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  • Automated carboxylesterase assay for the evaluation of ionic liquids' human toxicity.

Automated carboxylesterase assay for the evaluation of ionic liquids' human toxicity.

Journal of hazardous materials (2012-12-19)
Edite Cunha, Paula C A G Pinto, José P S Carvalho, M Lúcia M F S Saraiva
ABSTRACT

Aiming the prediction of ionic liquids' (ILs) human toxicity, an automated carboxylesterase activity assay was developed. The method was implemented on a sequential injection analysis (SIA) system and relied on the hydrolysis of 4-methylumbelliferyl acetate by the enzyme, to produce the fluorescent compound 4-methylumbelliferone (λ(exc)=365 nm; λ(em)=460 nm). The adopted approach was based on activity/inhibition assays in which the reduction of enzyme activity was denoted by a decrease of fluorescence. The activity assays were performed in aqueous media and in the presence of increasing concentrations of seven commercially available ILs and sodium bis(p-nitrophenyl) phosphate, a strong inhibitor of carboxylesterase. The inhibitory effects were quantified by means of the calculation of half maximal inhibitory effective concentration (EC(50)) values that were between 38 and 134 μmol L(-1). This allowed us to perform some considerations about the toxicity of the compounds. The less and more toxic ILs found in this study were emim [Ms] and tbph [Ms], respectively. The developed SIA methodology is robust and exhibits good repeatability in all the tested conditions (rsd<5.0%, n=10). At the same time a reduction of costs was attained by reduced consumption of reagents and minimal effluent production (2 mL per cycle).

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Esterase from rabbit liver, lyophilized powder, ≥30 units/mg protein
Sigma-Aldrich
4-Methylumbelliferyl acetate, esterase substrate
Sigma-Aldrich
Esterase from porcine liver, lyophilized powder, ≥15 units/mg solid
Sigma-Aldrich
Esterase from porcine liver, ammonium sulfate suspension, ≥150 units/mg protein (biuret)
Sigma-Aldrich
Esterase Isoenzyme 1 porcine liver, recombinant, recombinant, expressed in E. coli, ≥30.0 U/g
Sigma-Aldrich
Esterase from Bacillus subtilis, recombinant, expressed in E. coli, ≥10 U/mg
Sigma-Aldrich
Carboxylesterase 2 human, recombinant, expressed in mouse NSO cells, ≥95% (SDS-PAGE)
Sigma-Aldrich
Esterase Pseudomonas fluorescens, recombinant from E. coli, ≥4 U/mg
Sigma-Aldrich
Esterase from Bacillus stearothermophilus, ≥0.2 U/mg
Sigma-Aldrich
Carboxylesterase 1 isoform b human, recombinant, expressed in baculovirus infected BTI insect cells
Sigma-Aldrich
Carboxylesterase 1 isoform c human, recombinant, expressed in baculovirus infected BTI insect cells
Sigma-Aldrich
Esterase from Bacillus stearothermophilus, recombinant, expressed in E. coli, ≥4.0 U/mg