Contactless conductivity detection for screening myrosinase substrates by capillary electrophoresis

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 V-max and Michaelis-Menten constant K-m) 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 mu L and the incubation lasted only 5 min at 37 +/- 1 degrees C. Short-end injection of few tens of nanoliters (similar to 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 ((CD)-D-4) 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. (C) 2013 Elsevier B. V. All rights reserved.