Inhibition of Aggregation of a Biomimic Peptidolipid Langmuir

Inspired by a well-known fact that a stain reagent, Congo red (CR), binds well to .... Congo red was an extra pure reagent purchased from Wako Pure Ch...
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J. Phys. Chem. B 2007, 111, 14227-14232

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Inhibition of Aggregation of a Biomimic Peptidolipid Langmuir Monolayer by Congo Red Studied by UV-Vis and Infrared Spectroscopies Takeshi Hasegawa,*,†,‡ Yoshiko Sato,§ Tetsuo Okada,† Masami Shibukawa,| Changqing Li,⊥ Jhony Orbulescu,⊥ and Roger M. Leblanc⊥ Department of Chemistry, Graduate School of Science and Engineering, Tokyo Institute of Technology, 2-12-1 Ookayama, Meguro-ku, Tokyo 152-8551, Japan, PRESTO, Japan Science and Technology Agency, Sanbancho Building, 5-Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan, Department of Applied Molecular Chemistry, College of Industrial Technology, Nihon UniVersity, 1-2-1 Izumi-cho, Narashino, Chiba 275-8575, Japan, Department of Applied Chemistry, Saitama UniVersity, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan, and Department of Chemistry, UniVersity of Miami, P.O. Box 249118, Coral Gables, Florida 33124-0431 ReceiVed: July 26, 2007; In Final Form: September 26, 2007

A synthetic peptidolipid consisted of a hydrocarbon chain with a chain length of C18 and a peptide moiety of IIGLM terminated with an amine group, designated as C18IIGLM-NH2, has been employed as a biomimic model compound of amyloid peptide for exploring molecular interaction and orientation with the use of the Langmuir monolayer and Langmuir-Blodgett film techniques. Inspired by a well-known fact that a stain reagent, Congo red (CR), binds well to the amyloid-mimic part (IIGLM), inhibition of molecular aggregation of C18IIGLM-NH2 by interaction with CR was expected, and it has been investigated by use of surface pressure-area isotherm, surface dipole moment-area isotherm, Brewster-angle microscopy, and UV-vis/ infrared spectroscopies. It has been revealed that monomeric CR molecules whose long axis is parallel to the Langmuir monolayer surface are penetrating the C18IIGLM-NH2 Langmuir monolayer, which plays a role of inhibition of molecular aggregation via hydrogen bonding.

Introduction Chemical interactions can be categorized in terms of the interacting energy range. For example, covalent and van der Waals interactions are known to fall in the range of 4.8 and 0.01 eV, respectively.1 These two interactions have characteristics that their energy deviations are not so large (about (10%), irrespective of binding angle and distance between the adjacent atoms. On the other hand, hydrogen-bond (H-bond) interaction that is formed between a donor site and an acceptor one is largely different from these interactions. The donor site is a simple diatomic chemical group terminated with hydrogen such as O-H and N-H, while the acceptor site has at least one nonbonding π-electron cloud that is found in a lone pair electron such as CdO. In terms of H-bond acceptor, the π-conjugate in an aromatic ring can be an acceptor. The H-bond interaction varies a great deal in energy in a wide range of 0.4 ( 0.3 eV ( (75%) for weak and strong H-bond interactions, which depend on chemical groups, interacting angle between the donor and acceptor sites, and interacting distance. In this sense, the H-bond is a unique molecular interaction. Of another interest is that the energy of the H-bond is also influenced by the scale of the sites.1 If the donor/acceptor sites belong to long flexible chemical groups, the H-bond energy becomes more stable. Therefore, R-helix in a protein is a good * To whom correspondence should be addressed. E-mail: hasegawa@ chem.titech.ac.jp. † Tokyo Institute of Technology. ‡ Japan Science and Technology Agency. § Nihon University. | Saitama University. ⊥ University of Miami.

