NEUTRAL AND ACIDIC ASCORBATE OXIDASES FROM SATSUMA MANDARINE (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)(2)SO4 fractionation, ion-exchange and hydrophobic chromatograph y, and gel filtration. The neutral and acidic AAO were both diamers of two subunits with native molecular weights of 133 and 136 kDa, respec tively. Neutral AAO was stable in the range of pH 5 and 8 and exhibite d optimal activity at pH 5.5 and 45 degrees C. In contrast, acidic AAO was stable between pH 5 and 10 and exhibited optimal activity at pH 5 .5 and 50 degrees C. When L-ascorbate and D-iso-ascorbate were used as substrates, K-m values of neutral AAO were 2.98 x 10(-4) M and 3.36 x 10(-4) M and those of acidic AAO were 1.99 x 10(-4) M and 1.86 x 10(- 3) M, respectively. The activities of the two forms of AAO were totall y 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.