Active Superoxide Dismutase Copper Chaperone

Superoxide dismutase copper chaperone (SOD4) is an enzyme that in humans is encoded by the CCS gene. This gene encodes a member of the superoxide dismutase (SOD) protein family. SODs are antioxidant enzymes that catalyze the dismutation of two superoxide radicals into hydrogen peroxide and oxygen. Acroding to the report, in a weakly alkaline buffer solution (pH=8.2) with N-tris(hydroxymethyl) amino methane-HCL, pyrogallol can occur autoxidation in the air, then SOD can inhibit this reaction. Thus, we use this way to measued the activity of recombinant rat SOD4. The reaction was performed in adding 8 μl 5 mmol/L pyrogallol to 200 μl 50mmol/L Tris-HCl, rapidly mixing at 25 ℃, then read at 325 nm (using 50mmol/L Tris-HCl as blank control) in kinetic mode for 3 minutes using a microplate reader controlling the pyrogallol autoxidation rate at 0.70 OD/min. Different concentrations of recombinant rat SOD4 were added into 200 μl 50 mmol/L Tris-HCl, incubated for 20 min at 25 ℃, then adding 8 μl 5 mmol/L pyrogallol to each well, rapidly mixing and read at 325 nm in kinetic mode for 3 minutes. Under these conditions, the enzyme amount of 50% inhibition of pyrogallol autooxidation per minute is defined as a unit. The specific activity of recombinant rat SOD4 is 74 U/mg.