Mitochondrial Respiration

In this exercise, students examine several reactions of mitochondrial respiration. I have avoided this exercise for years for fear of the preparation required. Now that I've done it once, it isn't as bad as I had feared. The biggest requirement is a high-speed refrigerated centrifuge; our Biology Department has an old but very serviceable Sorvall that suits just fine. In advance of lab, I prepare beef heart cytosol, mitochondria, and a mixture of the two, as described below. Electron transfer is evidenced by bleaching of DCIP (2,6-dichloroindophenol) which, like methylene blue, is deep blue in its oxidized form and colorless when reduced.

In the first part of the exercise, students do tests to see what fuels are oxidized by "Beef Heart Extract". Oxidation is indicated by reduction (bleaching) of DCIP (stock solution is 0.5 mM, or 0.145 mg/mL). The buffer is 0.1 M potassium phosphate, pH 7.4. All tubes contain 2 drops of buffer and 1 drop of DCIP. Other components are as follows:

1. No fuel control: 6 drops of water
2. Citrate: 5 drops of water, 1 drop of 0.1 M sodium citrate
3. alpha-Ketoglutarate: 5 drops of water, 1 drop of 0.1 M alpha-ketoglutarate (sodium salt)
4. Succinate: 5 drops of water, 1 drop of 0.1 M sodium succinate
5. Malate: 5 drops of water, 1 drop of 0.1 M sodium malate
6. NADH: 5 drops of water, 1 drop of 10 mM NADH (7.09 mg/mL)

Reactions are started by adding 1 drop of "Beef Heart Extract" and mixing well. All tubes stand at room temperature for 10 minutes; only the succinate and NADH bleach the blue color of the DCIP, in a few minutes.

In the second part of the exercise, students identify the active cellular fraction carrying out the observed oxidations. Three tubes are set up like number 4 above (succinate) and three tubes like number 6 (NADH). In each set of three, one tube gets 1 drop of "Beef Heart Extract", another gets 1 drop of "Cytoplasm", and the third gets 1 drop of "Mitochondria". Only "Cytoplasm" does not bleach the DCIP.

In the third part of the exercise, students identify the specificity of inhibition by malonate (stock is 0.1 M sodium malonate). All tubes contain 2 drops of buffer and 1 drop of DCIP. Other components are as follows:

1. Succinate alone: 5 drops of water, 1 drop of succinate
2. Succinate + malonate: 4 drops of water, 1 drop of succinate, 1 drop of malonate
3. NADH alone: 5 drops of water, 1 drop of NADH
4. NADH + malonate: 4 drops of water, 1 drop of NADH, 1 drop of malonate

Reactions are started by adding 1 drop of "Mitochondria" and mixing well. All tubes bleach the DCIP except the second. (I've tried using rotenone, which selectively inhibits the NADH dehydrogenase activity, but I couldn't get inhibition. It's supposed to be very potent; maybe it's slow-binding or requires detergents to see the effect. Any suggestions?)

To wrap up the exercise, I ask the students (1) what fuels are used by the extract, (2) where the observed oxidizing activities are located, (3) what other reactants might be needed to see oxidation of citrate, alpha-ketoglutarate, or malate, (4) to compare the structures of succinate and malonate and suggest why malonate has the effect it does, and (5) to indicate on a diagram of the electron transport chain where DCIP is accepting electrons and which complex is inhibited by malonate.

Preparation of mitochondria: I ordered a fresh beef heart from a local grocery store, trimmed off most of the fat and fibrous stuff, and cut the muscle into chunks of about an inch and a half and stored them in a freezer. It seems to keep for a long time. To prepare mitochondria, I thaw 50-75 g of tissue in two and a half volumes of 0.1 M potassium phosphate at pH 7.4 and liquefy it using a standard kitchen blender. The thick suspension is centrifuged cold at 15,000 rpm for 10 minutes. The sedimented material forms a tightly-packed pinkish lower layer and a lightly-packed grayish upper layer, with a clear red supernatant fluid (cytosol); the upper sedimented layer contains mitochondria. The supernatant is removed carefully and the mitochondrial layer is rinsed off the lower layer with 20 mL of phosphate buffer.

The mitochondria are recentrifuged in one tube, balanced against some of the original cytosol. After centrifuging, cytosol ("Cytoplasm") is removed from its tube, carefully avoiding insoluble material from the top and bottom of the tube. The supernatant is removed from the mitochondrial pellet in the other tube and the mitochondria are suspended in 15 mL of phosphate buffer ("Mitochondria"). About 5 mL of suspended mitochondria is mixed with about 5 mL of cytosol to give what I call "Beef Heart Extract". The activities the students measure seem to survive at least a couple thaw-freeze cycles.