Zymographic Determination of Intrinsic Specific Activity of Oxidases in the Presence of Interfering Proteins

Methods Mol Biol. 2018:1853:207-221. doi: 10.1007/978-1-4939-8745-0_24.

Abstract

Zymography on peroxidase-entrapped gels enables the determination of the intrinsic specific activity (ISA) of H2O2-producing oxidases in the presence of interfering enzymes including catalase (decomposing hydrogen peroxide). To reveal the searched oxidase, the zymography gel is incubated first in the developing solution containing the appropriate substrate of the oxidase and an oxidizable chromogen such as o-phenylenediamine (o-PDA) peroxidase substrate. The zymographic gel, after image analysis, can be restained by Coomassie Blue for molecular weights, for the whole electrophoretic pattern and for the protein concentration, thus allowing for the determination of oxidase ISA.

Keywords: Amine oxidase; Bovine serum albumin; Catalase; Coomassie Blue; Entrapped peroxidase; Interfering enzymes; Intrinsic specific activity; Oxidase activity; Polyacrylamide gel electrophoresis (PAGE); Zymography.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Catalase / analysis
  • Catalase / chemistry
  • Cattle
  • Electrophoresis, Polyacrylamide Gel*
  • Enzyme Activation
  • Hydrogen Peroxide / chemistry
  • Oxidoreductases* / analysis
  • Oxidoreductases* / chemistry
  • Proteins* / analysis
  • Proteins* / chemistry
  • Rosaniline Dyes
  • Spectrophotometry / methods
  • Staining and Labeling
  • Substrate Specificity

Substances

  • Proteins
  • Rosaniline Dyes
  • Coomassie blue
  • Hydrogen Peroxide
  • Oxidoreductases
  • Catalase