Comparison of Myrosinase Activity Assays

Abstract

Plant thioglucoside glucohydrolase (myrosinase EC 3.2.3.1) catalyzes the hydrolysis of glucosinolates to isothiocyanate, glucose and sulfate. We have purified myrosinase from white mustard seed (Sinapis alba) in high yields and with a considerable specific activity in a single step by affinity chromatography on Con-A Sepharose. This myrosinase was suitable for use in our polarographic method, which allows the simultaneous determination of total glucosinolates and free glucose in cruciferous extracts. We compared four methods for measuring myrosinase activity for linearity, sensitivity, reproducibility and suitability for routine work, both for routine analyses of crude myrosinase extracts and for enzyme purification and characterization studies. The methods were: i) pH-Stat Assay (pHSA), which measures the rate of acid released during the hydrolysis of sinigrin substrate; ii) Spectrophotometric Coupled Enzyme Assay (SCEA), employing hexokinase and glucose-6-phosphate dehydrogenase (HK--G6PDH), which measures the rate of glucose released during sinigrin hydrolysis; iii) Direct Spectrophotometric Assay (DSA), in which the substrate hydrolysis rate was measured by following the decrease in absorbance at 227 nm; and, finally, iv) a new Polarographic Coupled Assay (PCA), involving the coupled enzymes glucose oxidase and catalase, which measures the rate of glucose released during substrate hydrolysis as O₂ uptake. Although pHSA and PCA showed comparable activities and were linear with increasing amounts of purified enzyme greater than 10 µg, these methods are not suitable for routine work because pHSA requires extensive sample dialysis and PCA shows poor sensitivity. DSA and SCEA gave 18% and 33% lower myrosinase activities, respectively, compared to pHSA; in addition, SCEA was nonlinear with increasing amounts of enzyme above 1 µg. As expected, the lower activity for DSA was due to the suboptimum substrate concentration while for SCEA, this result depends to the low concentrations of Mg++ and HK--G6PDH. In conclusion, DSA appears to be better than the other methods both for enzyme kinetic studies and, if used with care, for routine analyses of crude myrosinase.