Assignment

I need a lab report on study of mitochondrial metabolism and the mitochondrial electron transport chain.This report needs to be done in two part.First part contains the results,which i have mention in the file uploaded at the bottom of the page.From the result, we need to do a bar graph with the standard deviation error. You will need to produce a simple metabolic diagram outlining the metabolism of different substrates in relation to the TCA(tricarboxylic acid cycle) cycle. Second part of this report needs discussion.In this discussion,you need to provide us about 500 words about ETC(electron transport chain) and at which points the inhibitors exert their effects.And other 500 words discussing about the results we obtained from the lab which is uploaded(which u can find at the bottom of the page ). I have uploaded the file which contains information about the experiment and how we performed the experiment and the result we obtained from the experiment.

Cellular Biochemistry

Practical 1

A study of mitochondrial metabolism and the mitochondrial electron transport chain.

Introduction

In this experiment, mitochondria in which oxidative phosphorylation 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. Interrupting the chain with ferricyanide (hexacyanoferrate III) which will take electrons from cytochrome c, being reduced to ferrocyanide (hexacyanoferrate II) in the process. The concentration of ferrocyanide is determined colourimetrically.

3. Interrupting the chain at the unbiquinone (coenzyme Q) position, using tetrazolium reagent. The concentration of the formazan formed is determined colourimetrically.

Only the second technique will be used in this practical.

You will be working in groups of 3 and should agree on how you divide the work.

Preparation of mitochondria

Has been carried 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 ferricyanide K3[Fe(CN)6];
0.5 mg/mL cytochrome c;
ferric reagent.
Substrates(all at 50mM NA salts, pH 7.5).
Succinate;
Fumarate;
Malate
Pyruvate
Glutamate;
Aspartate;
B-hydroxybutyrate;
Lactate.

Inhibitors
Potassium cyanide (KCN) CARE-POISON;
Amobarbital CARE -POISON;
Malonate CARE-POISON;
Antimycin CARE-POISON

Please assemble all the assay tubes for any given experiment as a batch and then incubate simultaneously in the water bath. If assays are to be done individually, there will be no enough time during the practical to complete the work.

Experiment 1.

You are provided with a series of substrates that 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 one 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 Ferricyanide
0.25 mL cytochrome c
Total of 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 ferric reagent to each tube. Leave them to stand for 5-10 min on the bench for the colour to develop. Read the absorbances at 620 nm using a 1 cm plastic cuvette with water as a reference. The absorbance 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 Ferricyanide
0.25 mL cytochrome c
Total of 10 tubes.

Then proceed as above-pre-incubating prior to starting the reaction with 0.05mL mitochondria.

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

Results.

Experiment 1

                          Substrate    Absorbance
Succinate                                  0.659
Fumarate                                  0.501
Malate                    0.523
Glutamate                                 0.515
Aspartate                                  0.524
Pyruvate                                    1.362
B-hydorxybutyrate                     0.673
Lactate                 0.607

Experiment 2 Absorbance

                    Succinate       Pyruvate
Control             0.345           0.988
Inhibitor
Malonate           0.031           0.942
Cyanide            0.258           0.569
Amobarbital      0.24             0.363