example for having very stable H-bond interactions, although they are due to the intramolecular H-bond. In a similar manner, β-sheet in a protein is also in a highly stable state, which is further supported by a sequential array of amino acids, i.e., peptide. Peptide moieties commonly have a characteristic that they are spontaneously put together to form molecular aggregates via complementary H-bond interactions.2-5 The proteinaceous infectious particles (abnormal prion protein; one of the amyloids)6 are considered to be related to their extraordinarily strong molecular aggregation property, and they attract keen interest from a wide range of research fields. In our previous studies, one of the most notable peptide parts to account for the aggregation was chosen to be IIGLM, which was assembled in two synthesized peptidolipids: C18IIGLMOH6 and C18IIGLM-NH2.7 These peptidolipids can be spread on the water surface as a Langmuir monolayer, so the aggregation property can be analyzed by surface chemistry and spectroscopic techniques in a molecular-density controlled Langmuir monolayer.8 Through the studies, we have already clarified that both C18IIGLM-OH and C18IIGLM-NH2 yield tight molecular aggregates and fibrils on water, and the aggregation mechanisms are largely different from each other: the C18IIGLM-OH aggregates are based on the “parallel” β-sheet structure,6 while the C18IIGLM-NH2 aggregates are based on the “antiparallel” β-sheet structure,8 although the two compounds are slightly different only in the terminal small head group (-OH and -NH2). On the other hand, a stain reagent, Congo red (CR), is known to be a good binder to an amyloid peptide, and this technique is widely used for observation of fluorescence microscopic

10.1021/jp0759269 CCC: $37.00 © 2007 American Chemical Society Published on Web 12/06/2007

14228 J. Phys. Chem. B, Vol. 111, No. 51, 2007 images of amyloid-like fibrils.9 This binding property suggests that CR specifically binds to an amyloid-specific amino acid array. If this is true, CR is expected to be an inhibitor of molecular aggregation of the amyloid-mimic parts since the bound CR would disturb the formation of intermolecular H-bonded structure. In the present study, C18IIGLM-NH2 has been employed as a model compound to form aggregates, and interaction of its Langmuir monolayer with a CR aqueous solution has been analyzed by measurements of surface pressure-area isotherm, surface dipole-surface area isotherm, and Brewster angle microscope (BAM) images followed by UV-vis and infrared spectroscopic analyses of Langmuir-Blodgett (LB) films of the Langmuir monolayers. As a result, it has been revealed that CR in a “monomer form” interacts with C18IIGLM-NH2 effectively, and it remains in the Langmuir monolayer with a specific orientation even after compression of the Langmuir monolayer.

Hasegawa et al.

Figure 1. Surface pressure-area (π-A) isotherms of C18IIGLM-NH2 Langmuir monolayers prepared on pure water (black) and Congo red aqueous solutions of 1 × 10-7 (blue) and 1 × 10-5 M (red).

Materials and Methods Materials. Details of the synthesis of C18IIGLM-NH2 was described elsewhere,7 to which the reader is referred. Congo red was an extra pure reagent purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), and it was used as it was without further purification. Surface Pressure-Area Isotherm and Related Measurements. C18IIGLM-NH2 (Mr ) 779.17; relative molecular mass) was dissolved in a mixed solvent of chloroform and methanol (5:1 v/v) at a concentration of 0.375 mg mL-1 by applying sonication for 5 min in a bath-type sonicator, and it was kept overnight in a glass bin with a Mininert valve before use. A stock solution of 30 µL (1.44 × 10-8 mol of CR) was taken by a microsyringe and it was spread on a subphase solution in a Langmuir trough. The trough was a USI-System (Fukuoka, Japan) FSD-220 Langmuir trough equipped with a paper Wilhelmy plate hung on a microbalance. The Langmuir monolayer on water was compressed by a barrier at a compression rate of 5 × 10-2 nm2 molecule-1 min-1. The subphase was pure water or a CR aqueous solution of 1 × 10-5 or 1 × 10-7 M. The solution of 1 × 10-7 M in the trough (ca. 300 mL) contains about 3.0 × 10-8 mol of CR, which is comparable to the number of film molecules in the monolayer. Therefore, The concentration of 1 × 10-7 M is the lowest limit for the experiments. The measurements of surface dipole moment via surface potential measurements of the Langmuir monolayer were performed by use of an ionizing electrode (241Am) against a reference electrode (Ag/AgCl). The surface potential was converted to the perpendicular surface dipole moment.10,11 BAM images of spread Langmuir monolayers on a subphase were measured by use of a Nima Technology (Coventry, England) MicroBAM Brewster-angle microscope with a spatial resolution of 8 µm obtained by 662 nm laser of 30 mW. This equipment was installed over the Langmuir trough by a custommade metal block provided by Meiwafosis (Osaka, Japan). Preparation of Langmuir-Blodgett Film. A spread Langmuir monolayer on water was compressed up to a target pressure and the single Langmuir monolayer was transferred on a germanium substrate by the vertical dipping method.10,11 The germanium plate was purchased from Pier Optics (Gunma, Japan) with a size of 40 × 20 × 1 mm (1 mm is the thickness). After the surface of the plate was cleaned by chemical rinsing,12 the plate was used as the substrate of the LB film. The

