Purification and characterization of amine oxidase from pea seedlings

A novel, simple, and rapid procedure for the purification of pea seedling a mine oxidase is reported. The crude enzyme, obtained by ammonium sulfate fr actionation, was purified in two steps: the first one by anion-exchange chr omatography and the second one by affinity chromatography, The first chroma tography step was carried out on a diethylaminoethyl-cellulose column. By l owering the amount of protein loaded on the column and the buffer concentra tion it was possible to obtain an enzyme pure at 95% (sp act 1.2 mu kat/mg) . To achieve a higher degree of purification various affinity resins were p repared and tested. The resins were obtained by covalent immobilization of polyamines on Sepharose according to three different procedures. The best r esults were obtained with 6-aminohexyl-Sepharose 2B, prepared using CNBr as coupling agent, and eluting the enzyme by a solution containing 1,4-diamin ocyclohexane. This last compound was found to be a relatively strong compet itive inhibitor of the oxidative deamination of cadaverine catalyzed by pea seedling amine oxidase (K-i = 32 mu M). According to this procedure an ele ctrophoretically homogeneous enzyme, characterized by a specific activity o f 1.63 mu kat/ mg, was obtained. (C) 1999 Academic Press.