Purification of soluble acetylcholinesterase from sheep liver by affinity chromatography.

The purpose of this study was to develop a protocol for the purification of acetylcholinesterase (AChE, acetylcholine acetylhydrolase, E.C.3.1.1.7) enzyme and to extend a purification method for further enzyme characterization. A further aim was to study whether the edrophonium's pharmacologic action is due primarily to the inhibition or inactivation of AChE at sites of cholinergic transmission. The purification of a soluble AChE from sheep liver using affinity chromatography on Concanavalin A-Sepharose 4B and edrophonium-Sepharose 6B is studied. The affinity matrix was synthesized by coupling an inhibitor edrophonium to epoxy-activated Sepharose at flow rate of 0.5 ml/min. AChE is a pivotal enzyme in the cholinergic nervous system. Its primary function is to catalyze hydrolysis of released acetylcholine (ACh) and thus maintain homeostasis of this neurotransmitter in the central and peripheral nervous systems. Hence, AChE is important in both pharmacological and toxicological mechanisms. It was purified 842-fold with a specific activity of 21 U/mg protein. Sodium dodecyl sulfate (SDS) electrophoresis resulted in a monomeric molecular weight of 67.04 kDa, while on gel chromatography using Sephacryl S-200 under nondenaturing conditions to be 201.5 kDa. Based on the molecular weight obtained by gel filtration, the purified AChE was assumed to be a tetrameric form.

Spontaneous reactivation and aging kinetics of acetylcholinesterase inhibited by dichlorvos and diazinon.

Organophosphorus (OP) are among the most toxic of all substances that cause poisoning in food animals and are the most frequently encountered insecticides, commonly detected in agricultural products, animal-derived foodstuffs, environmental samples, and home use and represent a significant potential health risk. The first-order rate constants obtained for spontaneous reactivation (k(s)) was found to be higher in sheep compared to cattle, pig, and ranged between 0.133 to 0.323 hr⁻¹ and between 0.021 to 0.088 hr⁻¹ for dichlorvos (DDVP) and diazinon (DZN) respectively. Aging of phosphorylated acetylcholinesterase (AChE) follows the kinetics of a first-order reaction with rate constants of aging (k(a)) higher in cattle compared to sheep and pig, and ranged between 0.013 to 0.021 hr⁻¹ and between 0.009 to 0.01 hr⁻¹ for DDVP and DZN respectively. Half-time (t½) for spontaneous reactivation and aging are higher in DZN compared to DDVP and ranged from 2.3 to 85.3 hr (sheep), 3.2 to 76.3 hr (cattle), and 2.9 to 58.3 hr (pig), respectively.

Comparison of two storage methods for the analysis of cholinesterase activities in food animals.

Cholinesterases (ChE) are specialized carboxylic ester hydrolases that catalyse the hydrolysis of choline esters. They are classified into either acetylcholinesterase (AChE) or butyrylcholinesterase (BChE). Determination of ChE in the tissues is the appropriate tool for the diagnosis of organophosphorus and carbamate exposures. In general, a significant inhibition was seen in both AChE and BChE activities after 6 months of freezing at -80°C and after 3 months of freezing at -20°C. Linear regression of mean AChE and BChE was observed in all individual samples during the months of the two freezing methods. Bland and Altman plot of the ratios of the two freezing methods have showen the mean difference between the two freezing methods to be 8.8, and SD was 144.7 and -127.6 for upper and lower limits, respectively, for liver, while in muscle the mean difference was 1.5 and SD was 32.5 and -28.9 for upper and lower limits, respectively.

Comparative analysis of cholinesterase activities in food animals using modified Ellman and Michel assays.

This study investigated correlations between modified Ellman and Michel assay methods for measuring cholinesterase (ChE) activities. It also established a foundation for the applicability of measuring ChE activities in food animal species as biochemical biomarkers for evaluating exposure to and effects of organophosphorus and carbamate pesticides. Measuring ChE activities in blood and tissue is currently the most important method of confirming the diagnosis of such exposure. The study also characterized the level of ChE activity in the selected organs/tissues of these animals and determined the best organ/tissue in which to measure ChE activity. The ChE activities were found to be higher in cattle than in sheep and higher in erythrocytes than in plasma and serum. The anticoagulant heparin significantly affects AChE activity in plasma compared with ethylenediamine tetra-acetic acid (EDTA). Of the different tissues tested, the mean of ChE activities was found to be highest in tissue from liver, followed by lung, muscle, kidney, and heart for sheep and cattle. In pigs, the ChE activities tested higher in kidney, liver, lung, muscle, and heart. The highest activities of ChE were found in pigs, followed by cattle and sheep. There was no significant difference between the modified Ellman and Michel method, but the percentage coefficient of variance (%CV) values were higher when the Michel method was used.