Electrochemical study on the type of immobilized acetylcholinesterase inhibition by sodium fluoride
By Ovalle, Marcela; Stoytcheva, Margarita; Zlatev, Roumen; Valdez, Benjamin & Velkova, Zdravka
Published in Electrochimica Acta
2008
Abstract
Homogeneous cholinesterases inhibition by sodium fluoride was studied by many authors, using conventional techniques. Controversial results however were reported on the inhibition kinetics and mechanism. In this work electrochemical methods were applied to identify the type of sodium fluoride inhibition of the immobilized acetylcholinesterase taking advantage of the capabilities of the electrochemical biosensors. The acetylcholinesterase inhibition was evaluated by current measurement, at constant potential, of the mediated by K3[Fe(CN)6] and of the non-mediated oxidation of thiocholine iodide, produced by enzymatic acetylthiocholine iodide hydrolysis. Direct amperometric thiocholine detection was preferred for further studies, being more sensitive. The biosensor transducer response to thiocholine iodide was investigated by cyclic and hydrodynamic voltammetry. The reversibility and pH dependence (E1/2 = 1.08–0.06 pH) of thiocholine oxidation were demonstrated. The amperometric biosensor response to acetylthiocholine iodide obtained on a modified by acetylcholinesterase adsorption carbon electrode at +0.80 V/Ag, AgCl was characterised according to IUPAC recommendations. The enzyme reaction kinetic parameters: Imax, K M app, and KI were evaluated under kinetically controlled conditions, for various pH and temperature values. The reversibility of the inhibition was confirmed by dilution. Based on the kinetic analysis, the inhibition of the immobilized acetylcholinesterase by sodium fluoride was found to be of a competitive type.