SUIT-026 AmR mt D064

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SUIT-026 AmR mt D064

Description

SUIT-026 AmR mt D064.png

Abbreviation: RET

Reference: A - SUIT-026

SUIT number: D064_1S;2Rot;3D;4Ama

O2k-Application: AmR

MitoPedia: SUIT - Protocol for the evaluation of ROS production by RET in mtprep
SUIT protocol pattern: 1S;2Rot;3D;4Ama

SUIT-026 AmR mt D064 has been designed to study the RET-initiated ROS production in mitochondrial preparations (isolated mitochondria and tissue homogenate and permeabilized cells which are already permeabilized when they are added to the chamber). The protocol is focused on LEAK state for the S-pathway to provide the conditions of high membrane potential and redox power in the Q-junction. Under these conditions, in several samples has been described that a reverse flow of the electrons into the membrane arm and the quinone binding site of the Complex I occurs, promoting the production of ROS. The addition of rotenone provides excellent control to this mechanism because this compound blocks the point on which the electrons leak from the Complex I leading to decreased ROS production. If rotenone would have been added before S titration, it had inhibited the RET. The titration of ADP at saturating concentrations to reach OXPHOS allows us to harmonize this protocol with SUIT-006 O2 mt D022 for quality control and a full assessment of the coupling control. Addition of Ama leads to increased ROS production via inhibition of Complex III at the Qi site.

According to our results, the H2O2 production is linearly dependent on the O2 concentration in MiR05-Kit, therefore, during the measurements the O2 concentration has to be well-defined. In this protocol, we suggest not to go under ~100 µM O2. SUIT-018 AmR mt D041 has been designed to study O2 dependence of H2O2 flux in mitochondrial preparations.

Communicated by Komlodi T, Iglesias-Gonzalez J and Gnaiger E (last update 2019-07-05)
MitoPedia: SUIT

Steps and respiratory states

SUIT-026 AmR mt D064.png


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
Step State Pathway Q-junction Comment - Events (E) and Marks (M)
1S SL(n) S CII 1S
2Rot SL(n) S CII 1S;2Rot
3D SP S CII 1S;2Rot;3D
4Ama ROX 1S;2Rot;3D;4Ama
  • Rox is the residual oxygen consumption in the ROX state, due to oxidative side reactions, estimated after addition of Antimycin A (inhibitor of CIII). Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration (mt).



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Strengths and limitations

  • The conditions on which RET is observable in isolated mitochondria are not physiological but it has been suggested that corresponds to some pathological states.
  • This protocol must be run in parallel with SUIT-026 O2 mt D063 to have a respiratory control, since many fluorescence dyes can inhibit components of the ET system, thus affecting the respiration.
+ Short duration of the experiment.
+ Rotenone provides an excellent control step for RET.
- This protocol does not provide information about all the coupling control states (LEAK, OXPHOS and ET). However, it is possible to create a DLPU by additing an Event&Mark for the uncoupler titration (4U) after ADP (3D) to obtain ET-state.
- The addition of cytochrome c cannot be done in this SUIT protocol (SUIT-026 O2 mt D063 must be run in parallel to assess the mt outer membrane integrity as a quality control of the sample).
- Measurements with Amplex UltraRed assay cannot be carried out with liver homogenate.


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References

MitoPedia concepts: SUIT protocol, SUIT A, Find 


MitoPedia methods: Fluorometry