Assignment 2

1 .		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.
2 .		Begin an experiment in MitochondriaLab 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?
3 .		What do you think is happening to the concentration of H+, ADP, and ATP in this experiment? Repeat this experiment using oligomycin. What did you observe?
4 .		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 ADP and DNP each 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.