Makrecka-Kuka 2015 Biomolecules

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Makrecka-Kuka M, Krumschnabel G, Gnaiger E (2015) High-resolution respirometry for simultaneous measurement of oxygen and hydrogen peroxide fluxes in permeabilized cells, tissue homogenate and isolated mitochondria. Biomolecules 5:1319-38.

» PMID: 26131977 Open Access

O2k-Protocols contents

Makrecka-Kuka M, Krumschnabel G, Gnaiger E (2015) Biomolecules

Abstract: Whereas mitochondria are well established as the source of ATP in the process of oxidative phosphorylation (OXPHOS), it is debated if they are also the major sources of reactive oxygen species (ROS). Here we describe the novel approach of combining high-resolution respirometry and fluorometric measurement of hydrogen peroxide (H2O2) production, applied to mitochondrial preparations (permeabilized cells, tissue homogenate, isolated mitochondria). The widely used H2O2 probe Amplex Ultrared inhibited respiration in intact and permeabilized cells and should not be applied at concentrations above 10 µM. H2O2 fluxes were generally less than 1% of oxygen fluxes in physiological substrate and coupling states, specifically in permeabilized cells. H2O2 flux was consistently highest in the succinate-linked LEAK state, reduced with NS-linked convergent electron flow and in mitochondria respiring at OXPHOS capacity, and were further diminished in uncoupled mitochondria respiring at electron transfer-pathway capacity. Simultaneous measurement of mitochondrial respiration and H2O2 flux requires careful optimization of assay conditions and reveals information in functional OXPHOS analysis beyond these assays carried out separately.

Terminology and abbreviations edited according to Gnaiger 2019 MitoFit Preprint Arch (Gnaiger E).

Keywords: High-resolution respirometry, H2O2 flux, Amplex Red, HEK 293T, Mouse brain homogenate, Mouse cardiac mitochondria

O2k-Network Lab: AT Innsbruck Oroboros, AT Innsbruck Gnaiger E, LV Riga Makrecka-Kuka M


Labels: MiParea: Respiration, Instruments;methods 

Stress:Oxidative stress;RONS  Organism: Human, Mouse  Tissue;cell: Heart, Nervous system, HEK  Preparation: Permeabilized cells, Homogenate, Isolated mitochondria, Intact cells 

Regulation: Inhibitor  Coupling state: LEAK, ROUTINE, OXPHOS, ET  Pathway: N, S, NS, ROX  HRR: Oxygraph-2k, O2k-Fluorometer, O2k-Protocol 

O2k-Demo, O2k-MultiSensor, AmR, SUIT-009, SUIT-009 AmR mt D021, SUIT-009 AmR pce D019, SUIT-013, SUIT-013 AmR ce D023, SUIT-018, SUIT-018 AmR mt D031, SUIT-006 AmR mt D048, SUIT-003 AmR ce D017, SUIT-003 AmR ce D058, MitoFit 2021 AmR, MitoFit 2021 MgG, MitoFit 2021 Yeast 

MitoFit

MitoFit news 2015#10

Cited by

  • Cardoso et al (2021) Magnesium Green for fluorometric measurement of ATP production does not interfere with mitochondrial respiration. MitoFit Preprints 2021. doi:10.26124/mitofit:2021-0001
  • Sobotka et al (2021) Facts and artefacts on the oxygen dependence of hydrogen peroxide production using Amplex UltraRed in yeast cells. MitoFit Preprints 2021.