Assays for superoxide dismutase based on autoxidation of hematoxylin

This chapter discusses the assays for superoxide dismutase (SOD) based on the autoxidation of hematoxylin. The assay is based on the transformation of hematoxylin to its two-electron-oxidized product, hematein. The assay is sensitive and simple. The reaction product, hemarein, is relatively stable and has an absorption maximum and extinction coefficient similar to those of the commonly used O2– scavenger, cytochrome c. The reaction is inhibited by SOD and can be performed in the physiological pH range of 6.8–7.8, where SOD inhibits the reaction 90–95%. In addition, at pH values above 8.1, the autoxidation reaction is accelerated by added SOD, and the reaction becomes a positive assay for the enzyme, similar in principle to SOD assays based on the photochemical and enzymatic oxidation of dianisidine. The activity of purified SOD is determined by the xanthine oxidase cytochrome c method. Xanthine oxidase is isolated from unpasteurized cream. General protein is determined by the Bradford assay using bovine serum albumin (BSA) as a standard.