Enzyme activity assay

Superoxide dismutase (SOD), catalase and peroxidase enzyme activity were determined spectrophotometrically as described by Dhindsa et al.⁴², Aebi⁴³ and Castillo et al.⁴⁴ respectively. For these assays, 100 mg of leaf sample (flag leaf) was ground with 4 ml of extraction buffer (0.1 M phosphate buffer, pH 7.5, containing 0.5 mM EDTA) and filtered through 4 layers of cheese cloth. The filtrate was transferred to centrifuge tubes and centrifuged at 15000 rcf for 20 min. The supernatant was used as the enzyme extract and 100 μl of this extract was used for each enzyme activity assay. Briefly, SOD activity was determined by measuring the decrease in the absorbance of blue colored formazone and O2⋅− at 560 nm. For determining catalase activity, the reaction was started by adding H₂O₂ (12.5 mM) to the enzyme extract and decrease in the absorbance was measured at 240 nm for 1 min. Peroxidase activity was determined by increase in the optical density due to oxidation of guaiacol to tetra-guaiacol in the reaction mixture for 2 min, measured at 470 nm.