Mitochondria Lab Report

Experiment IV: Study of Krebs cycle using Mitochondria from Mung Bean Seedlings

INTRODUCTION
The mitochondria is a very important organelle in the plant cell because it carries out very important cellular reactions in the cell like the Krebs cycle and oxidative phosphorylation which is how the plants produce ATP from the pyruvate produced through glycolysis (Meyer and Millar, 2008). Glycolysis produces a net of 2 ATP for the plant which is not enough for the cell to function while the Krebs cycle and electron transport chain produces a net of 36 ATP which makes the mitochondria the power house of the plant cell (Meyer and Millar, 2008). Mitochondrion is also an important organelle because it contains its own genome and can be transcribed
and translated (Meyer and Millar). Mitochondria are isolated to study the Krebs cycle and the proteins that carry out the reaction (Wu and Scheffer, 1960).
In addition isolated mitochondria are studied to identify their proteomes and how development affects the proteome (Labbe et al., 2008). Study of isolated mitochondria has great application in the pharmaceutical industry through the discovery that some mitochondrial proteins stimulate apoptosis in the cells (Wang, 2001). This research has aided in finding cure for cancer and Parkinson’s disease among many other diseases (Labbe et al., 2008). Also, many drugs produced contain drug classes that can inhibit the function of mitochondria by preventing or altering a stage in ATP production in the Krebs cycle or electron transport chain or acting as an inhibitor of an enzyme that carries out a certain reaction in citric acid cycle or ETC (Dykens and Will, 2007). These drugs are therefore pretested
with isolated mitochondria to reveal any side effects the drug might have (Dykens and Will, 2007).
Most eukaryotic cells are compartmentalized which means that they have specific organelles to carry out specific cellular reactions (Wu and Scheffer, 1960). For example, in plants photosynthesis is carried out in the chloroplast while ATP production is carried out in the mitochondria. This makes it easy for this organelles to be isolated and their cellular reactions and functions to be studied (Wu and Scheffer, 1960). This involves breaking down the outer cell protection to release the cell organelles (Moller, 1996). In animal cells, gentle methods are applied to release cell content because animal cells only have an outer cell membrane. However, plant cells have a cell wall and a cell membrane, therefore more aggressive procedures like blending have to be used to homogenize the cell. These procedures have to be carried out in such a way to protect the membranes of each cell organelle and their contents therefore special mediums are used to maintain the right environment for the organelles (Wu and Scheffer, 1960). Etiolated seedlings are more favourable for mitochondria isolation (Brummond and Burris, 1954). These seedlings are grown in the dark which means they will have more mitochondria than chloroplast and their mitochondria would not contain much nuclease (Brummond and Burris, 1954).
Plants carry out two reactions for ATP production. Glycolysis is light dependent and takes place in the cytoplasm while Krebs cycle is light independent and takes place regardless of the presence of light (Brummond and Burris, 1954). Pyruvate (a 3 carbon sugar) enters the matrix of the mitochondrion where enzymes for the Krebs cycle are located. Krebs cycle is made up of 8 continuous reactions carried out by different enzymes characteristic to each reaction that convert pyruvate to citrate and finally to oxaloacetate to form a cycle with production of 4 NADH and 1 FADH₂ molecules (Meyer and Miller, 2008). The first reaction is the combination of pyruvate with Co-A to give acetyl Co-A and one NADH molecule. The other NADH molecules are formed when D-isocitrate is converted to α-ketoglutarate, α-ketoglutarate is converted to succinyl-CoA and malate is converted to oxaloacetate. FADH₂ is formed when succinate is converted to fumarate (Brummond and Burris, 1954). The NADH and FADH₂ molecules then enter the cristae of the inner membrane of the mitochondria where the electron transport chain is located (Pardee and Potter, 1949). The electron transport chain has four protein complexes and cofactors that oxidize NADH and FADH₂ and create an electron gradient to transport electrons along the four complexes and finally yield water and ATP molecules when the protons are passed through the ATP synthase (Pardee and Potter, 1949). Succinate dehydrogenase is the only enzyme found in both Krebs cycle and ETC and it is the enzyme that converts succinate to fumarate (Pardee and Potter, 1949).
The purpose of the experiment is to isolate mitochondria from mung bean seedlings using a waring blender and centrifuge to study the steps of the Krebs cycle and oxidative phosphorylation and observe the effects azide, succinate and malonate have on production of NADH and FADH₂ in the Krebs cycle.

