13
Purification of Mitochondria by Differential Sedimentation and Monitoring of Fractions for Specific Activity of Succinate Dehydrogenase
Abstract:
The main goal of this experiment was to find purified mitochondrial protein from liver. This was done by making a series of pellets and supernatants, and fractions were then made. After the fractions were made, they were treated with Bradford Reagent to indicate the amount of protein present. Then the fractions were then put through an assay procedure, where they were read in a spectrometer. The spectrometer gave the absorbance and %transmittance. From the absorbencies, a graph was made, along with an equation. When the absorbencies from another dilution were plugged into the equation four protein concentrations were found; homogenate = 14.749, nuclear = 2.609, mitochondrial = 12.896, and soluble = 6.041. From these the total proteins were found; Homo. = 265.5, nuclear = 13.045, mito. = 64.5, and soluble = 87.6. The next step was the making of standards. From the spectrometer readings from the standards calculations were made of the units of activity, units per ml, crude activity and specific activity. The fraction with the largest unit of activity (.3055) was the nuclear. The nuclear also had the largest units per ml (1.5275). The mitochondrial-10 had the largest crude activity (7.540). The nuclear had the largest specific activity (1.1709); however, there was a contamination of proteins in the nuclear fraction because the nuclear was not supposed to be the largest one. A way of improving this experiment was that it could have been done slower and more preparation could have gone into it. However, this experiment was very useful in describing where the largest amount of protein concentration was in a cell. Because where the highest concentration of protein was in a cell, that organelle was bound to be the ‘work house’ of the cell.
Introduction:
The main objective of this experiment was to isolate a purified sample of mitochondrial proteins from liver. This was done by a procedure called differential sedimentation. This was done by centrifuging the liquid, letting the more dense particles settle to the bottom of the tube faster. When the procedure was done a pellet was formed, along with a supernatant. Depending on what stage the experiment was at, the pellet will be consist of crude homogenate, nuclear, soluble, or mitochondrial. The crude homogenate was everything, including the nuclear, mitochondria and everything else in the cell. The nuclear was everything inside the nucleus, including the nucleoplasm and nuclear envelope. The mitochondrial was usually pure mitochondria. The soluble was everything except the nuclear and mitochondria.
At the initial stages of the experiment sucrose buffer was used to rinse off the liver during homogenizing. The sucrose buffer kept the environment at a pH of 7.0. Another reagent used was Bradford reagent, its purpose was to stain the proteins, and this was how the amount of protein was indicated. The bluer the more proteins were present, and vice versa. Another reagent used during the course of this experiment was in the assay medium. The assay medium included 10mM succinate, which acted as the substrate. Also 0.02mg/ml DPIP (2, 6-dichlorophenol indophenol), when oxidized it absorbs 600nm strongly, thus becoming colorless when it was reduced. This was why the best reading happens when there was very little DCPIP present. Also 10mM phosphate buffer was included, this controlled the pH at 7.4. 2mM of KCN were in there to block the electron transport chain from happening. 10mM of CaCl2 was there to increase the mitochondria’s permeability, so that the amount of SDH could be known. Albumin was present in the amount of 0.5mg/ml, since the permeability was increased; the albumin was there to keep the mitochondria from bursting.
The only new machine that was used during this experiment was the spectrometer. A spectrometer was an instrument that was used for measuring the amount of light of different wavelengths that are absorbed by a solution. The spectrometer was used to measure the %transmittance and absorbance (OD) of each fraction in the experiment.
Another main objective of this experiment was to look for the succinate dehydrogenase (SDH). As a coenzyme, SDH will catalyze the oxidation of the fumarate and succinate. And since KCN was present the electron transport chain was stopped and the FAD keeps the electrons and reroutes it to the DCPIP, thus changing its color. The SDH was found bounded to the inner membrane of the mitochondria, here the Krebs’ cycle occurs, the whole reaction takes place with pyruvate and in an aerobic environment.
Procedure:
One watch glass was obtained, along with a new razor blade and a graduated cylinder. The watch glass was used to zero out the scale, and it was also used because it served as a hard surface to cut on. Next a piece of liver was obtained and 2 grams were weighed out. The liver was then cut up on the watch glass. Next the liver was put into a homogenizer vessel along with 5ml of sucrose buffer. The sucrose buffer contained: .25M sucrose in 10mM phosphate buffer, which had a pH of 7.0, this helped keep the solution neutral through out the experiment. Then the tube was put into the homogenizer, and homogenized until there were no liver pieces left, about 15 times. The homogenized liver was then strained through a monolayer of cheesecloth into a test tube. After that 15ml of the sucrose buffer was put in the test tube, and stirred. This made a total of 20ml in the tube. Next 2ml were taken out of the tube, 1ml was put in one test tube and 1ml placed in another tube, leaving 18ml in the original test tube. This tube was then spun at 600g forces for a total of 10 minutes. This produced a pellet (#1) and a supernatant (#1).
First the supernatant (#1) was poured out into a new test tube. Then the supernatant was spun at 10,000g forces for 20 minutes. This produced a pellet (#2) and a supernatant (#2). The supernatant (#2) was named Soluble Fraction, and it was poured out and divided into two equal parts, 7.25ml went into one tube and 7.25ml went into another test tube. Then the tubes were placed into the freezer. Next the pellet (#2) was resuspended with 5ml of sucrose buffer and inverted to mix the pellet. This was named the Mitochondrial Fraction. It was divided into two equal parts, 2.5ml were placed in a tube and 2.5ml were placed in another tube. Then they were put into the freezer, until needed.