withdrawing rate of the substrate for the LB film deposition was 0.5 cm min-1. Infrared Spectroscopic Analysis. The molecular interaction through hydrogen bonding and molecular orientation changes was analyzed by employing multiple-angle incidence resolution spectrometry (MAIRS) coupled with a Fourier transformed infrared (FT-IR) spectrophotometer.13 The collection of infrared transmission single-beam spectra was performed on a Thermo Fisher Scientific (Madison, WI) Nicolet 6700 FT-IR spectrometer equipped with a liquid-nitrogen-cooled mercury-cadmiumtelluride (MCT) detector with an aperture fully opened.14 The laser modulation frequency for the interferogram collections was 60 kHz. The interferogram was accumulated 2000 times to improve the signal-to-noise ratio for every angle of incidence. For more details of the MAIRS analysis, the reader is referred to the literature.15,16 UV-Vis Spectroscopic Analysis. The UV-vis spectra were measured by use of an Otsuka Electronics (Tokyo, Japan) MCPD 7000 UV-visible spectrophotometer that has fiber optics and an ultra-high-sensitive CCD array cooled by a Peltier device. The sample room was a custom-made dark box in which versatile measurements including transmission measurements of LB films can be performed, and the transmitted light is collected through an integral sphere. For the visible spectrometric measurements, a quartz plate was used, which was purchased from the same provider as that for the germanium substrate. Results and Discussion Surface pressure (π)-surface area (A) isotherms of a Langmuir monolayer of C18IIGLM-NH2 were measured on an aqueous solution with various concentrations of Congo red. Representative isotherms are presented in Figure 1: the black curve is the result measured on pure water, the blue curve is measured on an aqueous solution of Congo red with a concentration of 1 × 10-7 M, and the red one is measured on the same subphase solution at a concentration of 1 × 10-5 M. Let us take a look at the isotherm measured on “pure water” (black curve). The surface pressure begins to increase from zero at a surface area of ca. 1.7 nm2 molecule-1, and the isotherm exhibits a short liquid-expanded region10,11 until the surface pressure goes up to ca. 5.0 mN m-1. Above 5.0 mN m-1, the isotherm looks like the condensed film region. The limiting molecular area is obtained as 1.34 ( 0.01 nm2 molecule-1 after

Biomimic Peptidolipid Langmuir Monolayer Aggregation

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Figure 2. BAM images of C18IIGLM-NH2 Langmuir monolayers observed on (a) pure water and (b) Congo red aqueous solutions of 1 × 10-7 M. The scale bar is for 1 mm.

repeated measurements three times. Although the isotherms varied a little in shape, the reproducibility of the limiting area was excellent with a very small experimental error as indicated by the deviation. In a similar manner, another isotherm was measured on a “CR aqueous solution” with the concentration of 1 × 10-7 M, which yielded a limiting molecular area of 1.44 ( 0.01 nm2 molecule-1 with good reproducibility. It is impressive that the isotherm on the CR solution has a significantly larger limiting molecular area by 7.5%, which suggests that the Langmuir monolayer is expanded by interaction with the CR solution. To investigate the difference of surface topography, BAM images (Figure 2) were measured for the two spread Langmuir monolayers on the pure water and the CR solution at a very low surface pressure (π ≈ 0) as indicated by an arrow in Figure 1. In the image measured on pure water, many dots that correspond to molecular aggregates are found (Figure 2a), whereas few dots are found in the image measured on the CR solution (Figure 2b). The homogeneous BAM image on the CR solution is considered to be the result of a specific molecular interaction between CR and the C18IIGLM-NH2 Langmuir monolayer. As a result of the interaction, CR should penetrate the Langmuir monolayer, so the limiting molecular area would be increased, which is confirmed by the enlargement of the limiting molecular area in Figure 1 (1.33 f 1.44 nm2 molecule-1). These results are therefore consistent with the initial expectation that CR inhibits molecular aggregation of an amino-acid array of IIGLM. To reveal the difference of the two cases in detail, surface potential (µ⊥) measurements were also performed along with the surface-pressure measurements as presented in Figure 3 in which the surface potential is converted to the perpendicular surface dipole moment to the surface,

