SOD Activity

SOD Activity

Fresh leaves (0.2 g) were homogenized with mortar and pestle using 100 mM potassium phosphate buffer (pH 7.0) containing 1% polyvinylpyrrolidone (PVP) (w/v) and 0.05% Triton X-100 (v/v). Later the centrifugation of homogenized material was done at 15,000× g for 20 min. The supernatant generated after centrifugation was utilized to assay the activity superoxide dismutase (SOD).

Activity of SOD was determined as per the protocol of Beyer and Fridovich, and Giannopolitis and Ries by monitoring the inhibition of photochemical reduction of nitro blue tetrazolium (NBT). Reaction mixture (5.0 mL) consisted of 50 mM Na₂CO₃ (pH 10.0), 5.0 mM HEPES (pH 7.6), 0.1 mM EDTA, 0.025% (v/v) Triton X-100, 13 mM methionine, 63 mM NBT and 1.3 mM of riboflavin. The extract of enzyme was illuminated for 15 min (360 μmol m² s⁻¹), and a set without illumination acted as a control to correct the turbidity of background absorbance. A unit of enzyme was expressed as the amount of the enzyme that inhibited the NBT reduction by 50% at 560 nm. The amount of the enzyme that inhibited the reduction of NBT by 50% at 560 nm was equal to one unit of SOD (van Rossum and van der Plas, 1997).

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