Zymographic Assay of Oxidases Using Peroxidase or Hemin Entrapped in Polyacrylamide Gel

This chapter describes a zymographic assay of oxidases which is based on a coupled peroxidase or hemin reaction. The enzymatic activity of oxidases (i.e., diamine oxidase/DAO, glucose oxidase, galactose oxidase) can be directly monitored on polyacrylamide gels containing horseradish peroxidase or hemin, in the presence of their specific substrates and ortho-phenylenediamine (OPDA), an oxidizable chromogen. In the presence of hydrogen peroxide, OPDA is oxidized to azo-aniline, which led to well-defined yellow-brown bands on gels, with intensities corresponding to the enzymatic activity of oxidases.

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