Estimation of Superoxide dismutase activity
SOD activity was analyzed by monitoring the inhibition of the photochemical reaction to nitroblue tetrazolium photoreduction (NBT) as described by Dhindsa et al. (1981). The composition of reaction mixture contains 1.5 ml phosphate buffer (pH 7.8, 100 Mm), water 900µl, methionine 200µl of 200 µM, NBT 100 µl, EDTA 100 µl (3Mm), sodium carbonate 100 µl (1.5 M), enzyme extract 300 µl and the riboflavin was added lastly (100 µl, 60 µM). For proper mixing, tubes were inverted 2-3 times and kept under fluorescent light. The lights were turned off after 15 minutes to stop the reaction and the tubes were immediately placed in dark. Absorbance was measured by UV/VIS spectrophotometer at 560 nm, and one unit of SOD activity (U) was defined as the amount of enzyme required to cause the 50% inhibition of the NBT rate. The results were expressed in Umg-1 protein.