Electrochemical evaluation of glutathione S-transferase kinetic parameters

Abstract

Glutathione S-transferases (GSTs), are a family of enzymes belonging to the phase II metabolism that catalyse the formation of thioether conjugates between the endogenous tripeptide glutathione and xenobiotic compounds. The voltammetric behaviour of glutathione (GSH), 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione S-transferase (GST), as well as the catalytic conjugation reaction of GSH to CDNB by GST was investigated at room temperature, T = 298.15 K (25 °C), at pH 6.5, for low concentration of substrates and enzyme, using differential pulse (DP) voltammetry at a glassy carbon electrode. Only GSH can be oxidized; a sensitivity of 0.14 nA/μM and a LOD of 6.4 μM were obtained. The GST kinetic parameter electrochemical evaluation, in relation to its substrates, GSH and CDNB, using reciprocal Michaelis–Menten and Lineweaver–Burk double reciprocal plots, was determined. A value of KM ~ 100 μM was obtained for either GSH or CDNB, and Vmax varied between 40 and 60 μmol/min per mg of GST.