Assignments for majors

The first screen that appears in MitochondriaLab shows you a biochemistry lab containing all the equipment you will need to perform your experiments. Click on each piece of equipment to learn more about its purpose. For your ease in completing each assignment, the background text relevant to the experiment that you will perform is italicized, instructions for each assignment are indicated by plain text, and questions or activities that you will be asked to provide answers for are indicated by bold text.

The following assignment is designed to help you become familiar with the operation of MitochondriaLab.

Assignment 1:
Getting to Know MitochondriaLab: Setting Up an Experiment

When you enter MitochondriaLab, you are provided with a reaction flask in which you will perform your reactions. These reaction flasks are a standard piece of glassware in a biochemistry lab because they allow biochemists to carefully control and measure reaction conditions within the flask. To begin each experiment, you will start with an empty reaction flask into which you will add isolated preparations of mitochondria. In real-life applications, these are typically mitochondria that have been isolated from mitochondria-rich animal tissues, such as liver or muscle, using cell fractionation techniques. Notice that you have two racks of test tubes to choose from. One rack of tubes contains seven different substrates that you can add to your flask (ADP, ascorbate/TMPD [tetramethyl-p-phenylene diamine], fumarate, glutamate, malate, pyruvate, and succinate). The second rack of tubes contains six different metabolic inhibitors of cellular respiration (antimycin, cyanide, DNP [2,4-dinitrophenol], malonate, oligomycin, and rotenone).

Notice that the chart recorder is connected to an oxygen-measuring electrode that inserts into a port on the left side of the reaction chamber. Substrates and inhibitors will be added to the flask through the port on the right side of the flask. Oxygen consumption in the flask will be determined as a measurement of the progress of reactions in the flask. The chart recorder will measure and plot oxygen concentration in the flask during the time course of your experiments.

  1. Begin your first experiment by developing a hypothesis to predict what will happen to oxygen consumption in the reaction flask after the addition of pyruvate. Develop a second hypothesis to predict how oxygen consumption will change in the flask upon the addition of pyruvate and ADP. Recall that pyruvate is an end-product of the reactions of glycolysis. Once you have formulated your hypotheses, set-up your simulated experiment as follows:

    1. Click on the To Experiment button in the lower left corner of the screen to enter MitochondriaLab. To begin an experiment you must first add mitochondria to the reaction flask. Do this by clicking on the Add Mitochondria button located below the reaction flask. A running ledger of the components that you added and the time that each component was added to the flask is shown in the lower right corner of the screen. Notice that the mitochondria have been added to a 2.0 ml suspension which consists of a solution buffered to the proper pH for these reactions. This solution contains ions and other components that are required by mitochondria to undergo cell respiration.

      The chart recorder at the left of the screen will be used to monitor the rate of cellular respiration by the mitochondria in the flask by plotting oxygen concentration (as a percentage of the starting concentration) on the x-axis against time in minutes on the y-axis. Oxygen concentration in the flask begins at 100%. Notice that the chart recorder is plotting oxygen consumption in blue ink. If desired, you can change the recording speed of the simulation by using the small buttons at the upper left corner of the screen. The default value is 4X.

    2. Allow the experiment to proceed for two minutes. Take note of any changes in oxygen consumption that occur during this time. Is this what you expect? Explain your observations. After two minutes, add pyruvate to the reaction flask by clicking on pyruvate from the substrates box (pyruvate should now be highlighted) then click the Add button. Notice that you added 20 ml of 500 mM pyruvate to the flask. Follow oxygen consumption in the flask for three minutes. What did you observe during this time? Based on what you already know about the reactions of cell respiration, explain your observations. Was this result what you predicted based on your hypothesis? Why or why not?

    3. Add ADP to the flask and follow oxygen consumption. What happened? Was this result what you expected based on your hypothesis? Why or why not. Explain your observations. Explain why oxygen consumption changes so dramatically following the addition of ADP while the addition of pyruvate alone results in slower consumption of oxygen.

