Cell Biochemistry and Biophysics

, Volume 75, Issue 3–4, pp 335–349 | Cite as

Recent Developments in the Probes and Assays for Measurement of the Activity of NADPH Oxidases

  • Jacek Zielonka
  • Micael Hardy
  • Radosław Michalski
  • Adam Sikora
  • Monika Zielonka
  • Gang Cheng
  • Olivier Ouari
  • Radosław Podsiadły
  • Balaraman Kalyanaraman
Original Paper


NADPH oxidases are a family of enzymes capable of transferring electrons from NADPH to molecular oxygen. A major function of NADPH oxidases is the activation of molecular oxygen into reactive oxygen species. Increased activity of NADPH oxidases has been implicated in various pathologies, including cardiovascular disease, neurological dysfunction, and cancer. Thus, NADPH oxidases have been identified as a viable target for the development of novel therapeutics exhibiting inhibitory effects on NADPH oxidases. Here, we describe the development of new assays for measuring the activity of NADPH oxidases enabling the high-throughput screening for NADPH oxidase inhibitors.


NADPH oxidase Reactive oxygen species Fluorescent probes High-throughput screening HPLC 











coumarin-7-boronic acod





cyt c3+

ferricytochrome c














electron paramagnetic resonance




hydroethidine (or dihydroethidium)


HE radical cation


high-performance liquid chromatography




horseradish peroxidase


high-throughput screening






nicotinamide adenine dinucleotide phosphate, reduced form


nitroblue tetrazolium


NADPH oxidase-2


NADPH oxidase-4


NADPH oxidase-5


oxygen consumption rate


phorbol 12-myristate 13-acetate


superoxide dismutase


1,3-benzoxazol-2-yl-3-benzyl-3H-[1–3]triazolo[4,5-d]pyrimidin-7-yl sulfide



This work was supported by a grant from NIH (R01 AA022986) to B.K. and from the French National Research Agency (ANR-16-CE07-0023-01) to O.O. and M.H. A.S. and R.M. were supported by a grant from Polish National Science Centre, No. 2015/18/E/ST4/00235.

Compliance with Ethical Standards

Conflict of Interests

The authors declare that they have no competing interests.


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Copyright information

© Springer Science+Business Media, LLC 2017

Authors and Affiliations

  1. 1.Department of Biophysics and Free Radical Research CenterMedical College of WisconsinMilwaukeeUSA
  2. 2.Aix Marseille Univ, CNRS, ICRMarseilleFrance
  3. 3.Institute of Applied Radiation Chemistry, Faculty of ChemistryLodz University of TechnologyLodzPoland
  4. 4.Institute of Polymer and Dye Technology, Faculty of ChemistryLodz University of TechnologyLodzPoland

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