Modified superoxide dismutase assay by pyrogallol auto-oxidation A modified superoxide dismutase assay protocol is shown in Table 2.2.

In order to calculate the enzyme activity SOD assay was performed with the enzyme sample. A 3ml reaction contained 50mM Tris-HCl buffer (pH 8.2), 1mM EDTA, 100l enzyme, 0.4mM pyrogallol and appropriate amount of distilled water. All reagents except pyrogallol was added into the blank and sample cuvettes (quartz) and mixed properly. The spectrophotometer was set to the time scan mode at 420nm. The scan time was set as two minutes. After keeping the cuvetts in the cuvette holder the absorbance was adjusted to zero by using auto zero function. Pyrogallol solution (200 l) was mixed in to the sample cuvette and the absorbance was recorded continuously for two minutes. Rate of the reaction (slope) was plotted using Microsoft Excel. Temperature of the room was maintained at 302oC throughout the entire study. Reagent Preparations: 1. Pyrogallol was dissolved in 0.01N HCl solution (in order to prevent autooxidation) and prepared freshly for each assay. 2. EDTA is difficult to soluble in water, so the pH of the solution was increased more than eight (using NaOH) in order to solubilise these salts. One unit of SOD activity was defined as the amount of enzyme that reduces the rate of autooxidation of pyrogallol by 50% at standard assay conditions. SOD activity can be calculated by the equation (Ma et al., 2009);

Where; Vp = Auto-oxidation rate of pyrogallol (control) Vs = Auto-oxidation rate of samples (with enzyme) Vt = Total reaction volume (ml) Vs = Volume of enzyme used for the assay (ml) n = Dilution fold of the SOD sample 0.5 = factor for 50% inhibition

Where Io = Rate of pyrogallol auto-oxidation without enzyme I = Rate of pyrogallol auto-oxidation with enzyme

Example; Table 2.2. Modified SOD assay protocol

The pyrogallol auto-oxidation was inhibited due to the scavenging of superoxide radicals (which is generated in pyrogallol auto-oxidation) by superoxide dismutase (SOD) as shown in Figure 2.6. The rate of auto-oxidation of the control reaction was 0.07592 Abs/Min and that of inhibited reaction (which contain enzyme) was 0.01836 Abs/Min. So the SOD activity can be calculated as follows;