title: Activation of phosphorothionate pesticides based on a cytochrome P450 BM-3 (CYP102 A1) mutant for expanded neurotoxin detection in food using acetylcholinesterase biosensors.
abstract: A novel enzymatic in vitro activation method for phosphorothionates has been developed to allow their detection with acetylcholinesterase (AChE) biosensors. Activation is necessary because this group of insecticides shows nearly no inhibitory effect toward AChE in their pure nonmetabolized form. In contrast, they exert a strong inhibitory effect on AChE after oxidation as it takes place by metabolic activation in higher organisms. Standard chemical methods to oxidize phosphorothionates showed inherent disadvantages that impede their direct use in food analysis. In contrast, a genetically engineered triple mutant of P450 BM-3 (CYP102 A1) could convert the two frequently used insecticides parathion and chlorpyrifos into their oxo variants as was confirmed by GC/MS measurements. The wild-type protein was unable to do so. In the case of chlorpyrifos, the enzymatic activation was as good as the chemical oxidation. In the case of parathion, the P450 activation was more efficient than the oxidation by NBS but neither activation method yielded an AChE inhibition that was as high as with paraoxon. The application of the method to infant food in combination with a disposable AChE biosensor enabled detection of chlorpyrifos and parathion at concentrations down to 20 microg/kg within an overall assay time of 95 min.
authors.affiliation: Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany.
authors.forename: Holger
authors.initials: H
authors.lastname: Schulze
authors.forename: Rolf D
authors.initials: RD
authors.lastname: Schmid
authors.forename: Till T
authors.initials: TT
authors.lastname: Bachmann
mesh_terms: D000110:Acetylcholinesterase / Q000187:drug effects / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D004390:Chlorpyrifos / Q000032:analysis / Q000378:metabolism; D002800:Cholinesterase Inhibitors / Q000032:analysis / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005506:Food Contamination / Q000032:analysis*; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015394:Molecular Structure; D009154:Mutation; D009251:NADPH-Ferrihemoprotein Reductase; D009946:Organothiophosphorus Compounds / Q000032:analysis* / Q000378:metabolism; D010084:Oxidation-Reduction; D010278:Parathion / Q000032:analysis / Q000378:metabolism; D010575:Pesticides / Q000032:analysis* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D002800:Cholinesterase Inhibitors; D009946:Organothiophosphorus Compounds; D010575:Pesticides; D010278:Parathion; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases; D000110:Acetylcholinesterase; D004390:Chlorpyrifos