ACETYLCHOLINESTERASE COMPLEXATION WITH ACETYLTHIOCHOLINE OR ORGANOPHOSPHATE AT THE AIR AQUEOUS INTERFACE - AFM AND UV-VIS STUDIES/

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 U V-vis, and the topography of the Langmuir-Blodgett films was determine d by tapping mode atomic force microscopy (TMAFM). For a better unders tanding of the complex formation mechanism between AChE and its substr ate, acetylthiocholine, the AChE monolayer was prepared and examined w ith TMAFM in two steps. The monolayer was first compressed on the subs trate-free buffered subphase. Once a surface pressure of 25 mN/m was r eached, the acetylthiocholine was injected into the subphase. The TMAF M images of a transferred monolayer, 6 min after the injection, show t he presence of an acetylcholinesterase-acetylthiocholine complex and a homogeneous monolayer composition. However, the images of a second tr ansferred monolayer at the same surface pressure, but 15 min after the injection, indicate the formation of a mixed monolayer due to the pre sence of both the enzyme-substrate complex and the free enzyme. Compre ssion of the AChE monolayer on a substrate subphase indicates that the hydrolysis reaction took place at the interface and ended before a su rface 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/so lid interfaces. The structural conformation of the enzyme is altered s ignificantly by the presence of the inhibitor. Large domains were obse rved rather than an organized acetylcholinesterase monolayer, and the spectroscopic properties indicate that the interaction between the ace tylcholinesterase and the paraoxon took place at the air/aqueous inter face.