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

  • Tien Canh Le
  • Mircea Alexandru Mateescu
  • Samaneh Ahmadifar
  • Lucia Marcocci
  • Paola Pietrangeli
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1853)

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.

Key words

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

Abbreviations

AO

Amine oxidases

APS

Ammonium persulfate

BSA

Bovine serum albumin

DAO

Diamine oxidase

GOx

Glucose oxidase

HRP

Horseradish peroxidase

ISA

Intrinsic specific activity

o-PDA

ortho-Phenylenediamine

PAA

Polyacrylamide

PAGE

Polyacrylamide gel electrophoresis

PBS

Phosphate buffer solution

SDS

Sodium dodecyl sulfate

TEMED

N,N,N′,N′-Tetramethylethylenediamine

Tris

Trihydroxymethylaminomethane

Notes

Acknowledgment

Financial support from NSERC (Natural Sciences and Engineering Research Council) of Canada (Discovery Program), from Fondation Courtois (Canada), from Regione Lazio of Italy (FILAS-RU-2014-1020), and from Joint Project Italia–Canada-Quebec (2017–2019), is gratefully acknowledged.

References

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Tien Canh Le
    • 1
  • Mircea Alexandru Mateescu
    • 1
  • Samaneh Ahmadifar
    • 1
  • Lucia Marcocci
    • 2
  • Paola Pietrangeli
    • 2
  1. 1.Department of Chemistry, Research Chair Allerdys and Centre Pharmaqam-BioMedUniversité du Québec à MontréalMontrealCanada
  2. 2.Department of Biochemical Sciences «A. Rossi-Fanelli»Sapienza University of RomeRomeItaly

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