    4. Click on the View Chart button to see an enlarged view of the plot. Use the slider bar at the right of the plot to move up or down the plot. Notice that the chart recorder labels the time when you added each component, the concentration of pyruvate added, and the concentration of oxygen (measured in nanomoles of oxygen molecules) at the time the pyruvate was added. You can export the values from this plot by clicking on the Add to Notes button in the lower left corner of the screen. A new browser window will appear with the data in your notebook. You can now print your data from this window or save your data to a disk.

    5. Click on the Return to Lab button then the To Experiment button to return to the experiment in progress. Reset the reaction flask by clicking on the Clean and Reset Chamber button below the flask to prepare the reaction flask for another experiment. Formulate a hypothesis to explain what you think will happen when you add succinate to the flask. Run the experiment by adding mitochondria to the clean flask, allow the reaction to proceed for one minute then add succinate to the flask. Did the results of this experiment validate your hypothesis? Why or why not? Explain why the addition of succinate produced the observed effect on oxygen consumption.

    6. Repeat the procedure in step e to perform individual experiments for each of the following substrates: fumarate, ascorbate/TMPD (tetramethyl-p-phenylene diamine), malate, and ADP. Note: Ascorbate and TMPD are synthetic electron donor molecules that can supply electrons to the electron transport chain. For each experiment, save all data to your notebook so you can compare results. Did the effect on oxygen consumption appear to be the same for each substrate? Describe any obvious differences in oxygen consumption that you observed with the different substrates? Based on what you know about the purpose of each substrate in the reactions of cell respiration, provide possible explanations the differences in oxygen consumption that you observed. What differences in oxygen consumption did you observe with the use of ADP as a substrate alone compared with pyruvate and ADP (step c)? Explain these differences.

Assignment 2:
The Catalytic Effect of Intermediates and Inhibitors on the Krebs Cycle

One of the important concepts established by many of Krebs' experiments was that the organic acids that are produced during the citric acid cycle can stimulate oxygen consumption. In addition, Krebs noted that these intermediates are generated in cyclical fashion and that each acid serves as the substrate for the next enzymatic reaction in the cycle. Therefore, once one organic acid is formed, the rate of subsequent reactions is dependent on the production of this organic acid. By following the production and catabolism of organic acids in the pathway it was possible to determine the sequence of enzymatic steps involved in the Krebs cycle.

The use of metabolic inhibitors was an essential aspect of the experiments which determined that the reactions of the Krebs cycle are cyclical. By adding an inhibitor and then measuring the accumulation of an intermediate, it can then be possible to determine the order or sequence of reactions in the cycle. The following assignment is designed to help you determine the sequence of reactions involved in the Krebs cycle by studying the effects of inhibitors on oxygen consumption.

  1. Malonate is an analog of one of the organic acids that is naturally produced during the Krebs cycle. Malonate acts as an inhibitor of the Krebs cycle. Your job is to determine which enzyme in the cycle is inhibited by malonate. Begin an experiment by adding mitochondria and malonate. Allow the reaction to proceed for one minute then add succinate. What happens to oxygen consumption in the experiment? To determine which step in the Krebs cycle is inhibited by malonate, try adding pyruvate to the experiment. Perform similar independent experiments by adding malate, fumarate, and ADP, to mitochondria containing malonate. Try other substrates as well. For each experiment describe what happens to oxygen consumption? What do these result tell you about the sequence of succinate, pyruvate, malate and fumarate relative to where malonate is acting? Based on your results, propose a order of reactions in which each substrate appears and suggest a step in the Krebs cycle that is inhibited by malonate. Consult a textbook if needed to verify your answer and to identify the enzyme inhibited by malonate.

    1. Once you have determined malonate's site of action, explain what would happen to the concentration of each of the following molecules: citrate, isocitrate, a-ketoglutarate, succinate, oxaloacetate, malonate, ADP, and ATP in the experiment if you treated these mitochondria with excess amounts of malonate?

