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.
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.
• Keywords: High-resolution respirometry, H2O2 flux, Amplex Red, HEK 293T, Mouse brain homogenate, Mouse cardiac mitochondria
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, ET Pathway: N, S, NS, ROX HRR: Oxygraph-2k, O2k-Fluorometer, O2k-Protocol
O2k-Demo, O2k-MultiSensor, Amplex UltraRed, 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