Superoxide Dismutase Assay
Method
Activity of SOD has been determined in two ways:
1. Inhibition by the enzyme of an O2- dependent reaction.
2. Pulse radiolytic methods (Rigo et al. 1975) See: Beauchamp and Fridovich (1971); Misra and Fridovich (1972); Tyler (1975).
The method employed at Worthington is essentially that of Winterbourn et al. (1975) and is based on the ability of superoxide dismutase to inhibit the reduction of nitro-blue tetrazolium by superoxide. One unit is defined as that amount of enzyme causing half the maximum inhibition of NBT reduction. The reaction velocity will depend largely on somewhat variable assay conditions such as light intensity and reaction temperature. Calibration of the method in individual laboratories is recommended.
Reagents
- 0.067 M Potassium phosphate buffer, pH 7.8
- 0.1 M Ethylene diamine tetraacetic acid (EDTA) containing 0.3 mM sodium cyanide
- 0.12 mM Riboflavin (store cold in a dark bottle)
- 1.5 mM Nitroblue tetrazolium (NBT) (store cold)
Cyanide is essential for inhibiting the respiratory process of mitochondria. Without the presence of cyanide, the data obtained would not specifically originate from Superoxide Dismutase (SOD), but rather from the enzymatic activities within the mitochondria themselves, which could potentially interact with Nitroblue Tetrazolium (NBT). The introduction of cyanide serves to redirect the reaction pathway from occurring within the mitochondria to being catalyzed by Superoxide Dismutase (SOD).
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