Differentiation between acetylcholinesterase and the organophosphate-inhibited form using antibodies and the correlation of antibody recognition with reactivation mechanism and rate.
Two types of polyclonal antibodies were generated from (a) a decapeptide sequence that includes the active site serine of acetylcholinesterase (anti-AChE10S) and (b) the identical decapeptide sequence phosphorylated at the active site serine of acetylcholinesterase (anti-AChE10SP). The anti-AChE10S antiserum was found to specifically recognize native, control, and vehicle-treated recombinant mouse AChE (rMoAChE) but did not recognize rMoAChE that was phosphorylated by the four organophosphate (OP) compounds tested. Conversely the anti-AChE10SP antiserum recognized phosphoserine rMoAChE that resulted from reaction with phosphorous oxychloride (POCl3) but did not recognize native or vehicle-treated rMoAChE. Anti-AChE10SP also did not recognize OP-AChE conjugates that resulted from the reaction of rMoAChE with other OP compounds that afford neutral or monoanionic phosphoserine groups thereby indicating a high specificity for a precise OP conjugate. Antisera recognition correlated well with the rates of enzyme inhibition, aging, and oxime-induced reactivation indicating these antisera can both quantify the extent and type of inhibition and also differentiate between select mechanisms of inhibition. The ability to discern mechanistic differences between native AChE and OP-AChE conjugates suggests that these antisera can be used to identify biomarkers of OP exposure in a mechanism-based approach.
George
Kathleen M
KM
Center for Environmental Health Sciences, The University of Montana, Missoula, Montana 59812, USA.
Schule
Travis
T
Sandoval
Lisa E
LE
Jennings
Lori L
LL
Taylor
Palmer
P
Thompson
Charles M
CM
D000110:Acetylcholinesterase / Q000737:chemistry*; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D015153:Blotting, Western; D000939:Epitopes / Q000737:chemistry; D007700:Kinetics; D051379:Mice; D008956:Models, Chemical; D009943:Organophosphorus Compounds / Q000737:chemistry*; D010091:Oximes / Q000737:chemistry; D017553:Phosphorus Compounds / Q000494:pharmacology; D010766:Phosphorylation; D010768:Phosphoserine / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D012694:Serine / Q000737:chemistry; D013997:Time Factors
D000939:Epitopes; D009943:Organophosphorus Compounds; D010091:Oximes; D017553:Phosphorus Compounds; D011994:Recombinant Proteins; D010768:Phosphoserine; D012694:Serine; C013196:phosphoryl chloride; D000110:Acetylcholinesterase