Neutral and acidic ascorbate oxidases from satsuma mandarin (Citrus unshiu Marc): Isolation and properties

The neutral and acidic forms of ascorbate oxidase (AAO) were isolated and purified from young fruits of satsuma mandarin by a combination of (NH₄)₂SO₄ fractionation, ion-exchange and hydrophobic chromatography, and gel filtration. The neutral and acidic AAO were both diamers of two subunits with native molecular weights of 133 and 136 kDa, respectively. Neutral AAO was stable in the range of pH 5 and 8 and exhibited optimal activity at pH 5.5 and 45°C. In contrast, acidic AAO was stable between pH 5 and 10 and exhibited optimal activity at pH 5.5 and 50°C. When L-ascorbate and D-isoascorbate were used as substrates, K_m values of neutral AAO were 2.98 × 10^?4 M and 3.36 × 10^?4 M and those of acidic AAO were 1.99 × 10^?4 M and 1.86 × 10^?3 M, respectively. The activities of the two forms of AAO were totally inhibited by metabisulphite and cyanide, substantially suppressed by higher concentrations of diethyldithiocarbamate and fluoride, and not inhibited at all by monovalent and divalent metal ions tested.