METHODS
Blend about 60g of mung bean stems with 40 ml of grinding medium for 20 sec and filter
Divide the filtrate equally into two 50ml centrifuge tubes and centrifuge at 5ᵒC for 10 min at 4000RPM
Spin in centrifuge for 10 minutes at 5ᵒC at 4000 RPM
Supernatant: divide equally supernatant into two 50 ml centrifuge with a balance
Cell pellet: Discard the pellet

Add 0.5 ml of mitochondrial suspension and read absorbance for 10 seconds at 30 sec interval
Prepare 8 reaction cuvettes according to table in lab manual
Pellet: using a rubber tipped rod, resuspend the pellet in 2 ml grinding medium and break up large pieces and add additional 2ml grinding medium and hold on ice.
Supernatant: discard the supernatant
Spin in centrifuge at 5ᵒC for 20 minutes at 12000 RPM

RESULTS
RCF = 1.117 × 10⁻⁵ n² r
RCF is the force applied to objects being centrifuged n is the number of revolutions per min r is the distance from the centre of the rotor to the molecules in the tube in cm rmin = 3.2cm
RCFmin = 1.117 × 10⁻⁵ × (4000 RPM) ² × 3.2cm = 571.9 × g rmax = 9.6cm n = 4000 RPM
RCFmax = 1.117 × 10⁻⁵ × (4000 RPM) ² × 9.6cm = 1715.7 × g r (distance between centre of rotor and molecules )(cm) | n (number of revolutions per min) (RPM) | RCF (× g) | 3.2 cm | 4000 RPM | 572 | | 12000 RPM | 5147 | 9.6 cm | 4000 RPM | 1716 | | 12000 RPM | 15441 | Table 1: table showing the rmax and rmin and the speed of the centrifuge and the relative centrifugal force of the centrifuge

RCF = 20,000 × g
If RCF = 1.117 × 10⁻⁵ n² rav rav = (3.2 + 9.6)/2 = 6.4 cm n = RCF1.117 ×10-5 ×rav = 200001.117×10-5×6.4 = 16726

Table 1 shows that as the distance from the centre of the rotor of the centrifuge increases, the relative centrifugal force increases. The minimum r has a RCF of 572 at a speed of 4000 RPM which is much smaller than the max r value of 1716 RPM. Also, a larger speed gives a greater centrifugal force.
Figure 1 shows that the 4 absorption curves slope downwards i.e. they have a negative slope. The absorption curve of the cuvette containing azide and sodium succinate has the decreasing slope with the largest downward slope. The first three absorption curves coloured green, red and blue start at almost the same point at time 0 while the last curve with malonate starts from about 0.72 which is lower than the other. The green and purple curves have about the same slope value downwards.

