Recommended Experiments with Isolated Mitochondria

In our teaching lab we encourage students to work with each other and to share insight, experience, and even experimental results. To facilitate such cooperation we have students work in teams of two, with two teams working together on the dissection and liver preparation and in conducting the experiments. Each polarographic station includes two chambers and recording systems. We assign one team to each chamber and the two teams start by conducting the same experiment. If one experiment fails, chances are the other will succeed and both teams can move on to the second experiment. If both experiments succeed the teams may use the better of the two records for data analysis.

Teams share respiratory medium and reagents and are expected to pay attention to what is going on in both chambers. Working this way should increase the chances of working out issues with the experimental methods. The philosophy is that four heads are better than two. It is also a lot more efficient for an instructor or teaching assistant to advise or assist a team of four than one or two individuals at a time. To begin a series of experiments with isolated mitochondria, the oxygraph system should be calibrated with the same respiration medium that will be used throughout the study. Our medium of choice consists of 70 mM sucrose, 220 mM mannitol, 2 mM HEPES buffer, 5 mM magnesium chloride, 5 mM potassium phosphate, 1 mM EDTA, and 0.1% fatty acid free bovine serum albumin, pH 7.4.

Unless noted, substrates and ADP should be added in 20 µl volumes and 10 µl volumes used for poisons. The optimum volume of mitochondria varies from one substrate to the next and with the quality of the preparation. To compare rates among experiments you will need to normalize for the volume of mitochondria added. Therefore you must be careful to draw up, deliver, and record the intended volume.

It is critical that the systems be calibrated and experiments conducted according to the principles described for calibration and use of our polarographic system for dissolved oxygen measurement. Following each addition of reagent or combination of reagents, record for a minute or two before adding the next reagent, so that a slope can be measured from the chart record. When you produce state III respiration you should obtain two slopes.

Required experiments

Experiments 1-4 have been the most reliable of the studies outlined here. They were designed to be conducted in sequence. Knowledge/experience gained from each experiment is applicable to conducting the next experiment. Results from experiments 1-4 will provide the basis for the research paper. After completing each experiment you are welcome (encouraged!) to try other reagents before cleaning out the chamber. If you plan ahead you may be able to address additional questions rather than simply try reagents randomly.

The pH of all aqueous reagents was adjusted to 7.

1.  Respiratory control on succinate and inhibition of electron transport

After calibrating the system add 15-25 µl mitochondria using a yellow tip pipettor, glass stopper removed, and triturate to suspend mitochondria without introducing air.  Replace the stopper and record for a minute or two.  Start each oxygraph run this way, varying only the amount of mitochondria added. Now, there cannot be respiration without a substrate, so if there is a continuing decline in chamber oxygen then something is providing fuel.  Is the rate of delivery of electrons from that fuel efficient enough to maintain a chemiosmotic gradient?  How would you check to see if a gradient is present?

To produce state IV respiration, add 20 µl 0.5M succinate using a Hamilton syringe, by injection through the hole in the glass stopper.  For each such injection make sure that the tip of the needle is well into the chamber (usually as far as it can go) and firmly push the liquid into the chamber.  Injecting slowly may result in poor mixing of reagent and yield equivocal results. It should take no more than a minute or two to obtain a measureable slope.

Produce state III respiration by adding 20 µl 0.01M ADP.  State III respiration on a limiting amount of ADP is transient. After the first slope change there should be a second steady state. The second slope change may not be as obvious as the first - view down the trace or use a straightedge to check. If you do not see an increase in oxygen consumption within 10-20 sec of adding ADP or do not see a definite reduction in oxygen consumption following state III respiration, then something is not right. For your paper you will need to calculate an ADP:O ratio on succinate.

Add 10 µl 30 µM antimycin.  There will be an initial rise in oxygen content because the vehicle (ethanol) raises the oxygen content in the solution.  Wait for a steady state. Add 10 µl each 0.5M ascorbate and 20 µl 30 mM TMPD in immediate succession (treat them as a single reagent).  Ascorbic acid maintains TMPD (an artifical electron donor) in a reduced state.  Reduced TMPD donates electrons to cytochrome c. 

Wait for a steady state, record long enough to get a measurable slope, then stop the chart record.  Clean out the chamber and stopper, and prepare for a second run.

Steady states are reached within moments of addition of reagents.  What conclusion can you draw, then, about the time it takes for mitochondria to establish a chemiosmotic gradient?

2.  Respiratory control on glutamate and inhibition of electron transport

Add 30-40 µl mitochondria, obtain a steady state.  We use a larger volume than for succinate-supported respiration because oxygen consumption is slower via the NADH pathway (think about why). Add 20 µl 0.5M glutamate, obtain a steady state.  Oxygen consumption may be high enough before adding substrate that you won't see a measureable change. Adding glutamate, however, provides a limitless source of substrate. Whatever supports respiration before adding glutamate does not likely allow maintenance of a gradient.

Add 20 µl 0.01M ADP, obtain a steady state, wait for a second steady state as you did for succinate, and record long enough to be able to measure the slope. You will need an ADP:O ratio on glutamate for your paper. Add 10 µl 10 µM rotenone – there will be an ethanol artifact.  Wait a couple of minutes for a steady state.