µ⊥ )

A∆V 12π

(1)

where µ⊥ is the perpendicular dipole moment (in Debye), A is the surface area, and ∆V is the measured surface potential (Volts). The results of the C18IIGLM-NH2 Langmuir monolayer on pure water and the CR solution of 1 × 10-7 M are largely different from each other in two aspects: (1) timing of the increase of the surface-dipole moment and surface pressure and (2) intensity and frequency of spike noise on the surface-dipole moment curve, both of which are discussed as follows. When the Langmuir monolayer is compressed on pure water, as indicated by black curves, increase of the surface pressure begins at 1.7 nm2 molecule-1 which is earlier than that of the surface dipole moment at 1.4 nm2 molecule-1. In general, stably compressed (soft) Langmuir monolayer exhibits that the surface pressure begins to increase when an increase of the surface dipole moment finishes, as found in Langmuir monolayers of

Figure 3. Surface dipole moment-area (µ⊥-A) isotherms (solid lines) overlaid with corresponding π-A isotherms (dashed lines) measured on pure water (black) and CR aqueous solution of 1 × 10-7 M (red).

fatty acids and closely packing molecules.17,18 In other words, the early increase of the surface pressure is a good indicator of a stiff Langmuir monolayer against the compression. On the other hand, the Langmuir monolayer on the CR solution exhibits a different result that the increase of the surface dipole moment begins very early, and the slope of the increase becomes large at 1.5 nm2 molecule-1 at which the surface pressure begins to increase. This sequence of increases is the same as that of the soft Langmuir monolayer, which suggests that the Langmuir monolayer prepared on the CR solution is much softer than that on pure water. These results are consistent with the BAM images. Another notable point in the surface dipole moment isotherms is that the isotherm measured on the CR solution has large spikelike peaks before the rapid increase. Although similar peaks are found in the isotherm on pure water, they are much weaker than those on the CR solution. Since the surface dipole moment reflects the orientation changes of a chemical group having a large dipole moment, the large spikelike peaks suggest repeated molecular orientation changes and relaxations. In other words, drastic molecular rearrangements happened during the Langmuir monolayer compression on the CR solution, which further suggests that molecular aggregation was loose on the CR solution. In this manner, the CR solution with the very low concentration of 1 × 10-7 M has been found to play a role of molecularaggregation inhibition in the C18IIGLM-NH2 Langmuir monolayer, and therefore another solution with a higher concentration is expected to give stronger inhibition. For this purpose, an aqueous solution of CR with a 100 times high concentration of 1 × 10-5 M was prepared in the trough and π-A isotherm measurement was performed with a C18IIGLM-NH2 Langmuir monolayer spread on the subphase. The isotherm is overlaid in Figure 1 by the red curve. In contrast to our expectation, however, the limiting molecular area was obtained as 1.39 ( 0.01 nm2 molecule-1 which is apparently smaller than that on the solution of 1 × 10-7 M (1.44 nm2 molecule-1). This result strongly suggests that CR molecules are associated at the concentration level of 10-5 M, while they are in a monomeric form at the concentration of 1 × 10-7 M. The associate molecules are not allowed to penetrate fully the Langmuir monolayer, so the Langmuir monolayer expansion is depressed. If this schematic model of molecular aggregation inhibition is true, the penetrated monomeric CR molecules should remain to interact with the C18IIGLM-NH2 molecules in the Langmuir monolayer, which breaks the intermolecular H-bond between