DISCUSSION
Eukaryotic cells are compartmentalized which makes it easy to carry out isolation of its organelles (Wu and Scheffer, 1960). During isolation, a process has to be carried out to break up first of all the tissues of the plant and secondly the cell membrane and/or cell wall (Wu and Scheffer, 1960). Plant cells have a cell wall and plasma membrane therefore, rigorous methods are used to break the tissue and tear the cell wall and cell membrane (Galbreith et al., 1995). This was why blending was used to ensure cell organelles were released. However, this procedure is carried out carefully so that the organelles are not also broken up and smashed. The organelles need to be studied as whole organelles and not ruptured organelles (Galbreith et al., 1995). After the homogenate have been produced, other factors have to be maintained to ensure the organelles are not destroyed (Moller et al., 1996). Firstly, because the organelle has been used to a particular osmotic balance with the cytoplasm of the cell which it resided in, it is important to maintain that osmotic balance in the homogenate so that the organelle does not undergo osmotic shock (Galbreith et al., 1995). In addition, rupturing the cell could expose the organelles to extreme temperature and pH causing especially from the components of the cell vacuole like phenolics and acids, etc (Moller et al., 1996). In mitochondria, this could disrupt the activity of enzymes that control the Krebs cycle, etc because enzymes are sensitive to temperature and pH and could cause the enzymes to be denatured (Galbreith et al., 1995). Also, when the cell is ruptured, some organelles that contain lytic and destructive enzymes can also be ruptured, releasing such enzymes that could cause harm to the mitochondria (Moller et al., 1996). Therefore, a specific medium has to be used when grinding the seedlings. In this experiment, the medium used contained 0.25M sucrose, 0.1 M potassium phosphate (pH 7.1), 0.1% BSA and 0.01 magnesium chloride. BSA in the medium prevents the mitochondria from phenolics that are released from the vacuole that may cause problems later by binding to them. Also, it protects the mitochondria from proteases that might want to degrade the mitochondria proteins by acting as a substitute to be degraded in the place of the mitochondrial proteins (Moller et al., 1996). The 0.25 M sucrose in the medium is an osmoticum. It maintains the appropriate osmotic environment to prevent swelling or bursting of the mitochondria due to hyper osmotic pressure (Moller et al., 1996). The potassium phosphate (pH 7.1) acts as a buffer to maintain the pH at around the pH of the cytosol of the plant cell which is about 7.5 (Moller et al., 1996). Also while blending the seedlings, it was important to stop blending after 10 seconds and wait for a while before the next 10 seconds. This was done to prevent extreme rise in temperature which might cause denaturing of enzymes (Moller et al., 1996). For the mitochondria isolation, etiolated mung bean seedlings were used (Brummond and Burris, 1954). It was important for these seedlings to be etiolated to achieve minimal chloroplast presence (Brummond and Burris, 1954). The mung bean seedlings had long yellow/white stems because they were grown underground were no light could be seen. This increases the amount of mitochondria present in the cell making it easier to successfully isolate the mitochondria (Brummond and Burris, 1954). Also as new plants, they contain the right amount of the hormones and cell proteins and organelles making it easy to study the mitochondria processes and cycles (Brummond and Burris, 1954). The method used for homogenization was blending with a waring blender. This method is good because it shreds the plant tissue, cell wall and plasma membrane properly to release its content under a short period of time (20 seconds in this experiment). On the other hand, this method could cause destruction of the mitochondria that is to be analyzed and therefore, there are fewer functional mitochondria released (Gross et al., 2011). Also, the blender can cause the temperature of the homogenate environment to increase and destroy some enzymes. The centrifugation of the homogenate was carried out in two steps (Gross et al., 2011). It was firstly centrifuged at 4000 RPM and then at a speed of 12000 RPM. This is called differential centrifugation (Pon and Schon, 2007). The homogenates contains both large organelles like nuclei and plasma membrane and smaller organelles like ribosomes and mitochondria (Pon and Schon, 2007). At lower speed, larger molecules are pelleted out (Pon and Schon, 2007). In the experiment, at 4000 RPM, the pellet contained the large organelles (nuclei, plasma membrane and intact cells) and the supernatant contained the mitochondria and other smaller organelles (Pon and Schon, 2007). At 12000 RPM, the pellet contained mitochondria with a fluffy layer of destroyed mitochondrial membrane and remains while the supernatant contained the smaller molecules because the mitochondrion is bigger than the other remaining organelles (Pon and Schon, 2007).
