MiPNet06.06 Chemical O2 background
Oxygraph assay of cytochrome c oxidase activity: chemical background correction. |
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Oroboros (2015-04-27) Mitochondr Physiol Network
Abstract: Kuznetsov AV, Gnaiger E (2010) Oxygraph assay of cytochrome c oxidase activity: chemical background correction. Mitochondr Physiol Network 06.06(07):1-4.
After sequential titration of ascorbate (added first) and TMPD (with or without added cytochrome c), the total oxygen flux increases due to (i) COX activity and (ii) autooxidation of ascrobate and TMPD. Correction for autooxidation is a routine procedure in high-resolution respirometry, but it requires precautions. We recommend, therefore, evaluation of chemical background effects by application of inhibitors of cytochrome c oxidase (cyanide, azide) subsequent to recording of flux with ascorbate and TMPD. After full inhibitioin of COX, a small non-COX component of biological oxidations and the large effect of autooxidation are recorded over a range of further declining oxygen concentration. After re-oxygenation, inhibited oxygen flux is recorded at oxygen levels above and within the oxygen concentration pertaining to the COX activity measurment. This provides a complete internal calibration of the chemical background, as a function of oxygen concentration.
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Labels: MiParea: Instruments;methods
Preparation: Enzyme, Oxidase;biochemical oxidation
Enzyme: Complex IV;cytochrome c oxidase
HRR: Oxygraph-2k, O2k-Manual
O2k-SOP