High-performance liquid chromatography-based method to evaluate kinetics of glucosinolate hydrolysis by Sinapis alba myrosinase.

Isothiocyanates (ITCs) are one of several hydrolysis products of glucosinolates, plant secondary metabolites that are substrates for the thioglucohydrolase myrosinase. Recent pursuits toward the development of synthetic non-natural ITCs have consequently led to an exploration of generating these compounds from non-natural glucosinolate precursors. Evaluation of the myrosinase-dependent conversion of select non-natural glucosinolates to non-natural ITCs cannot be accomplished using established ultraviolet-visible (UV-Vis) spectroscopic methods. To overcome this limitation, an alternative high-performance liquid chromatography (HPLC)-based analytical approach was developed where initial reaction velocities were generated from nonlinear reaction progress curves. Validation of this HPLC method was accomplished through parallel evaluation of three glucosinolates with UV-Vis methodology. The results of this study demonstrate that kinetic data are consistent between both analytical methods and that the tested glucosinolates respond similarly to both Michaelis-Menten and specific activity analyses. Consequently, this work resulted in the complete kinetic characterization of three glucosinolates with Sinapis alba myrosinase, with results that were consistent with previous reports.