Respiration Lab
Introductory information:
Answer all questions with asterisks below in the introductory information section of your lab notebook.
In this lab you will use a common mitochondrial preparation to visualize the electron transport and test the effect of DNP on electron transport in mitochondria. Electron transport is measured indirectly with the use of an artificial electron acceptor, DCIP. DCIP is blue in its oxidized state
(absorbing 600 nm light) but becomes colorless when reduced (DCIPH2). As electrons are transported, DCIP becomes reduced. We can therefore measure the rate of respiration by measuring the rate at which a solution becomes colorless.
*Before beginning, make predictions for each tube listed in Table 1 in the methods: (2 pts)
Tube 2:
Tube 3:
Tube 4:
Materials
• Cauliflower
• Homogenization Buffer HM (0.4 M Sucrose, 1 mM EDTA, 25 mM MOPS pH 7.8, 8 mM cysteine, 0.1% w/v BSA)
• Wash Buffer (0.4 M Sucrose, 1 mM EDTA, 10 mM MOPS pH 7.2, 0.1% w/v BSA)
• Assay Buffer (15 mM NaPO4 pH 7.2, 6 mM Sodium Azide)
• 1.8 mM DNP
• 0.2 M Succinate
• 0.05% w/v DCIP
Methods: (1 pt)
Mitochondrial Isolation
Quantities given are for one student group. Keep the mitochondrial fraction on ice.
1. Collect ~10 g of the outer 3–5 mm of chilled cauliflower florettes.
2. Grind in a chilled mortar with 10 ml of cold homogenization buffer.
3. Filter the homogenate through 2 layers of cheesecloth, and collect the filtrate.
4. Regrind the material trapped in the cheesecloth with 10 ml fresh cold homogenization buffer, filter, pool the filtrates.
5. Centrifuge filtrate; 5 min, 1500 x G, 4°C. Discard the pellet (nuclei and unbroken cells).
6. Centrifuge the supernatant; 20 min., 20,000 x G, 4°C. Discard the supernatant; keep the pellet
(mitochondria).
7. Thoroughly resuspend the pellet: add 1–2 drops of wash buffer and scrape the pellet from the bottom of the tube. Then add 5 mL wash buffer. This is the mitochondrial suspension.
Determination of the Effect of DNP on DCIP Reduction
The final volume of each tube is 3 ml.
Record the absorbance at 600 nm of each tube per unit time. To keep the timing of measurements consistent among your tubes and allow you to move quickly through the experiment, stagger the start of each tube. At the appropriate time, add mitochondria to a tube, invert quickly, and then measure initial absorbance.

Set up experimental tubes according to Table 1.
Table 1. Reagents for three cuvettes.
Tube Assay buffer
1(blank) 2 mL
2 2 mL
3
4
2 mL
2 mL
0.2 M succinate
0.3 mL
0.3 mL
-
0.3 mL
0.05%
(w/v) DCIP
-
0.1 mL
0.1 mL
0.1 mL water
0.2 mL
0.1 mL
0.4 mL
-
-
-
1.8 mM
DNP
-
0.1 mL
Mitochondria
0.5 mL
0.5 mL
0.5 mL
0.5 mL
1. Prepare duplicates of tubes 2–4.
2. Zero the spectrophotometer with tube 1 after adding the mitochondrial suspension. Do NOT add the mitochondrial suspension to the other tubes until you are ready to begin the reactions.
3. Begin the reaction in tubes 2-4 by adding 0.5 ml of mitochondria suspension. Stagger the start of each tube by 20 sec.
4. Read the absorbance of each tube immediately upon adding the mitochondria (this is called a time zero point) and then at 2-minute intervals for 12 minutes or until a stable absorbance, whichever comes first.
5. Calculate the change in absorbance for each tube over each time interval (by subtracting the absorbance at 2 min. from the absorbance at 0 min, etc.). Graph your data to show the change in absorbance as a function of time for each of the three conditions.
Results: In your lab notebook include graphs from #5 above (2 pts).
Further Questions:
A. Were your predictions at the beginning of the lab supported or not? Explain. (1 pt)
B. Compare the results from tubes 2 and 3. What effect does the addition of succinate have on the rate and extent of electron transport? Explain these results. (1 pt)
C. Compare the results from tubes 2 and 4. What effect does the addition of DNP have on the rate of and extent of electron transport? Use your understanding of DNP function to explain these results. (1 pt)
D. If you measured ATP production in this system instead of electron transport, how would tubes
2 and 4 compare? Explain your reasoning. (1 pt)