Experiment 1.
Part II
succinate
Part III
B-OH Butyrate
Succinate Fumarate malate pyruvate glutamate aspartate B-OH But lactate blank XXXXXXXXXX 1.0000000000000007E-2 5.0000000000000031E-2 1.0000000000000009E-3 7.0000000000000034E-2 1.0000000000000007E-2 XXXXXXXXXX 2.0000000000000014E-2 1.4999999999999998E-2 kcn antimycin amoba
itol malonate control 0.5 XXXXXXXXXX XXXXXXXXXX 3.0000000000000002E-2 0.44 kcn antimycin amoba
itol malonate control 0.1 9.0000000000000024E-2 0.05 7.0000000000000021E-2 6.5000000000000002E-2
BS2001-Metabolism. 2013
A study of mitochondrial metabolism and the mitochondrial electron transport chain.
Introduction
In this experiment, mitochondria in which oxidative phophorylation has been uncoupled from electron transport will be used. The electron transport chain (ETC) will be used as part of a linked assay system to measure the efficiency with which various substrates of the mitochondrial metabolic reactions are used by isolated mitochondria. The effects of various specific inhibitors of the ETC will also be studied.
The rate of the electron transport process can be measured in a number of ways:
1. Following consumption of 02 (the physiological substrate) using an oxygen electrode.
2. Inte
upting the chain with fe
icyanide (hexacyanofe
ate III) which will take electrons from cytochrome c, being reduced to fe
ocyanide (hexacyanofe
ate II) in the process. The concentration of fe
ocyanide is determined colimetrically.
3. Inte
upting the chain at the unbiquinone (coenzyme Q) position, using tetrazolium reagent. The concentration of the formazan formed is determined colimeterically.
Only the second technique will be used in this practical.
Preparation of mitochondria
Has been ca
ied out by the technical staff. Please ensure that the tubes containing the mitochondria remain on ice at all times!
Solutions provided
Tris/HCl buffer, pH 8.2,; 10mM Potassium fe
icyanide K3[Fe(CN)6]; 0.5 mg/mL cytochrome c; fe
ic reagent.
Substrates: Succinate, Fumarate, Malate, Pyruvate, Glutamate, Aspartate, B-hydroxybutyrate, Lactate (all at 50mM NA salts, pH 7.5).
Inhibitors ; Potassium cyanide (KCN) CARE-POISON; Amoba
ital CARE –POISON; Malonate CARE-POISON.
Experiment 1.
You are provided with a series of substrates which produce reduced co-factors which can in turn be oxidised by the ETC. Investigate the rate of the ETC using each of the substrates singly. Establish which is the most effective substrate. You will need to prepare a reagent blank.
Set up the incubation mixtures as follows, in labelled glass test tubes:
1.2mL Tris/HCl buffer, pH8.2
0.25 mL Substrate (or water in reagent blank)
0.5 mL 10mM Fe
icyanide
0.25 mL cytochrome c Total 9 tubes.
Place the tubes in a 37oC water bath for 5 min before starting the reaction. With the tubes still in the water bath, start the reaction by adding 0.05mL of the mitrochondrial preparation to each tube.
After exactly 5 mins stop the reaction by adding 2.5 mL of fe
ic reagent to each tube. Leave them to stand for 5-10 min on the bench for the colour to develop. Read the abso
ances at 620 nm using a 1 cm plastic cuvette with water as a reference. The abso
ance is a measure of the rate of reaction.
Experiment 2.
You are provided with 4 ETC transport inhibitors which act at different sites. Investigate the effects of each on 2 different substrates- one being succinate, the other being the most effective substrate as determined by experiment 1. You will need controls with no inhibitor for each of the substrates.
The assays are set up as follows:
1.2 mL Tris/HCl buffer, pH 8.2
0.12 mL Substrate
0.13 mL Inhibitor (or water in the controls)
0.5 mL Fe
icyanide
0.25 mL cytochrome c total 10 tubes.
The proceed as above-preincubating prior to starting the reaction with 0.05mL mitochondria.
NOTES. Please rinse all materials thoroughly after use.
Writing up. You will need to produce a simple metabolic diagram outlining the metabolism of different substrates in relation to the TCA cycle. You will also need to outline the ETC and at which points the inhibitors exert their effects.
Investigate the effects of each on 2 different substrates- one being succinate, the other being the most effective substrate which is B-OH Butyrate. So explain/discuss part II AND part III results for each inhibitor on the 2 different substrates.