5.7. Superoxide Dismutase (SOD) Activity

Superoxide dismutase activity was determined using the xanthine/xanthine oxidase system as O₂^●− generator, when it reacts with nitroblue tetrazolium (NBT), reducing it and producing formazan. This chemical species can be detected by spectrophotometry, when SOD inhibits the reduction of NBT [110].

An aliquot of 250 µL of the cell homogenate was mixed with 500 µL of homogenizing solution (phosphate buffer 0.1M, EDTA 60 mM and phenyl methyl sulfonyl fluoride PMSF); samples were centrifuged at 300 rpm, during 15 min at 4 °C. Supernatant was recovered and the precipitate was discarded. Working solution containing sodium-carbonate buffer (50 mM), xanthine (0.1 mM), NBT (0.025 mM), EDTA (0.1 mM,) xanthine oxidase (XO, 0.1 U mL⁻¹) and blank or sample were mixed. The absorbance was recorded at 560 nm every 30 s for 5 min (∆A560). SOD activity was expressed in Units mg⁻¹ of protein 10⁻⁴ cells.