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.
Getting to Know MitochondriaLab: Setting Up an Experiment
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.
- 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:
- 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.
- 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?
- 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.
- 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.
- 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.
- 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
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 Catalytic Effect of Intermediates and Inhibitors on 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
- 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.
textbook if needed to verify your answer and to identify the enzyme inhibited by
- 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: 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:
The Effect of Inhibitors on Oxidative Phosphorylation
- 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?
- 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.
- 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: 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.
Studying the Efficiency of Oxidative
Phosphorylation by Measuring the Rate of Oxygen Consumption
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.
- 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
- 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.
- 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).
- 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?
- 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
- 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
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.
- 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.
- 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.
- 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.
- 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.
- 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?
- 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?