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Figure 4. Infrared MAIRS-IP spectra of C18IIGLM-NH2 single Langmuir monolayer LB films prepared at 15 mN m-1 on (a) pure water and (b) CR aqueous solution of 1 × 10-7 M.

the C18IIGLM-NH2 molecules. To investigate the molecular interaction, a Langmuir monolayer of C18IIGLM-NH2 prepared on a CR aqueous solution of 1 × 10-7 M was transferred onto a germanium substrate at 15.0 ( 0.2 mN m-1 by the LB film technique for infrared MAIRS analysis. Infrared MAIRS simultaneously yields two spectra that separately capture inplane (IP) and out-of-plane (OP) molecular vibrations to the Langmuir monolayer surface. In this sense, the IP and OP spectra correspond to the conventional transmission and reflection-absorption (RA) spectra, respectively; however, it is notable that MAIRS requires no metallic surface for measurements of the OP spectrum. Since MAIRS enables us to have both spectra from an identical sample at a time, discussion of molecular orientation is made very easy without problems caused by use of different substrates required for the conventional techniques. Figure 4 presents infrared MAIRS-IP spectra of the two single Langmuir monolayer LB film prepared at surfaces of the pure water and the CR aqueous solution drawn by the blue and red curves, respectively. The amide I band that is mainly due to the CdO stretching vibration is split into two positions at 1678 and 1626 cm-1. This band split and the positions tell us that the molecules are organized in the antiparallel β-sheet structure.19-21 As the β-sheet is aligned parallel to the substrate surface in the LB film because of the hydrophilic character,6,7,22 the CdO and N-H groups in the β-sheet are both considered to appear strongly in the IP spectra. In fact, the CdO and N-H stretching vibration modes correspond to the bands at 1626 and 3282 cm-1, and the expectation has proved to be true. Also of note in the spectra of the Langmuir monolayer on the CR solution is that the N-H stretching vibration band about 3282 cm-1 is overlaid on a large broad band centered at the same position, while there is no broad band measured on the pure water surface. This suggests that the molecular organization through the antiparallel β-sheet formation is disturbed by interaction with CR molecules at the solution surface, and some variation of the hydrogen bonding is reflected as the broad band. Although the corresponding amide I band has no significant band broadening, the decrease of this band intensity to that on pure water attains 29%, even after the surface area correction (1.13 and 1.24 nm2 molecule-1 for the Langmuir monolayers on pure water and the CR solution, respectively). In this manner, inhibition of the molecular aggregation of C18IIGLM-NH2 molecules by CR has been proved by infrared spectroscopy. When the Langmuir monolayer was compressed up to 30 mN m-1, a portion of CR molecules in the Langmuir monolayer

Hasegawa et al.

Figure 5. Infrared MAIRS-IP spectra of C18IIGLM-NH2 single Langmuir monolayer LB films prepared at 30 mN m-1 on (a) pure water and (b) CR aqueous solution of 1 × 10-7 M.

Figure 6. Infrared MAIRS-OP spectra of C18IIGLM-NH2 single Langmuir monolayer LB films prepared at 15 mN m-1 on (a) pure water and (b) CR aqueous solution of 1 × 10-7 M. The same measurements were performed for the surface pressure of 30 mN m-1 as presented in (c) and (d).

were found to be removed from the Langmuir monolayer. Figure 5 presents infrared MAIRS-IP spectra of Langmuir monolayers at 30 mN m-1 on (a) pure water and (b) the CR solution. The spectra look similar to those in Figure 4. On closer inspection, however, the N-H stretching vibration band is relatively increased in intensity to the broad band, which suggests that antiparallel β-sheet formation proceeds by the Langmuir monolayer compression after the partial removal of CR. In accordance with this change, the amide I band on the CR solution is also improved, which is now less than that on pure water by 18%. These results suggest that molecular interaction between the Langmuir monolayer and CR molecules is relatively strong, and more than half of the penetrated CR molecules remain in the Langmuir monolayer even if it is compressed. This strong interaction is considered to be an important driving force of the inhibition of molecular aggregation of C18IIGLM-NH2. This discussion is supported by the MAIRS-OP spectra as presented in Figure 6. When the Langmuir monolayer is at a low surface pressure of 15 mN m-1, the antisymmetric and symmetric CH2 stretching vibration bands appear at 2927 and 2858 cm-1, respectively. These band locations are higher than those in the MAIRS-IP spectra (Figure 4) by 3-4 cm-1. The MAIRS shift4,16,23 in this region is known to indicate that the hydrocarbon chain is bent or folded in the Langmuir monolayer,

