HPLC-based kinetics assay facilitates analysis of systems with multiple reaction products and thermal enzyme denaturation

Abstract

Glucosinolates are plant secondary metabolites abundant in Brassica vegetables that are substrates for the enzyme myrosinase, a thioglucoside hydrolase. Enzyme-mediated hydrolysis of glucosinolates forms several organic products, including isothiocyanates (ITCs) that have been explored for their beneficial effects in humans. Myrosinase has been shown to be tolerant of non-natural glucosinolates, such as 2,2-diphenylethyl glucosinolate, and can facilitate their conversion to non-natural ITCs, some of which are leads for drug development. An HPLC-based method capable of analyzing this transformation for non-natural systems has been described. This current study describes (1) the Michaelis–Menten characterization of 2,2-diphenyethyl glucosinolate and (2) a parallel evaluation of this analogue and the natural analogue glucotropaeolin to evaluate effects of pH and temperature on rates of hydrolysis and product(s) formed. Methods described in this study provide the ability to simultaneously and independently analyze the kinetics of multiple reaction components. An unintended outcome of this work was the development of a modified Lambert W(x) which includes a parameter to account for the thermal denaturation of enzyme. The results of this study demonstrate that the action of Sinapis alba myrosinase on natural and non-natural glucosinolates is consistent under the explored range of experimental conditions and in relation to previous accounts.

Synthesis and spectral characterization of 2,2-diphenylethyl glucosinolate and HPLC-based reaction progress curve data for the enzymatic hydrolysis of glucosinolates by Sinapis alba myrosinase

“…Each analyte was individually standardized using the previously-described HPLC method [2], with minor modifications appropriate to the current related study [1]. Standard curves representing peak area vs. injection amount were generated for each wavelength of interest.…”

“…Enzymatic hydrolysis reactions of glucosinolates were conducted in aqueous buffer using a modified form of the established protocol [1,2]. The concentration of glucosinolate, concentration of Sinapis alba myrosinase, buffer pH, and incubation temperature were varied for a given experiment, which were conducted in triplicate.…”

“…This body of data is related to the methodological innovations and enzymological studies described in the related article, “HPLC-based kinetics assay facilitates analysis of systems with multiple reactions components and thermal enzyme denaturation” [1]; to improve clarity, compound numbering from the related article has been retained.…”

“…Reaction progress curve data was fit to the modified Lambert W (x) using nonlinear regression [1]. Initial rates were independently obtained from progress curves tracking analyte ([Gluc] t , [ITC] t , or [nitrile] t ) at a specific wavelength, then normalized for the concentration of myrosinase ( V 0 [Myr] −1 , min −1 ) [1]. Complete original datasets for each figure are provided as Supplementary material.…”

“…Klingaman, M.J. Wagner, J.R. Brown, J.B. Klecker, E.H. Pauley, C.J. Noldner, J.R. Mays,) [1]. This data article describes (1) the synthesis and spectral characterization data of a non-natural glucosinolate analogue, 2,2-diphenylethyl glucosinolate, (2) HPLC standardization data for glucosinolate, isothiocyanate, nitrile, and amine analytes, (3) reaction progress curve data for enzymatic hydrolysis reactions with variable substrate concentration, enzyme concentration, buffer pH, and temperature, and (4) normalized initial velocities of hydrolysis/formation for analytes.…”