Superoxide Dismutase Assays

Abstract : A number of studies support the idea that superoxide dismutase is one of the enzymes that modulates the threat of oxidative stress. This technical report presents detailed instructions for two methods presently being used at the Naval Submarine Medical Research Laboratory (NSMRL) to assay for superoxide dismutase. Both assays are of the indirect type consisting of two components: a superoxide generator and a superoxide detector. In the first method the generator produces the radical at a controlled rate, and in the absence of superoxide dismutase (SOD), the radical produced reacts with the detector. A unit of SOD then hs been defined as that amount which will reduce the rate produced by the generator to 50% of its control value. The second method, which is about 100-fold more sensitive, takes advantage of the biphasic nature of the production of the radical and its dismutation both spontaneously and by SOD. By allowing the generator to react for a specific time period before addition of the detector, a burst of reduction is obtained followed by linear rate after the detector reaches a steady state. The height of this burst in the presence and absence of SOD thereby gives a sensitive assay for which the unit (50% of control burst) is in the picomolar range. These assays are useful in determining SOD in a variety of tissue types such as erythrocytes obtained from Navy divers or cell culture samples that have been exposed to oxidative stress.