Oxygen electrode (oxygraph) recordings

If mitochondria are incubated in an oxygraph apparatus (oxygen electrode) in an isotonic medium containing substrate and phosphate, then addition of ADP causes a sudden burst of oxygen uptake as the ADP is converted into ATP:

The actively respiring state is sometimes refered to as "state 3" respiration, while the slower rate after all the ADP has been phosphorylated to form ATP is refered to as "state 4".

State 4 respiration is usually faster than the original rate before the first addition of ADP because some ATP is broken down by ATPase activities contaminating the preparation, and the resulting ADP is then re-phosphorylated by the intact mitochondria.

The ratio [state 3 rate] : [state 4 rate] is called the respiratory control index and indicates the tightness of the coupling between respiration and phosphorylation. With isolated mitochondria the coupling is not perfect, probably as a result of mechanical damage during the isolation procedure. Typical RCI values range from 3 to 10, varying with the substrate and the quality of the preparation. Coupling is thought to be better in vivo, but may still not achieve 100%.

It is possible to calculate a P:O ratio (the relationship between ATP synthesis and oxygen consumption) by measuring the decrease in oxygen concentration during the rapid burst of state 3 respiration after adding a known amount of ADP. It is necessary to subtract the basal respiration due to imperfect coupling and the recycling of ATP, as shown in the graph above. The change in concentration must then be multiplied by the chamber volume, so that the answer (in micro-atoms of oxygen) can be related to the quantity of ADP added. The quantity of oxygen in the chamber is calculated from published oxygen solubility data at the appropriate temperature. Using the figures from the graph above:

decrease in O2 (from graph) = 0.135 mM; chamber volume = 2.5ml

oxygen atoms consumed in state 3 = 0.135 * 2 * 2.5 = 0.68 micro-atoms (note the factor of 2, allowing for 2 atoms in an oxygen molecule)

injected ADP (20 microlitres of a 50mM solution) = ATP formed = 1 micromole in total

P:O = [ATP formed] : [oxygen consumed] = 1.48 (for succinate oxidation)

NAD-linked substrates give consistently higher values for the P:O ratio (about 2.5) compared with succinate (about 1.5). These results indicate that electrons from relatively poor reducing agents such as succinate (also acyl CoA and glycerol phosphate) enter part of the way along the respiratory chain, by-passing the first coupling site where energy is captured for ATP synthesis.

The sharp changes in slope after exhaustion of ADP and again after all the oxygen has been used up imply that mitochondria must have very high affinities for ADP and oxygen. The concentration of both these compounds is very low in most healthy cells, since they are efficiently scavenged by the mitochondria.

The addition of an uncoupling agent (such as dinitrophenol or CCCP) leads to a permanently high rate of respiration in the absence of ADP, until all the oxygen has been consumed.

Many natural and synthetic poisons block mitochondrial respiration. If mitochondria are incubated in an oxygraph experiment with substrates and inorganic phosphate, the interactions between inhibitors and uncouplers allow two major types of inhibition to be distinguished:

Mitochondrial respiration rates
additions / inhibitors none oligomycin cyanide
none low low zero
ADP high low zero
uncoupling agent high high zero

The conclusion to be drawn from this type of experiment is that cyanide prevents respiration by blocking the respiratory chain itself, so cyanide is effective whether or not ADP or uncoupler are added. Oligomycin on the other hand inhibits the ADP phosphorylation system, so it only blocks respiration in coupled mitochondria.

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