2.3 │ Antioxidant enzyme extraction and activity assays
The pre-weighed infected and non-infected roots were homogenized in 4 mL buffer (50 mM Tris pH = 8.5, 14.4 mM 2-mercaptoethanol) and 1% (w/w) insoluble polyvinylpolypyrrolidone and centrifuged at 6000g for 15 min at 4 °C to determine the SOD, CAT, POX, PPO and PAL (Jung et al., 2011). Total protein content of each enzyme extract was determined using bovine serum albumin as the standard (Bradford, 1976). The CAT activity was determined by measuring the rate of H2O2 conversion to O2 in absorbance at 240 nm and expressed as U mg−1 protein (Gratão et al., 2012). The POX activity was determined by the method described by Dazy et al. (2008). The photometric intensity of the reaction was measured using a spectrophotometer (470 nm) in a 40 mM hydrogen peroxide solution and expressed as units of enzyme activity per mg of soluble protein per min (U mg−1 protein). The activity of PPO was determined using the method of Constabel et al. (1998). Supernatant was added to substrate consisted of 5mgmL−1L-3,4-dihydroxyphenylalanine (L-Dopa). The assay solution consisted of 100mM NaPO4 (pH 7), 0.015% (w/v) sodium dodecyl sulfate, and catalase (280 U mL−1). The absorbance was measured at 490 nm and the PPO activity expressed as U mg−1protein. The activity of SOD was assayed by measuring its ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT) following the method of Van Rossum et al. (1997) and expressed as units of enzyme activity per mg of soluble protein per min (U mg−1protein). The PAL activity was assayed following the method of Beaudoin-Eagan and Thorpe (1985). The photometric intensity of the reaction was measured using a spectrophotometer (290 nm) with 15 mM phenylalanine as the substrate. PAL specific activity was expressed in U mg−1.