Step
|
State
|
Pathway
|
Q-junction
|
Comment - Events (E) and Marks (M)
|
1Q2
|
REN
|
|
|
1Q2
- To detect the redox state of the Q-pool a mimetic coenzyme Q2 is applied in suspension, which reacts both with the detecting electrode and the biological sites (CI, CII and CIII).
- Respiration in the REN state is due to the presence of residual endogenous substrates in the sample preparation.
- In the absence of endogenous substrates, this step can be used for calibration of fully oxidized Q.
|
1Rot
|
ROX
|
|
|
1Rot
- Rotenone is required to detect the fully oxidized Q-pool in the presence of isolated mitochondria (or permeabilized cells) and coenzyme Q2. Rotenone can inhibit the respiration supported by endogenous substrates that remained after the isolation procedure.
|
1S
|
SL
|
S
|
CII
|
1Q2;1Rot;1S
|
2D
|
SP
|
S
|
CII
|
1Q2;1Rot;1S;2D
|
(3Omy)
|
SL(Omy)
|
S
|
CII
|
1Q2;1Rot;1S;2D;(3Omy)
- Omy addition is skipped in SUIT-006 O2 mt D022
- Succinate, S ( type S-pathway to Q).
- Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin.
|
4U
|
SE
|
S
|
CII
|
1Q2;1Rot;1S;2D;(3Omy);4U
|
5Anox
|
|
S
|
CII
|
1Q2;1Rot;1S;2D;(3Omy);4U;5Anox
- Anoxia is a crucial step to detect the fully reduced Q-junction in the presence of coenzyme Q2 after the biological sample has consumed the O2 in the O2k-chamber for calculation of the Q redox state.
|
6Ama
|
ROX
|
|
|
1Q2;1Rot;1S;2D;(3Omy);4U;5Anox;6Ama
- 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).
- Since the previous step was anoxia, it is essential to reoxygenate the chamber before or after antimycin A titration to be able to measure Rox.
|