The hydrogen-bonding network formed between a triaminotriazine amphiphile (2C 18 TAZ, 1) and complementary barbituric acid (BA, 2) at the air-water interface is investigated by polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS). The molecular structure and orientation of the 1:1 hydrogenbonding network at the air-water interface is revealed in this study. Without the addition of BA to the subphase, the NH 2 scissoring of 2C 18 TAZ appeared in the spectrum as a broad negative absorption band between 1660 and 1605 cm -1 , indicating its perpendicular orientation to the air-water interface. When BA was added to the subphase, the NH 2 scissoring absorption band from the triaminotriazine moiety disappeared due to the complementary hydrogen bonding of BA to the 2C 18 TAZ monolayer. The formation of the rigid 1:1 hydrogen-bonding network also resulted in the disappearance of one of the ring quadrant stretch absorption bands of the 2C 18 TAZ molecule. New bands which are attributed to the vibration of BA can be clearly seen. Particularly, the CdO stretch from BA shows up in the spectra as two negative absorption bands around 1700 cm -1 . The negative signature of these two bands suggests that the BA molecules are oriented in the hydrogen-bonding network with the C-2 carbonyl positioned vertically toward the air, and the C-4 and C-6 carbonyls directed into the water subphase. This is consistent with formation of an assembly which optimizes the use of complementary hydrogen bonding between two components. Furthermore, the effect of competitive polar organic solvents in subphase, such as DMSO, on the hydrogen-bonding network has also been observed in this study. Compared to the previous IRRAS studies on the similar monolayers, the sensitivity of PM-IRRAS is obviously improved. PM-IRRAS will likely become a powerful analytical technique for the characterization of molecular structure and orientation of Langmuir monolayers at the air-water interface.

The vibrational spectrum of acetylcholinesterase (AChE) at the air−water interface in its free form and bound to either its substrate, acetylthiocholine, or organophosphorus (OP) inhibitor has been studied by polarization modulation infrared reflection absorption spectroscopy (PMIRRAS). The shape and position of the amide I band was used to gauge the surface orientation of α-helices and β-sheets. The measured secondary structure content indicated that the enzyme did not unfold for the surface pressures used (0−30 mN/m). At low surface pressures, a strong amide I band indicated that the average tilt axis of the helices was aligned parallel to the air−water interface. Upon further compression, the α-helix component was significantly reduced, because the tilt axis of the helix relative to the water surface achieved a perpendicular orientation. PMIRRAS was also used to investigate the effect of phospholipids on molecular organization and orientation of AChE at the air−water interface. The enzyme was found to be fully inserted into the lipidic film during compression. The hydrolysis and inhibition were studied at the air−water interface. Band frequencies associated with acetylthiocholine binding to the enzyme active site and formation of the reaction products were observed. The OP inhibitor, paraoxon, was observed to unfold the enzyme at the air−water interface, because only high-frequency components associated with the extended conformation were observed upon compression. The secondary structure of the AChE was reestablished 30 min after a reactivator, trimethyl bis-(4-formylpyridinium bromide) dioxime, was injected beneath the paraoxon-inhibited AChE. For the first time, an in situ study of the protein conformation is reported using the PMIRRAS technique, and direct supporting evidence that the enzyme did not lose its native secondary structure upon spreading at the air−water interface is provided.

The structural and electron-transfer properties of cytochrome c (Cyt c) Langmuir−Blodgett (LB) films have been studied on graphite electrode with tapping mode atomic force microscopy and cyclic voltammetry (CV). Cyt c in the LB films forms an ordered monolayer in which the individual proteins pack into a quasi-hexagonal structure. The monolayer undergoes a reversible electron-transfer reaction in phosphate buffer. The interactions of Cyt c with cardiolipin (CL) and phosphatidylcholine (PC) LB films have been studied. The LB films of CL and PC are both ordered on graphite, but their interactions with Cyt c are quite different. On a CL monolayer, Cyt c adsorbs spontaneously and the adsorbed protein preserves the electron-transfer reaction. However, on a PC monolayer, Cyt c does not adsorb.

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The hydrolysis reaction of acetylthiocholine catalyzed by the enzyme, acetylcholinesterase (AChE), was studied at the air/aqueous interface by spreading the enzyme as a monolayer and dissolving the substrate in the subphase. The reaction progress was monitored by time-dependent UV−vis, and the topography of the Langmuir−Blodgett films was determined by tapping mode atomic force microscopy (TMAFM). For a better understanding of the complex formation mechanism between AChE and its substrate, acetylthiocholine, the AChE monolayer was prepared and examined with TMAFM in two steps. The monolayer was first compressed on the substrate-free buffered subphase. Once a surface pressure of 25 mN/m was reached, the acetylthiocholine was injected into the subphase. The TMAFM images of a transferred monolayer, 6 min after the injection, show the presence of an acetylcholinesterase−acetylthiocholine complex and a homogeneous monolayer composition. However, the images of a second transferred monolayer at the same surface pressure, but 15 min after the injection, indicate the formation of a mixed monolayer due to the presence of both the enzyme−substrate complex and the free enzyme. Compression of the AChE monolayer on a substrate subphase indicates that the hydrolysis reaction took place at the interface and ended before a surface pressure of 25 mN/m was reached. Therefore, the topography of a monolayer prepared on a subphase containing the substrate resulted in a heterogeneous surface structure due to the presence of free enzymes and reaction products. UV−vis data confirmed the observations deduced from the TMAFM images. Furthermore, the effect of the organophosphate, paraoxon, on the enzyme was studied at the air/aqueous and the air/solid interfaces. The structural conformation of the enzyme is altered significantly by the presence of the inhibitor. Large domains were observed rather than an organized acetylcholinesterase monolayer, and the spectroscopic properties indicate that the interaction between the acetylcholinesterase and the paraoxon took place at the air/aqueous interface.

The surface topography of the enzyme acetylcholinesterase was studied at the air/aqueous and the air/solid interfaces using the Brewster angle and the atomic force microscopies, respectively. Surface potentials of the enzyme monolayer have been measured in conjunction with the surface pressure. The surface potential and the surface dipole moment data show that the orientation of the molecular dipoles occurs before the orientation of the hydrophobic groups of the acetylcholinesterase monolayer. The variations of the surface potential observed at large molecular area suggest the presence of domains in the film. The Brewster angle images confirm the formation of domains at the air/aqueous interface. The size of these domains increases with decreasing the molecular area. Furthermore, the Brewster angle microscopy allowed us to detect a reversible formation of the domains upon the compression and the decompression of the monolayer. On the other hand, the atomic force microscope images of the Langmuir−Blodgett films show that the enzyme molecules are more close-packed at a surface pressure of 25 mN/m than at 20 mN/m. Size measurements of the enzyme particles indicate that acetylcholinesterase has an ellipsoidal shape and that the tetramer form of this enzyme is the most abundant.

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