A novel approach for reliable activity determination of ascorbic acid depending myrosinases

Up to now, a wide array of methods for the determination of myrosinase activity has been described. These vary from the simple photometric estimation to highly sophisticated assays using radioactively labelled substrates. However, ascorbic acid-an effective activator of myrosinases-interferes with most of these enzyme tests. Unfortunately, in the past, such interferences were disregarded in many scientific examinations of myrosinases. Whereas such failings have less effects when the activation of myrosinases is not very distinctive, they are quite relevant in all cases where myrosinases are completely inactive in the absence of ascorbic acid. In this paper, the current methods for myrosinase determination are reviewed critically with special emphasis on putative interferences with ascorbic acid. Thereafter, an alternative and interference-free HPLC-based quantification method of the enzymatically produced glucose is presented. Due to the benzoylation of glucose, it becomes possible to quantify even those exiguous glucose concentrations, which are indispensable for correct determination of kinetic enzyme data in the presence of ascorbic acid. Using this new method, the activity of Tropaeolum majus myrosinase towards glucotropaeolin was analyzed. This enzyme shows a distinctive activation by ascorbic acid with maximal activation at a concentration of about 2 mM. (C) 2004 Elsevier B.V. All rights reserved.