Improvement of acetylcholinesterase-based assay for organophosphates in way of identification by reactivators.
Organophosphates present serious fulmination in several aspects of human life. Detection of organophosphates is frequently based on following acetylcholinesterase (AChE) inhibition. Although limit of detection and sensitivity for AChE-based assays seem to be intriguing, the identification of organophosphates is not currently efficient in this way. We introduce an improvement of AChE-based assay by reactivators using a selective come-back of AChE activity after previous inhibition. We have chosen four organophosphates: paraoxon-ethyl, paraoxon-methyl, trichlorfon, methamidophos as representative pesticides and the three most available reactivators: HI-6, obidoxime, pralidoxime. Reactivation was realized in the 96-wells photometric microplates and activity of human recombinant AChE was followed by reaction of Ellman's reagent with one of enzyme digestion product: thiocholine. Distinguishing of reactivation efficacy was judged by the independent two population t-test. The most significant identification was based on methamidophos inhibited AChE reactivation by HI-6 or pralidoxime and paraoxon-ethyl inhibited AChE by obidoxime; moreover, identification of trichlorfon and paraoxon-methyl was possible, too. Practical impact of described method is discussed.
Pohanka
Miroslav
M
Center of Advanced Studies, Faculty of Military Health Sciences, University of Defence, Hradec Kralove, Czech Republic. rau@atlas.cz
Jun
Daniel
D
Kuca
Kamil
K
D000110:Acetylcholinesterase / Q000032:analysis* / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D010755:Organophosphates / Q000032:analysis* / Q000378:metabolism*
D010755:Organophosphates; D000110:Acetylcholinesterase