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 differences.
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