title: Zymographic assay of oxidases using peroxidase or hemin entrapped in polyacrylamide gel.
abstract: 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.
authors.affiliation: Université du Québec à Montréal, Montréal, QC, Canada. mateescu.m-alexandru@uqam.ca
authors.forename: Mircea Alexandru
authors.initials: MA
authors.lastname: Mateescu
authors.forename: Carmen
authors.initials: C
authors.lastname: Calinescu
authors.forename: Tien Canh
authors.initials: TC
authors.lastname: Le
authors.forename: Rodolfo
authors.initials: R
authors.lastname: Federico
authors.forename: Bruno
authors.initials: B
authors.lastname: Mondovi
mesh_terms: D000180:Acrylic Resins / Q000737:chemistry; D002863:Chromogenic Compounds / Q000737:chemistry; D004396:Coloring Agents / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D057075:Enzyme Assays / Q000379:methods*; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000302:isolation & purification; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification; D006427:Hemin / Q000737:chemistry*; D006861:Hydrogen Peroxide / Q000737:chemistry; D057230:Limit of Detection; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification; D010655:Phenylenediamines / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification; D011487:Protein Conformation; D011700:Putrescine / Q000737:chemistry; D012394:Rosaniline Dyes / Q000737:chemistry; D013194:Staining and Labeling
chemical_list: D000180:Acrylic Resins; D002863:Chromogenic Compounds; D004396:Coloring Agents; D004800:Enzymes, Immobilized; D005656:Fungal Proteins; D010655:Phenylenediamines; D010940:Plant Proteins; D012394:Rosaniline Dyes; C016680:polyacrylamide gels; D006427:Hemin; C048139:Coomassie blue; C034193:1,2-diaminobenzene; D006861:Hydrogen Peroxide; D010088:Oxidoreductases; D011700:Putrescine