Abstract: The assay is based on continuous measurement of the fatty acid hydroperoxides based on the electrochemical reduction of the hydroperoxides at a Hg electrode. Soy-bean lipoxygenase (0.04 unit) was incubated at 25°C in 3 mL of aerated 0.05 M borate buffer (pH 9.2) containing 0.025% of Polysorbate 20. The reaction was initiated by adding 300 µM linoleic acid. The product was measured amperometrically by reduction at 0 V with a forced mercury-drop electrode vs. an SCE reference electrode and a Pt auxiliary electrode. The amperometric response was linear up to 200 µM linoleic acid 13-hydroperoxide formed. The method could be used to assay the enzyme in the presence of inhibitors.

Abstract: Antioxidant activity and/or the inhibition of lipoxygenases may contribute to the cell and tissue protective properties of flavonoids. Soybean lipoxygenase-1 (LOX-1), is a useful paradigm for lipoxygenases isolated from different sources, and can be used to devise assays of oxygenase inhibitors and antioxidants. Here we describe the voltammetric detection of methylene blue (MB) to assay for antioxidant and lipoxygenase inhibition activity. MB is formed by the catalytic action of hydroperoxide, produced by soybean lipoxygenase reaction on linoleic acid, on benzoyl-leuco methylene blue (BLMB). Every compound which blocks either the enzymatic reaction, lipoxygenase inhibitors, or the oxidation of BLMB, antioxidants, can thus be detected using this method. The assay involves reaction for a period of one hour of linoleic acid, lipoxygenase, BLMB and inhibitor, followed by injection into a flow-injection system and detection by square-wave voltammetry of the methylene blue produced. The problem of irreproducibility induced by adsorption at the solid platinum working electrode is alleviated using the method of alternate injections of control and assay solutions. Assays of several known lipoxygenase inhibitors were undertaken. All of these inhibitors were also, however, shown to be antioxidants. Extension of the assay to the rapid evaluation of novel therapeutic antioxidants and lipoxygenase inhibitors is proposed.