Makrecka-Kuka 2015 Biomolecules
 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. |
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 red 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 Complex II-linked LEAK state, reduced with CI&II-linked convergent electron flow and in mitochondria respiring at OXPHOS capacity, and were further diminished in uncoupled mitochondria respiring at electron transfer system 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. • 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: Intact cells, Permeabilized cells, Homogenate, Isolated mitochondria
Regulation: Inhibitor Coupling state: LEAK, ROUTINE, OXPHOS, ETS"ETS" is not in the list (LEAK, ROUTINE, OXPHOS, ET) of allowed values for the "Coupling states" property. Pathway: N, S, NS, ROX HRR: Oxygraph-2k, O2k-Fluorometer, O2k-Protocol
Amplex Red, O2k-Demo, O2k-MultiSensor, MitoFit news
MitoFit news 2015#10
- 2015-07-01: Light in the powerhouse of the cells - SUIT protocols for measurement of respiration and H2O2 production. » MitoFit news
Reprints
- features as a reprint in Breitenbach 2016 Biomolecules
