Assignment 3

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 in MitochondriaLab 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. Recall that the oxygen recorder is plotting molecular oxygen concentration as a percent of the starting concentration (100%). To determine the amount of molecular oxygen consumed while the mitochondria were in state 3, subtract the percent of oxygen present at the time when ADP was fully phosphorylated from the amount of oxygen present when ADP was first added to the experiment. Note: For your convenience, use the calculator in MitochondriaLab. Follow the steps listed next to determine the P/O ratio for pyruvate during state 3. 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. Enter the number of nmoles of molecular oxygen in the field below.

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 0.005 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. Enter the number of nmoles of atomic oxygen in the field below.

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 ml of 100 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. Enter the P/O ratio in the field below.

4 .		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. Enter your data in the field below.