Inhibition by Antioxidants of Alfalfa Enzyme Catalysis Inhibition of the Antioxidant Concentration Initial Reaction

The presence of the enzyme lipoxidase in alfalfa has been reported (3, 5, 6). However, the properties of this postulated alfalfa lipoxidase are quite different from the well known properties of soybean lipoxidase. Strain (5) showed that the alfalfa enzyme acts on oleic and ricinoleic acids, however it is known that soybean lipoxidase acts only on linoleic, linolenic and arachidonic acids. -Mitchell and Hauge (3) showed that the alfalfa enzyme was partially or completely inactivated by copper sulfate, basic lead acetate, sodium cyanide, thiourea and sodium fluoride. These reagents in relatively high concentrations have no effect on soybean lipoxidase. Recently, Walsh and Hauge (9) determined optimum temperature and pH and the cyanide inhibition of alfalfa suspensions. After comparing their findings with the properties of soybean lipoxidase, they came to the conclusion that the two enzymes are widely different. It is the purpose of this study to establish the identity of the alfalfa enzyme as a lipoxidase and to determine some of its important properties. The enzyme suspensions were prepared by pressing the leaves of fresh inature alfalfa (California common variety) in a hydraulic press. All experiments were done on this juice. Attempts at further purification were unsuccessful. Highly purified ammonium linoleate was the substrate. Reaction rates were measured by the standard Warburg techniques already described (4). In the first series of experiments, it was found that linoleate alone, linoleate + heated alfalfa juice and unheated alfalfa juice alone did not absorb any appreciable amounts of oxygen. The oxygen absorption value of 7.7 ,ul/min in the case of linoleate + alfalfa juice, therefore, suggests the presence of an enzyme system. Significant is the fact that ammonium oleate in the same concentration as linoleate and treated exactly the same way was not oxidized when reacted with the alfalfa juice. These observations indicate the presence of a lipoxidase similar to soybean lipoxidase which specifically catalyses the oxidation of unsaturated fatty acids having two methylene interrupted double bonds. In order to further compare the properties of the alfalfa enzyme with soybean lipoxidase, inhibitor and antioxidant studies were performed. Though an oxidizing enzyme, soybean lipoxidase is not known to possess a prosthetic group (1). Like soybean lipoxidase, the lipoxidases from mung beans (Phaseolus aureus), peas, wheat, peanuts and urd beans (Phaseolus mungo) are not inhibited by metal inhibitors or sulfhydryl agents (Siddiqi, unpublished results; 4).