Superoxide dismutase micro assay in biological material.

A micro assay for the rapid and convenient determination of superoxide dismutase activity in limited amounts of biological material was devised and successfully employed. The combination of the formazan derivative colour formation induced by reaction of O2theta with nitroblue tetrazolium and a suitable analytical polyacrylamide gel electrophoresis system was used. It was possible to show that the reactivity of soluble superoxide dismutases in polyacrylamide gels was proportional to the enzyme concentrations employed. Bovine erythrocyte Cu, Zn-superoxide dismutase (EC 1.15.1.1) (erythrocuprein) served as a standard throughout. To measure the degree of superoxide dismutase activity, a gel-scanning apparatus was usedThe integrated scanning curve of the unstained portions of the gel proved linearly proportional to the logarithm of the superoxide dismutase activity in the range between 10(-12) and 7 X 10(-11) mol of the bovine enzyme. Although the absolute integral is dependent on the different staining conditions, the slope of the superoxide dismutase calibration curve is highly reproducible. Superoxide dismutase added to crude liver and brain homogenates could be fully detected using this assay. Thus, biological material including nucleic acids, enzymes, lipids etc. do not inhibit this reaction.