SUIT-013

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SUIT-013

Description

Abbreviation: ce

Reference: A  »Versions

SUIT-category: ce

SUIT protocol pattern: linear

This protocol is ideal to study the oxygen dependence of H₂O₂ production in intact cells. SUIT-013 AmR ce D023 is ideal for demonstration of the oxygen dependence in baker´s yeast. For more details see: MiPNet18.06 Amplex-Yeast The respiration medium has to be choosen carefully, since the Amplex UltraRed assay leads to artefact formation with the components of the buffer.

Communicated by  Komlodi T  (last update 2019-01-22)

DLP applications

• SUIT-013 AmR ce D023
• SUIT-013 AmR pce D###??
• SUIT-013 AmR mt D###??

References

Steps and respiratory states

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
0DTPA				DTPA is an iron chelator, which decreases the chemical fluorescence background created by the Amplex UltraRed assay. Administration of DTPA into the O2k-chamber is recommended before all other chemicals because the iron chelation capacity of the compound is time-dependent (approx. 10-15 min). However, the experiments can be carried out in the absence of DTPA.
0SOD				SOD or superoxide dismutase converts the anion superoxide released by the mitochondria into H2O2, making it accessible to the Amplex UltraRed assay.
0HRP				HRP or horseradish peroxidase catalyses the conversion of Amplex UltraRed and H2O2 towards the fluorescent resorufin.
0AmR				AmR or Amplex UltraRed forms resorufin with H2O2 catalysed by horseradish peroxidase HRP.

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
ce1	ROUTINE			ce1 ROUTINE respiration in the physiological coupling state R. Externally added permeable substrates could contribute to this respiratory state.

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Coupling control

» Coupling control state

» ET capacity

» OXPHOS capacity

» LEAK respiration

Pathway control

» Electron transfer pathway

» Fatty acid oxidation pathway control state, F

» NADH electron transfer-pathway state, N

» Succinate pathway control state, S

» NS-pathway control state, NS

» Glycerophosphate pathway control state, Gp

» Complex IV single step, CIV

» Anaplerotic pathway control state

Main fuel substrates

» Glutamate, G

» Glycerophosphate, Gp

» Malate, M

» Octanoylcarnitine, Oct

» Pyruvate, P

» Succinate, S

Glossary

» List of SUIT states

» SUIT concept

Strengths and limitations

+ Reasonable duration of the experiment.

+ Quick demonstration of the oxygen dependence of H₂O₂ production.

+ No externally added substrates are required with yeast.

Compare SUIT protocols

MitoPedia concepts: MiP concept, SUIT protocol, Recommended 

MitoPedia methods: Respirometry, Fluorometry