Analysis of Lipoxygenase Activity and Products

Although LOX activity is important to the plant s defense against pathogens, there are negative aspects of the enzyme in foods. LOX activity and the resulting fatty acid hydroperoxide products initiate free radical chains that modify proteins (particularly residues of Trp, His, Cys, Tyr, Met, and Lys) as well as vitamins or their precursors (e.g., carotene and tocopherol). Evidence of such free radical reactions is often visibly observed as loss of carotenoid/chlorophyll pigments in improperly blanched frozen foods. Another consequence of these free radical reactions is the development of potent off-flavors, many of which originate from decomposition of the fatty acid hydroperoxide products. 

BASIC PROTOCOL 1 POLAROGRAPHIC MEASUREMENT OF OXYGEN UPTAKE Oxygen electrode equipment is used to determine oxygen uptake as a function of time. After preparing the enzyme (see Support Protocol) and calibrating the instrument, substrate and enzyme are added sequentially to a buffered solution, and the rate of oxygen uptake is monitored by a recorder. 

Analysis of Lipoxygenase Activity and Products Materials Sodium dithionite 0.1 M assay buffer, e.g. Na/K acetate, pH 4.5 to 5.5 Na/K 2-(/V-morpholino)ethanesulfonic acid (MES), pH 5.5 to 6.7 Na/K piperazine-A,A -bis(2-hydroxypropanesulfonic acid) (PIPES), pH 6.1 to 7.5 Na/K /V-2-hydroxyethylpiperazine-/V -2-ethanesulfonic acid (HEPES), pH 6.8 to 8.2 Na/K borate, pH 9 to 11 Tris-Cl, pH 7 to 9 appendix2A) LOX supernatant solution (enzyme see Support Protocol 1) 50 mM linoleic acid substrate solution (see recipe) 02SUBS-T1 (for type 1 LOX) or 02SUBS-T2 (for type 2 LOX) 

Additional reagents and equipment for determination of protein concentration 

Set up an oxygen monitor so that the circulating water bath and water-jacketed sample chamber are at 25°C. 

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