p-Nitrosodimethylaniline (RNO) bleaching bysoybean lipoxygenase-1. Biochemical characterization andcoupling with oxodiene formation

In this paper, a novel reaction catalysed by the soybean lipoxygenase-1 (linoleate:oxygen oxidoreductase, EC 1.13.11.12) (LOX-1) is reported: p-nitrosodimethylaniline (RNO) bleaching. RNO bleaching in the course of the LOX-1 reaction (with linoleate as substrate) was followed by monitoring photometrically at 440 nm the absorbance decrease of the test sample. The appearance of oxodienes, a class of compounds produced during the same reaction, was also studied. This was carried out by monitoring the absorbance increase at 285 nm in the course of the LOX-1 reaction. RNO bleaching and oxodiene formation by soybean LOX-1 were found to occur synchronously. They occurred only under conditions of limited oxygen; moreover, both reactions showed saturation kinetics as expected for enzyme-catalysed reactions, had similar dependence on pH and temperature (pH optima about 9, temperature optima about 45 °C) and were competitively inhibited by n-propylgallate (PG), a free radical scavenger. Finally, the occurrence of both reactions was found to depend on the presence of 13-hydroperoxy-linoleate, the primary product of the aerobic LOX reaction and was found to be not related with the β-carotene bleaching catalysed by the same enzyme. The possible molecular basis of the link between RNO bleaching and oxodiene formation in the course of the anaerobic soybean LOX-1 reaction is discussed.