Ascorbate oxidase of Cucumis melo L. var. reticulatus: purification, characterization and antibody production

Ascorbate oxidase activity and ascorbic acid content were followedduring the development of muskmelon (Cucumis melo L. var. reticulatus)fruits. The enzyme was highly expressed in ovaries and veryyoung fruit tissues, followed by a decrease in 10- and 20-d-oldfruits and an increase in 30- and 35-d-old fruits which coincidedwith early events of fruit ripening. Ascorbic acid content wasnegatively correlated with ascorbate oxidase activity. The enzymewas purified to homogeneity following ion exchange, affinityand gel filtration chromatographic trials. The purified enzymewas a glycoprotein of molecular weight 137 000 composed of twosubunits of molecular weight 68000, and formed by six isoenzymeswith isoelectric points in the range of pH 7.7 to 8.3. Its electronparamagnetic resonance and optical spectra were in agreementwith other copper proteins and the enzyme contained eight copperatoms per dimeric molecule. The K_m of the enzyme for ascorbicacid was 50 µM. Ascorbate oxidase activity was inhibitedby azide and by EDTA, two inhibitors of copper proteins. Optimalconditions for enzyme activity was pH 5.5, and a temperatureof 37 C. Polyclonal antibodies were produced against the purifiedprotein and immunoprecipitated ascorbate oxidase activity. Key words: Cucumis melo, muskmelon, ascorbate oxidase, fruit ripening