Estimation of Ascorbate peroxidase activity
The activity of ascorbate peroxidase enzyme assay was measured according to Nakano and Asada (1981). The composition of enzyme assay mixture consisted of 150 µL enzyme extract, 50 mM phosphate buffer (pH 7.0), 0.2 µM EDTA, 0.2mM ascorbate, and 2.0 mM H2O2 in a total volume of 3 ml. Ascorbate oxidation was measured by taking the absorbance at 290 nm by UV/VIS spectrophotometer at the moment of H2O2 addition and 1 min later. The difference in absorbance (ΔA290) was divided by the ascorbate molar extinction coefficient (2.8 Mm-1.cm-1). The enzyme activity was expressed as µmol of H2O2min-1 mg-1 protein, taking into account that 1.0 mol of ascorbate is required for the reduction of 1.0 mol of H2O2.