Assignment 3:
The Effect of Inhibitors on Oxidative Phosphorylation

DNP (2,4-dinitrophenol) and oligomycin are examples of inhibitors called uncoupling agents. Uncouplers inhibit ATP synthesis but allow the other reactions of cell respiration to proceed normally. Develop a hypothesis to predict the effects of each agent on oxygen consumption then test the effects of each uncoupler as follows:

  1. Begin an experiment with mitochondria, pyruvate, and ADP. Allow the reaction to proceed for one minute then add an excess amount of DNP by clicking on DNP and then clicking twice on the Add button. What happens to oxygen consumption? Will the addition of more ADP influence oxygen consumption? Add ADP and observe what happens. Explain your answers. How might DNP be acting on the reactions of oxidative phosphorylation to cause the change in oxygen consumption that you observed?

    1. What do you think is happening to the concentration of H+ ions, ADP, and ATP in this experiment? Repeat this experiment using oligomycin. What did you observe? Refer to a biochemistry textbook or discuss your results with your instructor to learn about how each uncoupling agent disrupts oxidative phosphorylation.

  2. To better understand the coupling effect, and the actions of DNP and oligomycin, perform an experiment with mitochondria, pyruvate, and ADP. Allow this experiment to proceed for two minutes then add oligomycin. What happened to oxygen consumption during this time? Follow this experiment for two or three minutes. Add ADP to the experiment. What happens? Try adding DNP. What happens to oxygen consumption now? Explain each of these results, why did each ADP and DNP produce the effects that you saw? Compare results from this and the previous experiment. What can you propose about the possible actions of each of these inhibitors on the reactions of oxidative phosphorylation? Consult a textbook to identify the site of action for each uncoupling agent.

Assignment 4:
Studying the Efficiency of Oxidative Phosphorylation by Measuring the Rate of Oxygen Consumption

Biochemists who study the efficiency of oxidative phosphorylation in isolated preparations of mitochondria are interested in examining the amount of ATP produced relative to the total amount of energy generated during the oxidation of substrates in reactions of cellular respiration. Recall, however, that ATP production depends on the electron flow produced by oxidizing substrates as well as the concentration of ADP in the mitochondrion. Because oxidative phosphorylation relies on this electron flow as well as ADP, oxidative phosphorylation is said to be "coupled" to respiration (electron flow from substrates to oxygen). One way to measure the efficiency of coupling is to determine what is known as a P/O ratio. The P/O ratio is a measure of the number of ATP molecules that are synthesized as a result of the energy from one pair of electrons that completes the chain by being transferred to one atom of oxygen. A P/O ratio can be calculated by dividing the number of molecules of ATP synthesized (or ADP consumed) by the number of oxygen molecules consumed. Depending on the substrates, inhibitors, and oxygen concentration and other parameters, biochemists often categorize oxygen consumption in isolated mitochondria into five energy states. Each state represents different rates of oxygen consumption depending on the components added to the preparation of mitochondria.

Generally a P/O ratio of approximately 3 (3 moles of ATP) is expected from the oxidation of one mole of NADH. This value is a measure of oxygen consumption during ADP phosphorylation in the ATP synthesizing step of oxidative phosphorylation catalyzed by ATP synthase. The P/O ratio for any given substrate can be measured. A P/O ratio indicates that a substrate has been oxidized to produce NADH and the value of the ratio depends on where the electrons from a given substrate or from NADH enter (known as coupling) the electron transport chain. When substrates dontate electrons that enter early in the chain (further from oxidative phosphorylation), these electrons are involved in more transfer reactions. As a result these electrons contribute more to the H+ gradient needed for oxidative phosphorylation then electrons coupled to the chain at later stages (thus resulting in a high P/O ratio). The following exercise is designed to help you understand the effects of different substrates on ADP and oxygen consumption by measuring ATP to oxygen ratios in your experiments. You will be measuring the amount of ADP that was phosphorylated to ATP to determine P/O ratios.

