Spectrophotometric Method for Determination of Lipoxidase Activity

Abstract

Lipoxidase specifically catalyzes the oxidation of methylene-interrupted unsaturated fatty acids such as linoleic, linolenic, and arachidonic acids and their esters, to their res(pective peroxides (3). Various techniques have been devised for the determination of lipoxidase activity, including a colorimetric method based upon coupled oxidation, a manometric method, and spectrophotometric methods (6). The spectro,photometric method was developed after Holman and Burr (4) and Bergstr6m (1) independently observed an increase in ultraviolet light absorption, at 234 m>, when lipoxidase acted upon essential fatty acids. The increase in UV-peak absorption was then related to the amount of peroxide formation which was found to be proportional to time and to enzyme concentration (7, 8). According to Holman and Bergstrom (3), both the manometric and the coupled oxidation methods give aberrant indices of lipoxidase activity because of variations in the degree of dispersion of fatty substrates. The spectrophotometric method, on the other hand, does not have these disadvantages because it employs more homogeneous substrates. Although satisfactory results are obtained when the method is operated at pH 9.0 or above, where the unsaturated fatty acids are present in a solu;ble form, nevertheless, this nonphysiological pH presents a major disadvantage of the spectrophotometric method. It was therefore concluded by Holman and Bergstr6m (3) that the question of the effect of pH on the inherent activity of the enzyme could not be solved until a water-soluble substrate could be found. The method described here is essentially a modification of the methods of Theorell et al. (8) and Tappel (7). The polyunsaturated fatty acid is solubilized by the addition of a detergent, and with this soluble substrate the activities of purified and crude lipoxidase are demionstrated over a wide range of pH. Also, lipoxidase activities in germinating mung beans, over a period of 80 hours, are presented.