Acetylcholinesterase Complexation with Acetylthiocholine or Organophosphate at the Air/Aqueous Interface:  AFM and UV−Vis Studies

Abstract

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.

Polarization-Modulated FT-IR Spectroscopy Studies of Acetylcholinesterase Secondary Structure at the Air−Water Interface

“…As the monolayer was compressed, the tilt axis of the helix had the tendency to be oriented perpendicular to the water surface, resulting in a linear decrease of the integrated intensity. This is in agreement with surface potential and surface dipole moment of the AChE monolayer data reported recently . The same qualitative trend was observed in the case of β-sheet conformation.…”

“…PMIRRAS data indicated the presence of both the substrate and the reaction product at the interface 10 min after AChE spreading. This agrees with the atomic force microscopy (AFM) images and the UV−Vis results reported recently 8 Normalized PMIRRAS spectra of AChE monolayer collected at the air−water interface at different surface pressures.…”

Moving Electrons Purposefully through Single Molecules and Nanostructures: A Tribute to the Science of Professor Nongjian Tao (1963–2020)

“…[99][100][101] For in situ investigations focusing on the reactions of molecular monolayers, AFM operating in the recently developed tapping mode was used effectively, in preference to STM, a pervasive trend at this time. [102][103][104][105][106] As the electrode potential changes, the surface charge density can be continuously changed, reaching values as high as 0.1 electron per surface atom. Another important contribution is the discovery of charge-induced order-disorder transition of an organic monolayer at a solidliquid interface.…”

“…g ., −10 9 to 10 9 V/m) using the applied electrode potential. These observations provide the basis for the conclusion that the local distribution of electron densities inside of a molecule can be modified and controlled by the electric field. , Because the local electron distribution of a molecule determines its reactivity, this discovery is significant in understanding or designing electric-field-induced redox reactions and even chemical reactions. − For in situ investigations focusing on the reactions of molecular monolayers, AFM operating in the recently developed tapping mode was used effectively, in preference to STM, a pervasive trend at this time. − …”

Responsive photonic crystal for the sensing of environmental pollutants

“…These compounds are potent irreversible inhibitors of acetylcholinesterase (AChE) [32,33]. Walker and Asher [34] developed a pioneering polymerized crystalline colloidal array (PCCA) photonic crystal sensing material that senses the parathion at ultra trace concentrations in aqueous solutions.…”