Identification of isoenzymes in cholinesterase preparations using kinetic data of organophosphate inhibition

Abstract

Commercial preparations of acetylcholinesterase (EC 3.1.1.7) and of cholinesterase (EC 3.1.1.8) were characterized by organophosphate inhibition. Cholinesterase activities were inhibited by varying organophosphate concentration and time of inhibition. Bimolecular rate constants were determined by plotting log activity vs inhibitor concentration or inhibition time. Inhibition of acetylcholinesterase from bovine erythrocytes by diethyl p-nitrophenyl phosphate (Paraoxon), diisopropylphosphorofluoridate (DFP), and N,N′-diisopropylphosphorodiamidic fluoride (Mipafox) in semilogarithmic plots showed a linear decay of activity. Inhibition of acetylcholinesterase from electric eel ( Electrophorus electricus) and of cholinesterases from horse serum and from human serum did not show linear characteristics, indicating the presence of more than one single enzyme in these preparations. The corresponding inhibition curves were resolved by subtraction of exponential functions. In each case two different activity components were identified and characterized in respect to partial activity, substrate specificity, and reactivity with organophosphorous compounds. The suitability of the method for application on crude homogenates is discussed.