Study of lipoxygenase assay using benzoyl leuco methylene blue

Abstract

A method to measure lipoxygenase (LOX) activities of large numbers of samples using benzoyl leuco methylene blue was investigated. Firstly, effect of buffer reagents and pH condition on the reaction of benzoyl leuco methylene blue to methylene blue (LMB colorimetric reaction) with tert-butyl hydroperoxide was determined. Optimal pH was determined to be 5.0 and the acetate buffer was the best reagent among phosphate, 2-morpholinoethanesulfonic acid, 2-amino-2-hydroxy-methyl-1,3-propanediol and glycine. Secondly, LOX activities of purified soybean LOX and crude extracts of soybean seeds were determined. Enzyme samples were incubated with linoleic acid, and the amount of linoleic acid hydroperoxide formed was determined by LMB colorimetric reaction. This method was successfully applied to these samples and was very useful to measure large numbers of samples, although when the concentration of extract is too high, it is thought that the turbidity may interfere the measurement of absorbance of samples.