Influence of plant secondary metabolites on in vitro oxidation of methyl ferulate with cell wall peroxidases from lupine apoplast

Ionically bound cell wall peroxidases (POXs) were liberated to intercellular washing fluids (IWFs) and isolated together with other proteins and metabolites present in the apoplast of white lupine (Lupinus albus L. var. Bac) root. After separation of proteins from low molecular weight compounds, activity of peroxidases was monitored in in vitro experiments. Oxidation of methyl ferulate with H2O2 was studied in multi-component mixtures of plant metabolites. Secondary metabolites identified in IWFs or other natural products playing important roles in different physiological processes were applied as modifiers of the dehydrodimerization process during oxidation reactions performed in vitro. These were isoflavones and their conjugates, lupanine representing quinolizidine alkaloids synthesized in lupine, or other natural products such as quercetin, ascorbic, and salicylic acid. The influence of these substances on the oxidation kinetics of methyl ferulate was monitored with liquid chromatography with ultraviolet detection (LC/UV), and identification of compounds was confirmed with the liquid chromatography/mass spectroscopy (LC/MS) system. On the basis of data collected, it was possible to reveal changes in the activities of cell wall POXs. Application of the LC system permitted us to monitor, independently, quantitative changes of two or more reaction products in the mixtures. In multi-component combinations, oxidation yields of methyl ferulate by POXs were modified depending on the actual composition of the reaction mixture. We conclude that various classes of plant secondary metabolites can modify the yield of methyl ferulate oxidation by hydrogen peroxide in the presence of POX, due to interactions with the enzymeʹs active site (genistein) or radical scavenging properties of metabolites present in the reaction mixture.