This document describes research conducted during 2009-2010 in the Kerman Group laboratory at the University of Toronto Scarborough to investigate the application of electrochemical techniques for the detection of acetylcholinesterase inhibitors in aqueous samples. Two main projects were completed and are discussed herein. The first project demonstrated that the new unmodified, nanostructured gold disposable electrochemical printed (DEP) chips produced by BioDevice Technology can compete with surface-modified electrode configurations to detect trace concentrations of insecticides. This was achieved through the measurement of acetylcholinesterase-catalyzed production of thiocholine after incubation of the enzyme with low concentrations of paraoxon (10 ppb) and carbofuran (8 ppb). The second project featured the novel application of a glassy carbon (GC) electrode to monitor the changes in availability of Thioflavin T (ThT) for oxidation at the electrode surface, which is non-linearly modulated by the presence of acetylcholinesterase and the enzyme’s pre-treatment with trace concentrations of paraoxon and carbachol.

Includes bibliographical references (p. 159-174).

Includes bibliographical references (p. 89-101).

Thesis (M. Phil.)--University of Hong Kong, 1975. / Photocopy of typewcript.

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Thesis (M.Phil.)--University of Hong Kong, 1975. / Also available in print.

Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2002. / Includes bibliographical references (leaves 151-175). Also available in electronic version. Access restricted to campus users.

Biochemical, anatomical, and histochemical studies were performed in the basal ganglia with an emphasis on the localization of the enzyme acetylcholinesterase (AChE). The existence of the enzyme in dopaminergic nigro-striatal neurons was demonstrated. Descending striato-nigral and pallido-nigral axons did not contain detectable amounts of AChE. A cell group called the nucleus basalis magno-cellularis, intimately associated with the globus pallidus, was found to contain high levels of AChE; furthermore, these neurons were shown to be the source of a cholinergic projection to the neocortex. In the striatum, large neurons containing high levels of AChE were found to be likely candidates as..the cholinergic neuron of the striatum. Cholinergic perikarya were found to be absent in the neocortex; nor were perikarya synthesizing large amounts of AChE found in the neocortex. An empirical hypothesis was formulated on the basis of these and other findings regarding cholinergic neurons: High levels of AChE are a necessary but not sufficient criterion for identifying cholinergic perikarya. / Graduate and Postdoctoral Studies / Graduate

This thesis describes the affinity purification of the enzyme acetylcholinesterase from the electric organ tissue of the electric eel (Electrophorus electricus) and the characterization of the enzyme by selective proteolytic cleavage monitored by sucrose gradient sedimentation, sodium dodecyl sulphate-polyacrylamide gel electrophoresis and gel chromatography. It describes conditions, using N-nmethylacri-dinium-Sepharose 2B, for the purification of the asymmetric forms of the enzyme from high salt extracts of electric tissue and for the purification of the globular form of the enzyme subsequent to treatment with the enzyme trypsin. In addition it describes for the first time the selective purification of either asymmetric or globular acetylcholinesterase from mixtures containing both forms of the enzyme. A distinction between autolytic and tryptic degradation of asymmetric acetylcholinesterase is described for the first time and two new forms of the enzyme generated by collagenase proteolysis of the asymmetric 18S and 14S forms are described. The species derived from the 18S form of acetylcholinesterase has a sedimentation coefficient of 21.IS and a Stokes radius of 12.9 nm while the 14S form gives rise to a 17.3S species with a Stokes radius of 11.1 nm. The changes in the sodium dodecyl sulphate-polyacrylamide gel electrophoresis migration pattern of acetylcholinesterase fragments following trypsin or collagenase proteolysis and the changes in sedimentation coefficient and Stokes radius with collagenase proteolysis are compared to identify a component with a molecular weight of 45,000 daltons on electrophoresis gels, that contributes greatly to the asymmetry but only minimally to the mass of the 18S and 14S forms of acetylcholinesterase. An appendix discusses some efforts at the purification of the individual subunits of the 18S and 14S forms of acetylcholinesterase and describes several observations made on the proteolytic instability of even highly purified asymmetric acetylcholinesterase. / Science, Faculty of / Chemistry, Department of / Graduate

Amphioxus (Branchiostoma floridae) cholinesterase 2 (ChE2) hydrolyzes acetylthiocholine (AsCh) almost exclusively. We constructed a homology model of ChE2 on the basis of Torpedo californica acetylcholinesterase (AChE) and found that the acyl pocket of the enzyme resembles that of Drosophila melanogaster AChE, which is proposed to be comprised of Phe330 (Phe290 in T. californica AChE) and Phe440 (Val400), rather than Leu328 (Phe288) and Phe330 (Phe290), as in vertebrate AChE. In ChE2, the homologous amino acids are Phe312 (Phe290) and Phe422 (Val400). To determine if these amino acids define the acyl pocket of ChE2 and its substrate specificity, and to obtain information about the hydrophobic subsite, partially comprised of Tyr352 (Phe330) and Phe353 (Phe331), we performed site-directed mutagenesis and in vitro expression. The aliphatic substitution mutant F312I ChE2 hydrolyzes AsCh preferentially but also butyrylthiocholine (BsCh), and the change in substrate specificity is due primarily to an increase in kcat for BsCh; Km and K ss are also altered. F422L and F422V produce enzymes that hydrolyze BsCh and AsCh equally due to an increase in kcat for BsCh and a decrease in kcat for AsCh. Our data suggest that Phe312 and Phe422 define the acyl pocket. We also screened mutants for changes in sensitivity to various inhibitors. Y352A increases the sensitivity of ChE2 to the bulky inhibitor ethopropazine. Y352A decreases inhibition by BW284c51, consistent with its role as part of the choline-binding site. Aliphatic replacement mutations produce enzymes that are more sensitive to inhibition by iso-OMPA, presumably by increasing access to the active site serine. Y352A, F353A and F353V make ChE2 considerably more resistant to inhibition by eserine and neostigmine, suggesting that binding of these aromatic inhibitors is mediated by π-π or cation-π interactions at the hydrophobic site. Our results also provide information about the aromatic trapping of the active site histidine and the inactivation of ChE2 by sulfhydryl reagents.