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Difference between revisions of "MiPNet06.06 Chemical O2 background"

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{{Template:OROBOROS support page name}}
{{Publication
{{Publication
|title=Kuznetsov AV, Gnaiger E (2001) Oxygraph assay of cytochrome c oxidase activity: Chemical background correction. Mitochondr Physiol Network 06.06.
|title=[[Image:O2k-Manual.jpg|right|70px|link=O2k-Manual|O2k-Manual]] Oxygraph assay of cytochrome ''c'' oxidase activity: chemical background correction.
|info=[[Media:MiPNet06.06 ChemicalBackground.pdf|'''Open Access-MiPNet06.06''']]; [http://www.bioblast.at/index.php/File:MiPNet06.06_ChemicalBackground.pdf Versions] [[Image:O2k-Protocols.jpg|right|150px|link=http://www.oroboros.at/?o2k-protocols|O2k-Protocols contents]]
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/1/1b/MiPNet06.06_ChemicalO2Background.pdf |Bioblast pdf]] Β» [http://www.bioblast.at/index.php/File:MiPNet06.06_ChemicalO2Background.pdf Versions]
|authors=OROBOROS
|authors=Oroboros
|year=2001
|year=2015-04-27
|journal=Mitochondr Physiol Network
|journal=Mitochondr Physiol Network
|abstract=Autoxidation of reduced compounds (such as ascorbate, TMPD, cytochrome c) causes a chemical background oxygen flux, which is a function of oxygen concentration and has to be subtracted fom total oxygen flux. The new DatLab software provides on-line correction for instrumental and chemical background.
|abstract=Kuznetsov AV, Gnaiger E (2010) Oxygraph assay of cytochrome ''c'' oxidase activity: chemical background correction. Mitochondr Physiol Network 06.06(07):1-4.
:Β» Product: O2k-Catalogue: [[O2k-MultiSensor]], [[O2k-Core]], [[O2k-Catalogue]]


FIGURES HERE (2figs)!!
|mipnetlab=AT_Innsbruck_Oroboros
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'''O2k-Protocol for chemical background correction '''
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Oxygraphic determination of cytochrome c oxidase activity in the presence of TMPD, ascorbate and cytochrome c requires consideration of chemical background oxygen consumption. Several compounds are readily oxidized by molecular oxygen when dissolved in water. This leads to a significant chemical oxygen consumption in the absence of any respiring biological sample. The rate of this autoxidation represents a chemical background which strongly depends on experimental conditions, such as temperature, chemical composition, pH and oxygen concentration. Moreover, the rate of autoxidation may be significantly catalyzed by metal traces and metal-containing proteins (e.g. cytochrome c). Therefore, high-resolution respirometric measurement of oxygen flux ultimately depends on chemical background correction analyzed under experimental conditions over the entire oxygen range.
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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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:>> O2k-Protocols:[http://www.oroboros.at/?o2k-protocols Overall contents]
:>> Product: [http://www.oroboros.at/?oxygraph OROBOROS Oxygraph-2k], [[O2k-Catalogue_OROBOROS| O2k-Catalogue]]
|mipnetlab=AT_Innsbruck_OROBOROS
}}
}}
{{Labeling
{{Labeling
|area=Instruments;methods
|area=Instruments;methods
|preparations=Enzyme, Oxidase; Biochemical Oxidation
|preparations=Enzyme, Oxidase;biochemical oxidation
|instruments=Oxygraph-2k, Protocol
|enzymes=Complex IV;cytochrome c oxidase
|instruments=Oxygraph-2k, O2k-Manual
|additional=O2k-SOP
|additional=O2k-SOP
}}
}}
== Further information ==
'''References'''
[[Renner_2002_Mol_Biol_Rep| Renner K, Kofler R, Gnaiger E (2002) Mitochondrial function in glucocorticoid triggered T-ALL cells with transgenic Bcl-2 expression. Mol Biol Rep 29: 97-101.]]
:>> [http://www.oroboros.at/?chemical_background MiPNet06.06]
* Version 7: 2010-08-23
* Version 1: 2001
[[Category:OroboPedia]]

Latest revision as of 21:19, 13 February 2020


                  


O2k-Open Support

MiPNet06.06 Chemical O2 background


Publications in the MiPMap
O2k-Manual
Oxygraph assay of cytochrome c oxidase activity: chemical background correction.

Β» Bioblast pdf Β» Versions

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.

Β» Product: O2k-Catalogue: O2k-MultiSensor, O2k-Core, O2k-Catalogue


β€’ O2k-Network Lab: AT_Innsbruck_Oroboros


Labels: MiParea: Instruments;methods 



Preparation: Enzyme, Oxidase;biochemical oxidation  Enzyme: Complex IV;cytochrome c oxidase 


HRR: Oxygraph-2k, O2k-Manual 

O2k-SOP