2.4.2. Assay of Superoxide Dismutase (SOD)

The activity of superoxide dismutase (SOD) was determined using the method of Kakkar et al. [30]. The initial supernatant prepared was purified by precipitating the protein using 90% ammonium sulphate before an assay of enzyme activities. The fraction was then dialysed against 0.0025 M tris-HCl buffer (pH 7.4) and the supernatant was used as the source of enzyme. The content of the assay solution was made up of 1.2 mL of sodium pyrophosphate buffer, 0.1 mL of phenazine methosulphate (PMS), 0.3 mL of nitro-blue tetrazolium (NBT), 1.3 mL of distilled water and 0.1 mL of the enzyme source. The supernatant was kept inside the vials at 30 ◦C for 90 s and the reaction was stopped by adding 1 mL of glacial acetic acid. 4.0 mL of n-butanol was added to the reacting mixture and mixed properly, then allow to stand for 10 min and the upper portion of the butanol layer was decanted. Absorbance was measured at 560 nm against a blank of n-butanol. A system that lacks enzyme was used as the control and one unit of activities of the enzyme was define as its concentration needed to inhibit 50% production of chromogen per minute. Specific activity was expressed as Ug−<sup>1</sup> protein. 1 IU = change in absorbance/min/extinction coefficient (0.021).
