Automated evaluation of the inhibition of glutathione reductase activity: application to the prediction of ionic liquids' toxicity

Abstract

This work is based on the automated evaluation of the influence of several ionic liquids (ILs) on the activity of immobilized glutathione reductase (GR), as an indicator of their toxicity. The reactions involved are the reduction of oxidized glutathione (GSSG) by GR, followed by the oxidation of the formed product (reduced glutathione – GSH) by the sulfhydryl reagent 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) to form a yellow derivate 2-nitro-5-thiobenzoic acid (TNB), measurable at 412 nm. The bioassay was performed in a SIA system where the ILs were submitted to a simple and precise on-line dilution strategy before evaluation of their toxicity. The activity assays were implemented in strictly aqueous media and in the presence of increasing concentrations of seven commercially available ILs. EC₅₀ values between 0.24 and 2.55 M were obtained for the tested compounds, revealing distinct inhibitory effects. The least and most toxic ILs found in this study were emim[Ms] and emim[BF₄], respectively. The automated methodology is an interesting analytical tool for the evaluation of GR activity in ILs proving to be robust while presenting good repeatability, with RSD less than 5%, in all the assay conditions, leading also to a reduction of the consumption of solvents as well as of effluent production (2 mL per cycle).