Conformational studies of acetylcholinesterase at air-water and air-solid interfaces and development of an optical fiber for detection of organophosphorous compounds
Date of Award
Doctor of Philosophy (Ph.D.)
First Committee Member
Roger M. Leblanc, Committee Chair
A fiber optic biosensor based on acetylcholinesterase (AChE) as molecular recognition element was constructed to detect highly toxic organophosphorous compounds that are currently used as pesticides. The core fiber was coated with labeled AChE using Langmuir-Blodgett film as a technique of immobilization. A fluorescent signal generated by fluorescein isothiocyanate (FITC) labeled AChE was detected when the fiber tip was dipped into an aqueous solution while this signal vanished in the presence of the inhibitor (paraoxon). Interfacial (surface pressure and surface potential-area isotherms), spectroscopic (UV-vis, fluorescence and vibration mode) properties and surface topography of AChE at air-water and air-solid interfaces were determined. Size measurements indicated that AChE has an ellipsoidal shape and that the tetramer form of this enzyme is the most abundant. Polarization modulation infrared reflection absorption spectroscopy (PMIRRAS) was utilized to investigate the AChE conformation in its free form and bound to, either its substrate, acetylthiocholine, or organophosphorous and carbamate inhibitors. The shape and position of the amide I were used to gauge the surface orientation of alpha-helices and beta-sheets at different surface pressures. At low surface pressures, a strong amide I band indicated that the average tilt axis of the helices was aligned parallel to the water surface. Upon further compression, the tilt axis of the helix reoriented perpendicular to the water surface.Hydrolysis reaction of acetylthiocholine catalyzed by AChE was monitored by time dependent PMIRRAS, UV-Vis and atomic force microscopy (AFM) at the air-water and air-solid interfaces. Band frequencies associated with the acetylthiocholine binding to the enzyme active site and formation of the reaction products were observed. The AFM images showed the presence of an AChE-acetylthiocholine complex six minutes after the substrate injection, while the presence of both the enzyme-substrate complex and free enzyme was observed 15 minutes after the injection. On the other hand, the organophosphorous inhibitor (paraoxon) was observed to unfold the enzyme. Only high frequency components associated with the extended conformation were observed while the inhibition by carbamate was reversible and the decarbamyolation occurred readily. The secondary structure of the AChE was re-established thirty minutes after a re-activator, [trimethyl bis-(4 formylpyridinium bromide) dioxime, was injected beneath the paraoxon-inhibited AChE.
Chemistry, Analytical; Chemistry, Physical
Dziri, Leila, "Conformational studies of acetylcholinesterase at air-water and air-solid interfaces and development of an optical fiber for detection of organophosphorous compounds" (1999). Dissertations from ProQuest. 3696.