Purification and characterization of cytosolic ascorbate peroxidase from komatsuna (Brassica rapa)

In komatsuna (Brassica rapa) leaves, the cytosolic ascorbate peroxidase was the major enzyme, but the chloroplastic stromal ascorbate peroxidase accounted for approximately 20% of the total activity, judging from the separate assay of ascorbate peroxidase isozymes reported by Amako et al. [11]. The cytosolic ascorbate peroxidase was purified to electrophoretic homogeneity from komatsuna leaves. The purified enzyme was a monomer with a molecular mass of 28 kDa using gel filtration and SDS-PAGE. The enzyme activity was maximal at pH 6.5 and 38 degrees C, and stable between pH 6.5 and pH 7.5 to 35 degrees C. The inhibition of the enzyme by cyanide and azide showed that it is a hemoprotein. This enzyme was not a glycoprotein. When the enzyme was diluted with the ascorbate-depleted medium, the half inactivation time was approximately 80 min. The komatsuna cytosolic ascorbate peroxidase cross-reacted by western blotting with the monoclonal antibody raised against Euglena cytosolic ascorbate peroxidase.