Determination of Lipoxygenase Activity in Plant Extracts Using a Modified Ferrous Oxidation-Xylenol Orange Assay MARIA DEL CARMEN PINTO,ADRIA Ä N TEJEDA,ANTONIO L. DUQUE, AND PEDRO MACı ´AS* Departamento de Bioquı ´mica y Biologı ´a Molecular, Facultad de Ciencias, Universidad de Extremadura, 06071 Badajoz, Spain The ferrous oxidation-xylenol orange (FOX) assay method for determination of lipid hydroperoxides is based on that under acidic conditions Fe 2+ is oxidized to Fe 3+ , which then oxidizes xylenol orange to a product that absorb at 550 nm. The procedure has been adapted for determination of lipoxygenase activity in plant extracts. This enzyme is responsible for generation of off-flavors in vegetal foods, bleaching of pigments, and a lot of oxidative degradations. It is of interest to check the initial lipoxygenase activity in vegetal foods before the processing, using an assay that is rapid, reproducible, and easily adaptable to high throughput format. The enzymatic assay is based on a discontinuous determination of lipoxygenase activity using the FOX reagent for colorimetric determination of hydroperoxides accumulated in the medium by a period of incubation that is established by the addition of the extract (start of the reaction) and the addition of FOX reagent (finish of the reaction). The procedure is capable of detecting lipoxygenase activity in a number of vegetable homogenates, being especially useful for a rapid visual evaluation of this enzymatic activity. KEYWORDS: Lipoxygenase; xylenol orange; enzymatic activity determination INTRODUCTION Lipoxygenase is a non-heme iron dioxygenase that is ubiquitous in plants and animals and catalyzes the dioxygenation of polyunsaturated fatty acids (PUFAs) containing a (1Z,4Z)- pentadiene system such as, for instance, linoleic acid, R-linolenic acid, or arachidonic acid. The enzyme may have a specific location in plants and produces the formation of hydroperoxy PUFAs with a temporal differentiation of activity, the involve- ment in storage lipid metabolism and the formation of jasmonic acid being their main physiological functions (1). In vegetables, lipoxygenase possesses, in addition to dioxygenase activity, a hydroperoxidase activity that produces the cooxidation of suitable substrates (2). With the aim to prevent oxidative degradations or pigment bleaching produced by peroxidase activity, it is of great interest to check lipoxygenase activity before or during the industrial processing of vegetal foods. In addition, the measurement of lipoxygenase activity may con- stitute a valid procedure to determine the efficiency of thermal treatments during vegetal foods preparations. Typically, the methodologies used for the measurement of lipoxygenase activity are the spectrophotometric assay based on the measurement of the absorbance at 234 nm produced by hydroperoxy lipid product or the oxygraphic assay based on the evaluation of oxygen consumption using an oxygen electrode (3). Both methods, although accurate, are time-consuming, need specialized equipment, and are unsuitable for rapid screening of multiple samples. Alternatively, other methods have been described for determination of plant lipoxygenase activity; for instance, the use of MBTH (3-methyl-2-benzothiazolinone), coupled with DMAB [3-(dimethylamino)benzoic acid] in the presence of hemoglobin, which catalyzes the reaction of detection of linoleic acid hydroperoxide, constitutes an assay method capable of detecting lipoxygenase activity in vegetable homogenates, but the quasi-lipoxygenase activity of hemoglobin needs precise control of the experimental conditions (4). A colorimetric method for determination of lipoxygenase activity applicable to high throughput assays, based on that a lipid hydroperoxide can oxidize F 2+ to Fe 3+ , which in acidic medium oxidized xylenol orange to a colored forms that absorbs in the region of 500-600 nm has been reported (5). Although the procedure was validate by the use of a solubilized prepara- tion of platelet 12-lipoxygenase, the method could result in false positive and requires a complete adaptation for their use in plant extracts. A modified ferrous oxidation-xylenol orange (FOX) assay has been used for detection of lipid hydroperoxides in plant tissues (6). Recently, the FOX assay, with some modifica- tions, has been applied to the screening of 5-lipoxygenase, obtaining rapid and precise results with a high level of sensitivity using insect cell lysates or commercial human 5-lipoxygenase as source of enzyme (7). On the other hand, has been reported that the assay FOX for peroxides determination is optimized if sulfuric acid is substi- tuted by perchloric acid, resulting in an increase in the sensitivity and reproducibility, due principally to a better maintenance of * Corresponding author. E-mail: pedrom@unex.es. 5956 J. Agric. Food Chem. 2007, 55, 5956-5959 10.1021/jf070537x CCC: $37.00 © 2007 American Chemical Society Published on Web 06/30/2007