Biomimic Peptidolipid Langmuir Monolayer Aggregation which is consistent with the Langmuir monolayer architecture based on the antiparallel β-sheet attached to the water or substrate surface (Figure 9). These two bands appear strongly in the spectrum of the Langmuir monolayer prepared on pure water (Figure 6a), while they are depressed in that on the CR solution (Figure 6b). When these two bands are strong in an OP spectrum, molecular tilt angle can be evaluated to be large because of the surface selection rule as the RA spectrometry.13,15,24,25 As monitored by BAM, there is no doubt that the self-aggregation of C18IIGLM-NH2 molecules is very strong on pure water even at the surface pressure of zero, while the aggregation is highly inhibited on the CR solution. The strong aggregation plays a role of molecular organization in a short range to form crystalline. In a long range, however, the pieces of the crystalline would randomly aggregate to form disordered film or fibrils,6 which accounts for the large two bands in Figure 6a, when the surface selection rule of the MAIRS-OP spectrum15,16 is taken into account. On the other hand, the random aggregation of the crystallines is largely inhibited on the CR solution like a Langmuir monolayer of a fatty acid. Therefore, a moderate Langmuir monolayer compression up to 15 mN m-1 would help the Langmuir monolayer ordered, which accounts for Figure 6b. The reader may also be interested in the strong band at 2981 cm-1 in Figure 6b. This is rarely found in this region, but it is known to be related to a mechanically stressed methyl group in an ordered molecular architecture.22 In the present case, some methyl groups as the terminal groups of the leucine and isoleucine moieties are in very narrow space in the antiparallel β-sheet formed by the Langmuir monolayer compression after the softening by CR. Therefore, the appearance of this band strongly supports that the C18IIGLM-NH2 molecules are packed in an ordered way at 15 mN m-1. When the Langmuir monolayer on pure water is compressed up to 30 mN m-1, the Langmuir monolayer having the randomly oriented crystallines would be rearranged to have better orientation as presented in Figure 6c. On the other hand, when the Langmuir monolayer on the CR solution is compressed to the same surface pressure, a different change is expected. The penetrated CR molecules in the Langmuir monolayer via direct interactions with the C18IIGLM-NH2 molecules would disturb the β-sheet formation of C18IIGLM-NH2. In this fashion, Figure 6d is also accountable reasonably. These infrared studies have experimentally proved that the interaction of the Langmuir monolayer molecules with the CR ones inhibits the spontaneous aggregation of C18IIGLM-NH2 in a Langmuir monolayer. At this stage, however, it is not clear whether the CR molecules are in associated or in monomeric form. To examine this issue, three CR samples that have different concentrations (different degrees of molecular association) were prepared for analysis by UV-vis spectrometry: (1) a 1 × 10-7 M CR aqueous solution, (2) a 1 × 10-5 M CR aqueous solution, and (3) a cast film of CR deposited on a quartz plate. The samples (1) and (3) were expected to have CR in a monomer and a heavily associated form, respectively, whereas the sample (2) represents a middle state of the two extreme states. UV-vis absorption spectra of the three samples are presented in Figure 7. Variation of the spectra has characteristics as follows. (1) Only the CR solution with the concentration of 1 × 10-7 M has the shorter wavelength band at 338 nm, whereas a solution with a higher concentration exhibits the band at 345

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Figure 7. UV-vis absorption spectra of CR aqueous solutions of 1 × 10-7 (blue) and 1 × 10-5 M (red), and a CR cast film (black) deposited on a quartz plate. The upper spectrum is a second derivative of the spectrum of the 1 × 10-7 M CR solution.

