engleski

Quantification of the butyrylcholinesterase active sites by organophosphorus compounds

The kinetics of interaction of the horse serum butyrylcholinesterase (EC 3.1.1.8) with DEPQ (7-(O,O-diethylphosphinyloxy)-1-methylquinolinium methyl sulphate) and haloxon (Di-(2-chloroethyl)-3-chloro-4-methyl-coumarin-7-yl phosphate) was studied in order to establish experimental conditions for quantification of butyrylcholinesterase active sites in a commercial preparation of the enzyme. Rapid inhibition of butyrylcholinesterase by covalently binding inhibitors as organophosphorus compounds allows determination of the quantity of the enzyme active sites in samples. The phosphorylated enzyme conjugate should be stable and resistant to spontaneous hydrolysis. Horse butyrylcholinesterase was inhibited by haloxon and DEPQ in excess over enzyme concentration. From first order kinetics the overall inhibition rate constants were evaluated (ki (haloxon) = 1.2 x 107 min-1M-1, ki (DEPQ) =3.1 x 108 min-1M-1). The rate of spontaneous hydrolysis of phosphorylated butyrylcholinesterase by haloxon was 1.5 x 10-3 min-1. Half time of the spontaneous hydrolysis (t 0.5 = 462 min) was long enough to allow determination of the number of the active sites by haloxon. In quantification of number of active sites in enzyme sample stoichiometric subnanomolar concentrations of organophosphates and enzyme were used. The concentration of horse butyrylcholinesterase active sites in 2mg/ml solution of the enzyme preparation was 190 nM detected by DEPQ and haloxon.

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