The following exercise is designed to help you understand the effects of different substrates on ADP and oxygen consumption by measuring ATP-to-oxygen ratios in your experiments. You will be measuring the amount of ADP that was phosphorylated to ATP to determine P/O ratios. To calculate a P/O ratio, you need to know the following parameters: (1) the reaction is performed at 25 °C, (2) the volume of the reaction flask is 2 ml, and (3) there are 237 nanomoles (nmoles) of molecular oxygen (O2)/ml of reaction volume at 25 °C.

  1. For this assignment, we will consider two energy states of mitochondria. State 4 represents the rate of oxygen consumption due to the substrate alone while state 3 represents oxygen consumption during ADP phosphorylation. Begin an experiment by adding mitochondria, pyruvate and ADP. Follow this experiment for several minutes.

    1. The mitochondria will go into state 3 when ADP is added. Click on the View Chart button. Note that once all of the ADP has been phosphorylated to ATP this will be labeled on the plot as ADP Phosphorylated. At this point the mitochondria are now in state 3. Take note of the amount of oxygen consumed while the mitochondria were in state 3 by subtracting the amount of oxygen consumed (recorded in nanomoles of atomic oxygen) at the time when ADP was fully phosphorylated from the amount of oxygen consumed when ADP was first added to the experiment. Note: For your convenience, use the calculator in MitochondriaLab. Follow the steps below to determine the P/O ratio for pyruvate during state 3.

      (1) To determine the number of nmoles of molecular oxygen present in the reaction flask, multiply 237 nmoles/ml molecular oxygen by 2 ml (the size of the reaction flask). Because P/O values are calculated based on the amount of atomic oxygen consumed, you must now convert nmoles of molecular oxygen into nmoles of atomic oxygen by multiplying nmoles of molecular oxygen in the reaction flask by 2 (recall that there are two oxygen atoms in each molecule of oxygen). This value now represents nmoles of atomic oxygen in the flask.

      (2) To determine nmoles of atomic oxygen consumed in state 3, multiply the percent of molecular oxygen consumed in state 3 by the number of nmoles of atomic oxygen in the flask. (For example, if you observed that 5% of oxygen was consumed during state 3, multiply 0.05 by the number of nmoles of atomic oxygen in the flask that you calculated in step (1) above.) This value represents nmoles of atomic oxygen consumed in state 3.

    2. To calculate the P/O ratio in state 3, you must also determine the amount of ADP in nmoles. Recall that you added 20 m l of 10 mM ADP to the reaction flask. Convert this to nmoles first. To determine the P/O ratio, divide nmoles of ADP added by nmoles of atomic oxygen consumed (determined in step 2).

    3. Repeat this experiment using malate, succinate, and ascorbate/TMPD as the substrates. Calculate P/O ratios for each substrate in state 3 and compare these ratios. Refer to a textbook to use diagrams of the Krebs cycle and electron transport chain to trace each substrate to where the substrate itself or electrons from its oxidation enter (are coupled to) the reactions of cellular respiration. Do the ratios make sense to you?

    4. As you learned in the first assignment, Getting to Know MitochondriaLab, ascorbate/TMPD is not a natural substrate combination. From the P/O ratio, you should be able to narrow down the possible location at which TMPD donates electrons to the reactions of cellular respiration.

  2. Repeat experiment 1 with glutamate and then fumarate. Are the state 3 ratios different for the two substrates? You will have to look up the glutamate dehydrogenase reaction to determine whether the P/O ratio makes sense, because glutamate is not a direct substrate of the Krebs cycle; it enters the reactions of cellular respiration at a different stage. By what pathway does glutamate donate energy to the reactions of cellular respiration? Trace the path of energy from fumarate. What is the difference between the glutamate and fumarate pathways? What is the rate-limiting step in each reaction pathway?

Assignment 5:
Group Exercises

Certain poisons act as metabolic inhibitors by binding to molecules in the electron transport chain thereby preventing electron transport and blocking production of the H+ gradient required for oxidative phosphorylation. Binding can occur in a reversible or irreversible fashion. Rotenone is a poison that works as an irreversible inhibitor of the electron transport chain. Rotenone is frequently used to selectively kill undesirable species of insects and non-native fish, such as grass-carp, that can cause extensive destruction of plant species in lakes and reservoirs.

