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In mitochondria pyuvate is made from glycolysis and is imported into the mitochondria and oxidised by O2 to CO2 and H2O. 15 times more ATP is made than produced by glycolysis. Mitochondria use both pyuvate and fatty acids for fuel. Both pyuvate and fatty acids are transported across inner mitochondrial membrane and converted to acetyl coA by enzymes in mitochondrial matrix. These enzymes are then oxidized via the citric cycle. This cycle converts acetyl coA to CO2. This oxidation produces high energy electrons that are carried by NADH and FAH2. These electrons are transported to the inner membrane where they enter the electron transport chain. Electrons are transferred from NADH to O2 by multi-subunit inner membrane complexes I, III, & IV, plus coenzyme Q and cytochrome c. Each complex contains electrons that pass sequentially through a series of electron carriers. These electrons are found in the inner membrane and are essential to the process of oxidative phosphorylation. The citric cycle is part of the aerobic metabolism and does not itself use oxygen. Only in the inner mitochondrial membrane and the final catabolic reactions is where oxygen is directly consumed. The generation of ATP by oxidative phosphorylation via the electron transport chain depends on the chemiosmotic process.(1) The aims of the following experiments taken were to investigate the respiratory electron transport, coupling and respiratory control using whole isolated mammalian mitochondria. Before experiments were taken a preliminary experiment was done to ensure what samples we would be using. First part involved detecting the oxidation with DCIP using 3 fractions all from chicken liver with fraction 3 containing most of the mitochondria. Results showed the control sample showing signs of bleaching but it was not relevant with mitochondria. Also showed rates of fraction 2 were not linked to mitochondria. This showed the experiment was not an accurate way of measuring mitochondria. In the first experiment mitochondrial extracts of rat liver and chicken liver were used. Measurements of oxygen uptake were used to observe the overall rate of oxidative metabolism. In the absence of ADP the oxygen uptake caused by oxidation of endogenous substrates is slow. The addition of substrates such as succinate or ÎÂ±-ketoglutarate shows an effect on the respiration rate. Adding ADP causes the oxygen uptake to proceed at an increased rate until all of the added ADP is converted into ATP. This conversion causes the oxygen uptake to return to the basal rate. This experiment shows this stimulation of respiration is stoichiometric this because the addition of twice as much ADP causes double the amount of oxygen uptake at the increased rate. The chemiosmotic theory is used to describe the enzymatic reactions that involve a chemical reaction and a transport process. The P/O ratio is this number of molecules of ATP formed per pair of electrons carried through electron transport. Values of P/O ratio range from 1.5-3. A p/o ratio of 3 is for mitochondrial oxidation of NADH. The p/o ratio of succinate to O2 is 1.5. (2) (3)(4)
The oxygen uptake is determined by recordings of an oxygen electrode. The experiments that will be taken involve isolated rat liver and chicken liver mitochondria being incubated in a medium of phosphate and substrates,then the addition of ADP will cause the increase of oxygen uptake as the ADP is converted into ATP. A graph of time against amount of oxygen will be produced. State 3 is known as the actively respired state compared to the slow rate being referred to as state 4. The ratio [state 3 rate]: [state 4 rate] is called the respiratory control index. This index shows the tightness of the coupling between respiration and phosphorylation.RCI values range from 3-10, this is varied by the substrate used and the preparation taken of the experiment.( 3)
A second experiment to be looked into is determining the protein concentration of mitochondria extracts. A colorimetric method known as a Bradford assay. Bradford assay is very fast and accurate method of identifying the maount of protein. It is used to determine the protein content of cell fractions and accessing protein concentration.
Results to include in the Document
To prepare the details required for this report, it is best to analyse the data that you collected. However, if you prefer you can analyse the model data that will be made available to all 2B01 students.
When you submit your report, attach the following Figures/Tables:
ONE Table or Figure that describes how you used your data to calculate the concentration of protein in each of the samples that you studied;
ONE Figure that describes how you used your data to calculate the rate of oxygen consumption for the samples that you studied.
