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

• Published in: Analytical biochemistry Vol. 516; pp. 37 - 47
• Main Authors: Klingaman, Chase A., Wagner, Matthew J., Brown, Justin R., Klecker, John B., Pauley, Ethan H., Noldner, Colin J., Mays, Jared R.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.01.2017
• Subjects: Chromatography, High Pressure Liquid - methods; Glucosinolate; Glucosinolates - chemistry; Glycoside Hydrolases - chemistry; Hot Temperature; HPLC; Hydrogen-Ion Concentration; Isothiocyanate; Kinetics; Michaelis–Menten; Myrosinase; Plant Proteins - chemistry; Progress curve; Protein Denaturation; Sinapis - chemistry; Isothiocyanate; Michaelis–Menten; Glucosinolate; Progress curve; Myrosinase; HPLC
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2016.10.010

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.