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.
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.
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?
首页
