Enzymatic determination of fenitrothion by cholinesterase and acetylcholinesterase on fluorogenic substrates

Abstract

The action of the enzymes acetylcholinesterase and cholinesterase on the substrates indoxylacetate and 2-naphthyl acetate was studied kinetically. Both enzymes convert the substrates to highly fluorescent products (3-hydroxy-indole and 2-naphthol, respectively). The kinetic curves present the initial rate as the variation of fluorescence for a unity of time (ΔF/Δt) against the substrate concentration. This enzymatic reaction was investigated in presence of the inhibitor organophosphate pesticide fenitrothion. From the kinetic curves the enzymatic parameters and enzyme and substrate concentrations used for the calibration curve can be obtained. Four simple spectrofluorimetric methods for the enzymatic determination of fenitrothion were developed showing detection limits between 5.5 to 19.5 μmol/l, depending on the enzyme and the substrate used. The precisions (R.S.D.) of the methods were between 1.6 and 9.6%. A comparative study of the kinetic enzymatic parameters and the detection limits was performed. A good correlation was obtained between inhibition constants and the detection limit.