Add ADP again.  Record for about a minute.   You know that steady states are reached within moments.  Is there any reason to wait longer? Add 20 µl 0.5M succinate – a steady state should be evident within a few seconds, and within a minute or two you should see a second slope change.  Allow enough time to see it, but don't let the chamber run out of oxygen.  What might be the reason for the second steady state? Add 10 µl 0.2M KCN.

3. Uncoupling agents

Produce state IV respiration on succinate, as you did for experiment one. Add 10 µl 1 mM FCCP, a potent uncoupler of oxidative phosphorylation. Record the slope, for comparison with the slope that you previously recorded for state III respiration on succinate.  For the research paper you will need to compare state IV, III, and uncoupled rates on succinate. Do you expect any change after the mitochondria reach a steady state?  Is there any reason to let the record continue until the oxygen is depleted?

***AFTER USING FCCP OR ANY UNCOUPLING AGENT, THOROUGHLY RINSE THE SYRINGE, STOPPER, AND CHAMBER SEVERAL TIMES*** Even a small concentration of uncoupling agent will ruin the next experiment.

4. Inhibition of oxidative phosphorylation

You must thoroughly rinse out the chamber, glass stopper, and syringes before beginning this (or any) experiment.  If any uncoupling agent remains to contaminate the medium, for example, you will not have a successful experiment.

Produce state IV respiration on succinate. Add 10 µl 0.1 mM oligomycin and record a slope after witnessing the artifact.  Is there any indication that this antibiotic is either an uncoupling agent or an electron transport inhibitor?  Minor changes in slope can be considered to be negligible. Now design an experiment to determine if oligomycin inhibits oxidative phosphorylation (hint:  it would inhibit ATP synthase, if that is the case).

Assuming that you design and perform an appropriate experiment, what sort of controls might be needed (hint:  what agent should you add to determine if the mitochondria were exercising respiratory control?  That is, how can you confirm that there was an intact gradient, and the ETS is restricted by it?

If you are quick, or (better) if you thought about this problem before you came to lab, you can complete the experiment without having to start over.

Optional experiments

The following experiments need not be conducted in sequence. Their success rate depends on the quality of the mitochondria preparation and to some extent the skill of the investigators.

5.  Electron transport inhibition continued

Add a volume of mitochondria that produced good slopes with glutamate as substrate. Add 5 µl 0.5M succinate, confirm the slope increase. Without too much delay, add 10 µl 0.5M malonate, obtain a slope. Malonate is a competitive inhibitor. What would you expect to be the effect of successive additions of small amounts of malonate on the slope of oxygen consumption. Repeat the malonate addition to ensure that you have obtained near maximum inhibiton. Add 10 µl 0.01M ADP.

After determining whether or not ADP sped up respiration, add 20 µl 0.1M NADH or 20 µl 0.5M glutamate (recall that in an earlier experiment the addition of glutamate did not speed up state IV respiration a great deal).

A little reflection will reveal that this part of the study is the counterpart to the second experiment in which NADH-supported respiration was blocked and inhibition overcome by adding succinate. It often gives us very good results, but just as often the preparation does not respond to adding NADH or glutamate.

Add 20 µl 0.01M ADP, record slope changes if evident. If you obtain responses to glutamate and ADP additions, then add 10 µl 30µM antimycin. Add ascorbate + TMPD. 

6.  ADP:O ratio on ascorbate + TMPD

This experiment will work best with very well coupled mitochondria (RCR for NADH supported respiration of 4 or better). You may have noticed that the RCR was lower on succinate than on glutamate. The trend continues on the combination of ascorbate and TMPD, in fact the mitochondria behave as if they are nearly uncoupled.

Knowing that TMPD donates electrons to cytochrome c, what is the expected ADP:O ratio on ascorbate + TMPD?  Test this expectation by putting 15 µl mitochondria into state IV respiration on ascorbate + TMPD, then adding 10 µl ADP. If possible, calculate an ADP:O ratio.  You may have to stretch your imagination in order to detect a slope change.  Use a straightedge to line up the slopes.

7.  Site of action of antimycin

This elegant little experiment should narrow the possibilities for the location at which antimycin binds the ETS. Start with 15-25µl mitochondria and add FCCP.  The uncoupling agent should put the mitochondria in a state of rapid respiration once a substrate is added. Add succinate, confirm a slope, then inhibit respiration with malonate. Add glutamate, confirm a slope, then inhibit respiration with antimycin. Add ascorbate + TMPD.

You already knew that antimycin inhibits respiration on succinate. With this new information, can you determine with some precision where antimycin binds?

8. Mixed actions of 2,4-dinitrophenol (DNP)

The mixed actions of DNP are sometimes evident and sometimes not. It all depends on the quality of the preparation.

Repeat the third experiment, but substitute 10 µl 200 mM DNP for FCCP. DNP has mixed effects on mitochondria.  One action should be apparent right away, provided the mitochondria are still exercising a significant degree of respiratory control.  The second will be delayed and gradually take effect.

Still "alive?"

Feel free to try anything else, especially if you have unanswered questions.  A typical mitochondria preparation should give good results for 2 1/2 to 3 hours. When they begin to deteriorate they uncouple. State IV rates will increase and the preparation becomes less responsive to ADP.