Superoxide dismutase assay (SOD)

The method used to assay the SOD activity was described by Marklund, (1985)³¹. The method is based on the capacity of pyrogallol to autoxidize, a process highly dependent on superoxide (O₂-), which is a substrate for SOD. Briefly, to 15 mL of each sample, 215 mL of a mixture containing 50 mM Tris buffer, pH 8.2, 1 mM EDTA and 30 mM CAT was added. Subsequently, 20 mL of pyrogallol was added, and the absorbance was immediately recorded every 30 s for 3 min at 420 nm using a UV–visible Shimadzu spectrophotometer. The inhibition of autoxidation of pyrogallol occurs in the presence of SOD, whose activity can be indirectly assayed spectrophotometrically. A calibration curve was generated with purified SOD as a reference to calculate the activity of SOD present in the samples. One SOD unit is defined as the amount of SOD necessary to inhibit 50% of pyrogallol autoxidation, and the specific activity is reported as SOD units/mg protein.