Antioxidant Enzyme Assays in Leaf Tissue

Superoxide dismutase, POX (EC 1.11.1.7), CAT, GSH POX (GPX), APX, GR, glutathione-S-transferase (GST), monodehydroascorbate reductase (MDHAR, EC 1.6.5.4) and DHAR were determined in an homogenate of 100 mg (FW) of leaf tissues, prepared in 1 ml of 50 mM potassium phosphate buffer (pH 7.0), containing 10% (w/v) polyvinylpyrrolidone (PVP), 0.25% (v/v) Triton X-100, 1 mM phenylmethylsulfonyl fluoride(PMSF) and 1 mM ASC, by using a MagNA Lyser (Roche, Vilvoorde, Belgium). SOD activity was determined according to Dhindsa et al. (1982) (link) by measuring the inhibition of NBT (nitroblue tetrazolium) reduction at 560 nm. POX activity was determined by the oxidation of pyrogallol (ε₄₃₀ = 2.47 mM^-1 cm^-1; Kumar and Khan, 1982 ). CAT activity was assayed according to the Aebi (1984) by monitoring the decomposition of H₂O₂ at 240 nm. APX, MDHAR, DHAR, and GR activities were measured by the methods of (Murshed et al., 2008 (link)). GST activity was estimated by measuring the conjugation of GSH with excess 1-chloro-2,4-dinitrobenzene (CDNB) at 340 nm (ε₃₄₀ = 0.0096 μM^-1 cm^-1; Habig et al., 1974 (link)). GPX activity was assayed by measuring the decrease in NADPH absorbance measured at 340 nm (ε₃₄₀ = 6.22 mM^-1cm^-1; Drotar et al., 1985 (link)). All activity measurements were scaled down for semi-high throughput using a micro-plate reader (Synergy Mx, Biotek Instruments Inc., Winooski, VT, USA), and optimized to obtain linear time and protein concentration dependence.