nm. Since the resolution of the spectrometer is 1 nm, the band shift by 7 nm is obvious. (2) The band at about 500 nm exhibits a longer wavelength component at ca. 550 nm (determined by the second derivative technique) when the concentration is increased. (3) The spectrum of the CR solution of 1 × 10-7 M has a hidden component at 423 nm, which is revealed by the second derivative analysis. This component is very minor in the spectrum for 1 × 10-5 M and it is absent for the cast film. Since the very thin solution of 1 × 10-7 M can have CR molecules in the “monomer” form, the minor band found at 423 nm should be related to the monomeric CR molecule. On the other hand, as the component at 549 nm is increased in intensity when the concentration is getting higher, the band component should be related to CR molecules in “associated” forms. Another marker band at 338 nm may be less useful than the new component at 549 nm, since the band shift is less apparent. Nonetheless, the coincidence of these two bands is useful for finding associated molecular species. After study of the fundamental characteristics of UV-vis spectra of CR in different association states, a single Langmuir monolayer LB film of C18IIGLM-NH2 prepared on the CR solution of 1 × 10-7 M has been measured as presented in Figure 8. The measurements were repeated three times at different positions of the LB film, and the reproducibility was quite good except for noise, although the molecular density of CR was very low in the Langmuir monolayer LB film. The spectrum has a broad band at about 520 nm. The peak position seems shifted to a longer wavelength from the CR solutions. Nonetheless, the bandwidth becomes larger to the right side compared to that of the CR solution at 1 × 10-7 M. Therefore, this apparent peak shift should be caused by the appearance of the band of the associated species (550 nm), which is supported by the band location of the accompanying marker band at 339 nm. In this manner, these two bands tell us that the LB film has associated CR molecules in or on the Langmuir monolayer. The more important band in this spectrum, however, is the very sharp band at 421 nm, which was not obviously found in the spectra of CR solution and cast films, either. The band location being close to that of the band component (423 nm) found in the spectrum of the CR solution at 1 × 10-7 M strongly suggests that the LB film possesses monomeric CR species. The sharpness of the band also tells us that the band comes from monomeric species since associated species can have many

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Hasegawa et al. substrate (LB films) were analyzed by infrared spectrometry, and it has been confirmed that the molecular aggregation was largely diminished by penetration of CR in the Langmuir monolayer. Although the LB films possessed both associated and monomeric CR species on and in the Langmuir monolayer, the aggregation inhibition was mainly caused by the monomeric CR species with a specific molecular orientation, in which the long axis of the molecule is aligned parallel to the film surface. Acknowledgment. This work was financially supported by a Grant-in-Aid for Scientific Research (B) (No. 16350048) from the Japan Society for the Promotion of Science. In addition, this collaborative work was financially supported by Cooperative Research Projects under the Japan-U.S. Cooperative Science Program co-organized by the Japan Society for the Promotion of Science and the National Science Foundation (U.S.A.) to whom the authors’ (T.H. and R.M.L.) thanks are due.

Figure 8. UV-vis transmission spectrum of a single Langmuir monolayer LB film of C18IIGLM-NH2 deposited on a quartz substrate prepared from a Langmuir monolayer on the CR aqueous solution of 1 × 10-7 M at 15 mN m-1.

Figure 9. Schematic image of molecular aggregation inhibition of the molecules by insertion of the oriented CR molecules represented by the squares. Interdigitated arrows indicate that the peptidolipid forms the “antiparallel” β-sheet structure. The yellow rectangle indicates a schematic of a monomeric CR molecule, which breaks some portions of H-bond interactions indicated by dashed lines.

forms, which would yield a broad band. The reason only this band is so strongly enhanced in the spectrum should be attributed to the molecular orientation of the monomeric CR molecules in the film. According to the calculated results by Edwards and Woody,26 the band at 339 nm has a transition moment parallel to the short axis of CR, while the bands at 420 and 500 nm have transition moments parallel to the long axis of the molecule. Since the measurements of the LB film were performed by transmission spectrometry, the strong appearance of the 421 nm band strongly suggests that the monomeric CR molecules are aligned in a way that the longer axis of CR is parallel to the film surface as presented in a schematic (Figure 9). Conclusion Through the isotherm analyses, CR has been found to inhibit molecular aggregation of the C18IIGLM-NH2 molecules on the water surface. The transferred Langmuir monolayers on a solid

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