In the experiment, DCPIP was used to detect the presence of NADH and FADH₂ molecules which is produced by the Krebs cycle reactions. The presence of NADH and FADH₂ reduces the DCPIP from its blue dye form to a colourless form. Figure 1 shows that most of the curves are curved downwards i.e. a negative slope. Since the absorbance is at 610 nm, this means that as time passed, the blue colour reduced and became more colourless which signifies the presence of NADH and FADH₂ as time proceeded in all four cuvettes. The first cuvette was the control cuvette and contained the reaction buffer, DCPIP, azide and Na succinate. The second cuvette contained the reaction buffer, DCPIP and Na succinate, the third cuvette contains the reaction buffer, DCPIP and azide while the last cuvette contained reaction buffer, DCPIP, azide, Na succinate and malonate. In the Krebs cycle, sodium succinate provides an excess of succinate for the Krebs cycle. Succinate is an intermediate of Krebs cycle and is also used in complex II of the electron transport chain to generate a proton gradient. Malonate inhibits the oxidation of succinate to fumarate and since that is an intermediate reaction in the Krebs cycle, it prevents the completion of the Krebs cycle and reduces the production of FADH₂ (Pardee and Potter, 1949). This means that DCPIP should not be reduced as much and therefore the cuvette containing malonate should not have much colour difference from the colour at the start and therefore the slope of its absorption curve should be very small (very close to a straight line) (Pardee and Potter, 1949). The azide inhibits electron transport in complex IV of the electron transport chain (Bowler et al., 2006). It binds between to heme proteins in the complex, specifically binding between the heme iron and cub in the oxygen reduction site of complex IV (Bowler et al., 2006). This prevents the reduction of oxygen (the electron collector) to water. Therefore, electron transport is not complete and NADH and FADH₂ which carries the electrons to the electron transport chain cannot transfer their electrons to the ETC since the azide prevents the function of the ETC (Bowler et al., 2006). This would cause a very large amount of NADH and FADH₂ to be in the assay because it is being produced in the Krebs cycle but not being used. Therefore, the cuvette containing azide should be very close to colourless at the end of the reaction time (Bowler et al., 2006). As seen from figure 1, the control assay which contains azide and sodium succinate had a very steep decreasing absorption curve which means it turned almost colourless at the end of the reaction time. This backs up the theory explained that azide causes excess of NADH and FADH₂ in the assay (Bowler et al., 2006). Also, the excess of succinate from sodium succinate causes more production of NADH and FADH₂ than regular. The cuvette containing only azide compared to the control is not as decreasing as the control containing azide and sodium succinate, meaning that the presence of sodium succinate causes very large production of FADH₂ because of the role it plays in the Krebs cycle. The assay containing only sodium succinate is not as steep as the control assay because it only contains excess of succinate. Since there is no inhibitor in the Krebs cycle or the electron transport chain, preventing the NADH or FADH₂ form being produced, however this means that the NADH and FADH₂ continue to the electron transport chain where they are then oxidized back to NAD+ or FAD so an excess is not in the solution since it is being used. The assay containing sodium succinate, azide and malonate does not decrease as steeply as the control because of the presence of malonate. This supports the theory explained previously since FADH₂ is not produced and the Krebs cycle is not completed. The absorption curve for this assay starts at a lower absorption because the assay did not have as much mitochondrial suspension as the other three since it had almost finished and therefore it starts at absorption of around 0.72 while the others start at about 0.96.
Compartmentalization in eukaryotes is very advantageous because it organizes functions to specific parts of in the cytoplasm which makes it easy to study each cellular process of the cell individually and draw correct inferences without being disturbed by other cellular processes occurring in the cell (Wu and Scheffer, 1960). Biochemists use this property of eukaryotic cells to their advantage to isolate organelles in the cell and study their functions in other to research specific diseases and find cures to the diseases (Wu and Scheffer, 1960).
In conclusion, mitochondria are a very important organelle in cells because it undergoes cellular respiration for the cell to provide energy in the form of ATP for the cell. Krebs cycle takes place in the matrix of the mitochondria and through this, NADH and FADH₂ molecules are produced which then provide electrons for the electron transport chain where ATP is produced. Azide and malonate are inhibitors of cellular respiration in cells by either acting as a competitive inhibitor for succinate dehydrogenase that converts succinate to fumarate or by preventing reduction of oxygen to water in the ETC. Lastly, the study of the mitochondria is important for biochemist in the pharmaceutical industry for manufacturing medicine and finding sources and cures to illnesses.

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