Purification of the Main Isozyme of Ascorbate Oxidase in Vigna mungo Sprouts and Mechanism for its Inhibition by Salts

Abstract

Ascorbate oxidase (AAO, EC 1.10.3.3. ) is widely distributed in plants and is responsible for the oxidation of ascorbate during the storage and processing of vegetables. We found two AAO isozymes in the sprouts of Vigna mungo. The main isozyme was separated by DE-52 cellulose column chromatography and purified by Sephacryl S-200 gel filtration. The molecular mass of the isozyme II was 125kDa by Sephacryl S-200 gel filtration. The optimum pH value was 4.50, and the Km value for ascorbic acid was 4.50×10^-5M. The enzyme activity was inhibited by various salts in the order of sodium citrate>NaCl>KCl>sodium malate>NH₄Cl>(NH₄)₂SO₄. Sodium citrate and NaCl inhibited the enzyme activity competitively with respective Ki values of 25mM and 28mM.