Summary Table: Complete the Summary Table, included below, which reports on measurements on the rates of respiration by rat liver mitochondria.
Rate of State 3 Respiration
(ÎÂ¼atom O. min-1)
Rate of State 4 Respiration
(ÎÂ¼atom O. min-1)
Respiratory Control Ratio
Rat Liver mitochondria + 2-oxoglutarate
Rat Liver mitochondria + succinate
Discussion details should fit within the box provided below; Figures may be added if you wish, but all of the information you include should fit within the box provided. In your Discussion you should highlight conclusions from your experiments, and answer the following questions:
How do you explain the differences between expected and observed results for the experiments with Fractions 1-3?
How do your experimentally determined P:O ratios compare with those you anticipated from the theory behind the experiments? What might be the origin of any differences?
From the experiment the p/o ratios were calculated for both succinate and 2-oxoglutarate. The result of 2-oxoglutarate showed a ratio of 3.04 and succinate of 3.14. Both these results show to be higher than the range it should be. The typical ranges vary between 1.5 for succinate and 2.5 for 2-oxoglutarate. NAD-linked substrates give higher values of P:O around 2.5 this is shown to be correct for oxoglurate result being 3.04. Succinate is an FADH2 linked substrate values of p/o ratio are around 1.5. A value of 3.14 is shown in this experiment.
The succinate graph shown on figure 1 shows the oxygen used during incubation with rat liver mitochondria and succinate. The graph shows a slow consumption of oxygen when mitochondria was added. When 0.1 ml of ADP was added there was a stimulation of oxygen consumption causing a decrease from around 1 min to 1.8 this is called state 3.Adding 0.1ml of ADP allows respiration to turnover within mitochondria causing Reduced NADH going to NAD+ and oxygen being consumed and proton motor force being generated causing ATP to go through causing a decrease in rate and slowing down. Adding 0.1ml of ADP allows us to calculate background rate of oxygen consumption of state4. 0.2 ml ADP were then added and graph shows an increase again and then slowing off. Adding two ADP causes a rapid rate this can be shown on figure this is because the total formation of ADP into ATP. The size of change is directly proportional to ADP added. The graph shows when the first substrate was added to the isolated mitochondria there was a change in rate of oxygen consumption this is known as steady state. State 4 is a steady state this is because energy from the substrate is shown to maintain a chemiosmotic gradient. When ADP was added to the mitochondria in state 4 the oxygen consumption increases. In state 4 respiration the electron transport system is restricted by the gradient. The rate of energy loss from the gradient determines the rate of oxygen consumption in state4.
Electron transport from NADH to oxygen stores more energy compared to electron transport from succinate to oxygen. When succinate is added it moves 6 protons across membrane for every oxygen atom consumed the P/O ratio drops to around 1.5 but this is not shown with the results produced. One reason could be the liver fractions were diluted before use this could have affected the P/O ratio and final results. Succinate is shown not to be a good reducing agent this is because less energy is released when it is oxidised. Results of respiratory control range around 3-10. Results showed both substrates ranged within those values showing how tightly coupled they are. A ratio of 5 is said to be a reasonable condition for isolated mitochondria. Results from the second experiment show fraction 4 with rat liver mitochondria had a higher concentration of 2.12 compared to the fractions containing chicken liver. This could be the way each of the fractions were prepared. Rat liver was fresh compared to chicken livers that were frozen and from a supermarket. Fraction 4 had everything each of the other fractions contained. Fraction 1 also had a slight higher concentration of 1.44 micrograms/ microlitre. This high concentration could be it was spun first time at a medium speed of 14,000 compared to other fractions of chicken liver and was kept supernant. In this experiment model data was used this is because absorbances calculated were higher than the absorbances with bovine serum therefore protein content couldnââ‚¬â„¢t be calculated from the graph and therefore concentration. The model data was diluted by 5 fold this could be another reason. Fraction 4 with the highest concentration was spun 3 times both in superanent and in pellet therefore this could have influenced this fraction having a higher concentration.