquid expanded phase to the liquid condensed phase is marked by the area per molecule change from 35 to 23 (28) Joseph, Y.; Besnard, I.; Rosenberger, M.; Guse, B.; Nothofer, H.-G.; Wessels, J. M.; Wild, U.; Knop-Gericke, A.; Su, D.; Schlo¨gl, R.; Yasuda, A.; Vossmeyer, T. J. Phys. Chem. B 2003, 107, 7406. (29) Setiawan, L. D.; Baumann, H.; Gribbin, D. Surf. Interface Anal. 1985, 7, 188.
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Scheme 1. Network Formed at the Air-Water Interface by the Cysteinyl Peptidolipid
Å2/molecule. The steep and linear curve before the collapse corresponds to the liquid condensed state of the Langmuir film. The extrapolation of the linear part of the curve to zero surface pressure is near 28 Å2/molecule, which is close to the value of 32 Å2/molecule calculated from the CPK model. We then ran compression-decompression cycles of this peptidolipid in the oxygen-saturated subphase at a surface pressure of up to 30 mN m-1, as shown in Figure 9. Moving the barrier to its original position gives rise to a first decompression curve that does not overlap with the initial compression curve; nevertheless, further compression and decompression showed a near overlapping of the curves, which is quite different from the hysteresis observed for C18-Cys-Gly-Cys-NH2 (see Figure 4). This is due to the fact that for the control peptidolipid, after the surface pressure reaches 30 mN m-1 in the first cycle, a void still exists in the Langmuir film. Thus, the relaxation of the Langmuir film does not result in the overlapping of the curve, and compression leads to the equilibrium state of the Langmuir film in which the compact arrangement of the amphiphiles is achieved; in other words, the film is very stable in this sequence, so reversibility could be established. This result is quite reasonable in view of the inactive properties of the amino acid residues in the control peptidolipid. The epi-fluorescence micrographs of the control peptidolipid C18-Ser-Gly-Ser-OH were obtained on both subphases, i.e., argon-
saturated (pH 5.8) and oxygen-saturated (pH 7.8) subphases at a surface pressure of 25 mN m-1. As expected, under both conditions, the peptidolipid molecules form very homogeneous films during compression, and no domain could be identified during compression of the Langmuir film. On the basis of the different surface behaviors and micrographs of the two peptidolipids, we conclude that the polymerization takes place in the Langmuir film of C18-Cys-Gly-Cys-NH2 via formation of the disulfide bonds by cysteinyl groups. Through flushing of the subphase with oxygen and with basic conditions, the oxidization of the SH group is realized at the air-water interface, leading to formation of intermolecular disulfide bonds. Scheme 1 illustrates a network that could be established for this type of cross-linking. This would definitely enhance the rigidity of the Langmuir film as shown in the ESEM micrographs (see Figure 6) which therefore will make it amenable to application in areas that require high stability for the peptidolipid Langmuir film. Acknowledgment. We thank Dr. Matt Lynn for running ESEM, and this research was supported by National Science Foundation Grant CHE-0091390. LA0527700
