Purification of arachidonate 5-lipoxygenase from potato tubers

Abstract

The chapter briefly describes the assay, the purification, and the properties of arachidonate 5-1ipoxygenase (EC 1.13.11.34) from potato tubers. Lipoxygenases (EC 1.13. 11. 12) are a group of closely related enzymes that appear to be widely distributed in plants and mammalian tissues. One of the most physiologically important lipoxygenases and the subject of greater interest in recent years is 5-lipoxygenase, which catalyzes the first committed step in leukotriene (LT) biosynthesis. Information on the mechanistic details of this enzyme-catalyzed reaction is scant because of limitations in obtaining sufficient quantity of the purified enzyme in stable form from mammalian sources. Therefore, potato tubers are frequently employed as the source of large amounts of 5-1ipoxygenase. The mechanism of dioxygenation of polyunsaturated fatty acids (PUFA) catalyzed by lipoxygenases involves the following three main steps: (1) Abstraction of a hydrogen atom from the double allylic methylene carbon atom (the first and rate-limiting step), (2) conjugation of double bonds followed by rearrangement of the radical electron, and (3) addition of molecular oxygen (this occurs at a diffusion-controlled rate under ambient O2 concentrations). Based on this mechanism, it is possible to determine the lipoxygenase activity by measuring the following: (1) absorbance of conjugated diene at 235 nm, (2) O2 uptake by means of a Clark oxygen electrode, and (3) product formation by means of high-performance liquid chromatography (HPLC) or thin-layer chromatography (TLC). Although each one of these methods can be used for monitoring enzyme activity during the course of purification of 5 -lipoxygenase from potato tubers, the polarographic method is found to be the most convenient and reproducible. © 1990, Elsevier Inc. All rights reserved.