The following exercises are designed to help you determine which complex of the electron transport chain is a possible binding site for rotenone and other metabolic inhibitors, as well as examining where other substrates enter the electron transport chain. Before you begin these assignments, you may need to refer to a biochemistry textbook to view a detailed figure of the electron transport chain complexes I, II, III, and IV, and the electron transport molecules involved in each complex. Work together in a group of four students to complete the following assignments.

  1. Begin an experiment with mitochondria, ADP, and glutamate. Allow the experiment to proceed for one minute then add rotenone. Note: Keep a record of all of your experiments in your lab notebook so you can refer back to your results as you interpret the results form other experiments. What happened to oxygen consumption? Now add ADP to the flask. What happened to oxygen consumption after adding ADP? Is this what you expected? Explain your answer.

    1. Ascorbate can bind to a complex in the electron transport chain and donate electrons to the chain. Use ascorbate to help you pinpoint where rotenone is blocking the chain by repeating the same experiment with mitochondria, ADP and glutamate, then wait one minute, add rotenone, wait another minute then add ADP and ascorbate/TMPD to the experiment. What happened to oxygen consumption? Can you determine which complex of the electron transport chain is bound by ascorbate? Based on these results and knowledge of where ascorbate binds, can you determine which complex (I, II, III, or IV) is likely to be inhibited by rotenone. Continue to the next experiment to pinpoint the site of action of ascorbate and rotenone.

    2. Succinate can also bind to a complex in the electron transport chain and donate electrons to the chain. Use succinate to help you determine where rotenone is blocking the chain by repeating the same experiment as above with mitochondria, ADP and glutamate, then wait one minute, add rotenone, wait another minute then add ADP and succinate to the experiment. What happened to oxygen consumption? Can you determine which complex of the electron transport chain is bound by succinate? Based on these results and knowledge of where succinate binds, can you determine which complex (I, II, III, or IV) is likely to be inhibited by rotenone. Refer to a biochemistry textbook to determine which complex of the electron transport chain is bound by succinate if necessary. Based on your results and knowledge of where succinate binds, which complex (I, II, III, or IV) is likely to be inhibited by rotenone. Continue to the next experiment to pinpoint the site of action of rotenone, ascorbate, and succinate.

    3. Antimycin is another metabolic inhibitor that acts on the electron transport chain. Use antimycin to help you pinpoint where rotenone is blocking the chain by repeating the same experiment as step b but after adding rotenone, add ADP and antimycin to the experiment. What happened to oxygen consumption? Which complex of the electron transport chain is bound by antimycin? Based on your results and knowledge of where antimycin binds, which complex (I, II, III, or IV) is likely to be inhibited by rotenone. Are all of your results consistent enough to help you pinpoint the binding site of rotenone? Once you have determined which complex is bound by rotenone, discuss your answer with your instructor to find out the specific molecules affected by rotenone, ascorbate, succinate, and antimycin.

  2. Repeat experiment b above but after adding succinate and ADP wait one minute then add cyanide as a final step in the experiment. Cyanide blocks the electron transport chain in the same fashion as carbon monoxide (CO) the colorless, odorless gas produced when many organic materials are burned. What happened to oxygen consumption in this experiment? Repeat the same experiment for each of the other experiments you conducted above. For each experiment, what happened to oxygen consumption? Is this what you expected? Why or why not? Can you add substrates to bypass the cyanide block? Try this experiment with all of the substrates available. What did you observe? Explain your results then determine which complex of the electron transport chain is blocked by cyanide?

  3. Based on what you know about the electron transport chain and oxidative phosphorylation, explain how adjusting the pH of your reaction flask can be used to artificially bypass the effects of these inhibitors? What would you add to the reaction flask if you were actually going to perform this experiment? Based on what you know about the actions of oligomycin from earlier experiment, how might this experiment be affected by the addition of oligomycin?