title: Contactless conductivity detection for screening myrosinase substrates by capillary electrophoresis.
abstract: Myrosinase is a unique enzyme that catalyzes the hydrolysis of glucosinolates (GLS) to isothiocyanate (ITC), glucose and sulfate. Isothiocyanates display a diversified very interesting biological activity. In this study, capillary electrophoresis (CE) was used for the first time for evaluating myrosinase kinetics (maximum velocity Vmax and Michaelis-Menten constant Km) and to assess the affinity of a variety of substrates toward this enzyme. The pre-capillary approach was chosen since it is very simple to conduct. For this, the enzymatic reaction was performed in a micro-vial. The reaction mixture volume was of only 100 μL and the incubation lasted only 5 min at 37±1°C. Short-end injection of few tens of nanoliters (~25 nL) of the reaction mixture was performed which decreased analysis time without using any electroosmotic modifier. The sulfate produced was detected and quantified with a contactless capacitively coupled conductivity detector (C(4)D) allowing the evaluation of myrosinase kinetics. This study shows, that capillary electrophoresis with contactless conductivity detection can be very useful for monitoring myrosinase activity. Comparing to the conventional spectrophotometric method (1982), the CE method developed here is simple, automated, economic, rapid (incubation for few minutes) and robust. Results compared very well with those reported in literature using the conventional method. Moreover, the affinity of a variety of natural and synthetic glucosinolates toward this enzyme has been assessed for the first time.
authors.affiliation: Institut de Chimie Organique et Analytique (ICOA), Université d'Orléans, CNRS FR 2708, UMR 7311, Orléans, France. Electronic address: reine.nehme@univ-orleans.fr.
authors.forename: Reine
authors.initials: R
authors.lastname: Nehmé
authors.affiliation: Institut de Chimie Organique et Analytique (ICOA), Université d'Orléans, CNRS FR 2708, UMR 7311, Orléans, France.
authors.forename: Hala
authors.initials: H
authors.lastname: Nehmé
authors.affiliation: Institut de Chimie Organique et Analytique (ICOA), Université d'Orléans, CNRS FR 2708, UMR 7311, Orléans, France.
authors.forename: Grégory
authors.initials: G
authors.lastname: Roux
authors.affiliation: Institut de Chimie Organique et Analytique (ICOA), Université d'Orléans, CNRS FR 2708, UMR 7311, Orléans, France.
authors.forename: Deimante
authors.initials: D
authors.lastname: Cerniauskaite
authors.affiliation: Institut de Chimie Organique et Analytique (ICOA), Université d'Orléans, CNRS FR 2708, UMR 7311, Orléans, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Morin
authors.affiliation: Institut de Chimie Organique et Analytique (ICOA), Université d'Orléans, CNRS FR 2708, UMR 7311, Orléans, France.
authors.forename: Patrick
authors.initials: P
authors.lastname: Rollin
authors.affiliation: Institut de Chimie Organique et Analytique (ICOA), Université d'Orléans, CNRS FR 2708, UMR 7311, Orléans, France.
authors.forename: Arnaud
authors.initials: A
authors.lastname: Tatibouët
mesh_terms: D004553:Electric Conductivity; D019075:Electrophoresis, Capillary; D005961:Glucosinolates / Q000032:analysis* / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism*; D017879:Isothiocyanates / Q000032:analysis; D007700:Kinetics; D013053:Spectrophotometry; D013379:Substrate Specificity; D013431:Sulfates / Q000032:analysis
chemical_list: D005961:Glucosinolates; D017879:Isothiocyanates; D013431:Sulfates; D006026:Glycoside Hydrolases; C022739:thioglucosidase
keywords: C(4)D; CPA; Contactless capacitively coupled conductivity detector (C(4)D); Enzyme kinetics; GLS; Glucosinolates; K(m); Michaelis–Menten constant; Myrosinase; Pre-capillary electrophoresis; V(max); contactless capacitively coupled conductivity detector; corrected peak area; glucosinolate; maximum velocity