Superoxide Dismutase Assay

Activity of SOD has been determined in two ways:

1. Inhibition by the enzyme of an O^2- dependent reaction.

2. Pulse radiolytic methods (Rigo et al. 1975) See: Beauchamp and Fridovich (1971); Misra and Fridovich (1972); Tyler (1975).

Method: 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)

Enzyme

Prepare stock solution at one mg/ml.

Procedure

Pipette into a series of tubes:

EDTA/Cyanide	0.2 ml
NBT	0.1 ml
Enzyme	*
Phosphate buffer	q.s. to 3.0 ml

Include several tubes with no enzyme as controls.

* A series of samples ranging from 0.1-10 micrograms is recommended. A tube containing approximately 100 micrograms will generally produce maximum inhibition. Place the tubes in a light box providing uniform light intensity. (A foil-lined box approximately 4' long X 8" X 6" with an internally mounted 40 W fluorescent bulb has been used successfully). Incubate the tubes for 5-8 minutes to achieve a standard temperature. At zero time and at timed intervals add 0.05 ml riboflavin. Incubate all tubes in the light box for 12 minutes and at timed intervals read A₅₆₀. Determine percent inhibition of NBT reduction. Plot percent inhibition versus amount of enzyme in test. Determine the amount of enzyme resulting in one half of maximum inhibition.

Calculation