2.4.11 Antioxidant Enzyme Assays and Protein Determination
For superoxide dismutase, catalase and guaiacol peroxidase extractions, the shoot tissues (0.1 g) were homogenized with a 50 mM sodium phosphate buffer (pH 7.8) with 1 mM ethylenediaminetetraacetic acid (EDTA) and polyvinylpolypyrrolidone (1%). For determination of APX activity, 2 mM of ascorbate was added into the sodium phosphate buffer. The samples were centrifuged at 15.000g for 15 min.
Superoxide dismutase activity was determined according to the method of Beauchamp and Fridovich (1971). Reaction was initiated by the addition of 2 μM riboflavin to 1 ml reaction medium containing 50 mM potassium phosphate buffer (pH 7.8), 0.1 mM EDTA, 13 mM L-methionine, 75 μM nitro blue tetrazolium (NBT), and 50 μl extract. The absorbance values at 560 nm were determined after the mixture was exposed to white light at 375 μmol m−2 s−1 for 10 min. One unit (U) of SOD activity was defined as the amount of enzyme needed to bring about 50% inhibition of the NBT photoreduction rate.
Catalase activity was determined by measuring the decrease in reaction time of 1 ml at 240 nm for 5 min, containing 50 mM potassium phosphate buffer (pH 7.0), 30 mM H2O2 and 20 μl enzyme extract. Catalase activity was calculated using the 39.4 mM−1cm−1 epsilon coefficient for H2O2 (Bergmeyer and Graßl, 1983).
Ascorbate peroxidase activity was determined with decrease at 290 nm (Nakano and Asada 1987). APX activity was determined by measuring a 1 ml reaction mixture containing 50 mM potassium phosphate buffer (pH 7.0), 250 μM ascorbate, 5 mM H2O2 and 20 μl enzyme extract. APX activity was calculated using the 2.8 mM−1cm−1 epsilon coefficient for ASC.
Guaiacol peroxidase activity was measured by increase in absorbance at 470 nm (25°C, ε = 26.6 mM-1cm-1) in a 100 mM potassium phosphate buffer (pH 7.0) containing 0.1 mM EDTA, 5 mM guaiacol, 15 mM H2O2 and 50 μl of enzyme extract (Urbanek et al. 1991).
Glutathione reductase activity was determined spectrophotometrically according to Foyer and Halliwell (1976). GR was assayed by the fall in absorbance at 340 nm as NADPH was oxidized. The assay contained 50 mM Tris-HCl (pH 7.8), 150 μM NADPH, 500 μM oxidized glutathione (GSSG) and 50 μl extract. The activity of GR was calculated using an extinction coefficient of 6.22 mM−1cm−1 for NADPH at 340 nm.
Antioxidant enzyme activities were presented on a protein basis. Protein content was determined according to Bradford (1976), using BSA as a standard.