Then pellet #1 was resuspended with 20ml of sucrose buffer and inverted. It was then spun at 600g forces for 10 minutes. The supernatant was poured out and discarded. The pellet (#3) was resuspended with 5ml of sucrose buffer and inverted. This was labeled the Nuclear Fraction. Then it was divided into two equal parts, 3ml in one tube and 3ml in another tube. Then they were placed in the freezer, until needed.
One sample from each fraction was taken out of the freezer and placed on a test tube rack, and allowed to thaw. Eight 1.5ml test tubes were gathered and labeled 1 through 7. Then dilutions were made in each tube, and they were called the standards. The reagents that were added to each tube included Bovine Serum Albumin (BSA), which was in a concentration of 200mg/ml, distilled water, and the assay. In tube #1, it had 1ml of BSA and no water added to it, the dilution factor was zero, so the concentration was 1000micrograms/ml, and the amount in assay was 100 micrograms. In tube #2, 0.8ml of BSA and 0.2ml of water was added to the tube, the dilution factor was 4:5, with a concentration of 800microgram/ml, and an assay of 80micrograms. In tube #3, there was 0.6ml of BSA and 0.4ml of water added to the tube; the dilution factor was 3:5, with a concentration of 600microgram/ml and an assay of 60micrograms. In tube #4, there was 0.4ml of BSA and 0.6ml of water added to the tube, the dilution factor was 2:5, with a concentration of 400microgram/ml, and the assay was 40microgram. In tube #5, 0.2ml of BSA and 0.8ml of water was added to the tube, the concentration was 200 micrograms/ml, the assay was 20micrograms, with a dilution factor of 1:5. In tube #6, 0.1ml of BSA and 0.9ml of water was added to the tube, with a dilution factor of 1:10, and a concentration of 100microgram/ml, the assay was 10 micrograms. In tube #7, just 1.0ml of water was added to the tube, this was the control (blank).
Next seven spectronic tubes were gathered and numbered 1 through 7. Then 0.1ml of each standard that was in the test tubes was pipetted into the corresponding spec tube. Then 5.0ml of the Bradford reagent were added to each tube, and stirred with a piece of Parafilm on top, so as not to get contaminated. Each tube was allowed to sit for five minutes, to allow the color to develop. The Bradford reagent indicated the amount of protein present in the solution in each tube. This was indicated by the color of blue that was shown in each tube. The more blue, the more protein that was present. The less blue, the less protein. One tube was prepared with just Bradford reagent, this became the blank. The blank was used to zero out the spectrometer, so that the spectrometer would read 100 and 0. The spectrometer was set to 595nm. The spectrometer was used to measure the absorbance and % transmittance for each of the fractions. Before each tube was placed in the spectrometer, a Kim wipe was used to clean off any fingerprints off the tube that could foul up the readings. Also before a tube was placed in the spectrometer the blank was used to zero out the machine. After the data was taken for each tube it was plotted on a graph, with the BSA concentrations on the x-axis and the absorbance on the y-axis. Then a straight line was plotted in between all of the points; this became the standard curve. This graph was used later on in the experiment.
Next eight more 1.5ml tubes were gathered. And reagents were added to each tube, the reagents included, the fractions previously made, and distilled water. Tube 1 had a dilution factor of 1:10, with 0.5ml of the homogenate fraction with 4.5ml of distilled water added to the tube. Tube 2 had a dilution factor of 1:20, with 0.5 homogenate fraction and 9.5ml of distilled water added together. Tube 3 had a dilution factor of 1:5, with 0.5ml of nuclear fraction and 2.0ml of distilled water. Tube 4 had a dilution factor of 1:10, with 0.5ml of nuclear fraction and 4.5ml of distilled water. Tube 5 had a dilution factor of 1:5, with 0.5ml of mitochondrial fraction and 2.0ml of water. Tube 6 had a dilution factor of 1:10, with 0.5ml of mitochondrial fraction and 4.5ml of distilled water. Tube 7 had a dilution factor of 1:5, with 0.5ml of soluble fraction and 2.0ml of distilled water. Tube 8 had a dilution factor of 1:10, with 0.5ml of soluble fraction and 4.5ml of distilled water. When the reagents were added to each tube, pieces of Parafilm were placed on top of the tubes and inverted, this was to protect the hands from unnecessary contamination from the chemicals that were used. Next 0.1ml of the diluted samples were put in the next set of spec tubes. Along with the samples, 5.0ml of the Bradford reagent were put in the tubes too. Then the tubes were allowed to sit for five minutes, to let the color develop. The spectrometer was set to 595nm. The blank from the previous section of the experiment was used to zero out the machine; also Kim wipes were used. The data gathered from the spectrometer was used to compare with the graph that was given from the previous section.
Next an assay was done on the fractions for the succinate dehydrogenase (SDH). First the spectrometer was set to 600nm. Then 2.8ml of the assay medium was pipetted into a spectrometer tube, and placed in a 37OC water bath. The assay medium included 10mM succinate, which was the substrate, 0.02mg/ml DCPIP, which acted as the indicator dye that changed from purple to clear when reduced, 10mM phosphate buffer, which kept the pH at 7.4, 2mM KCN, which helped block the electron transport chain, 10mM CaCl2, which increased the mitochondrial permeability, and 0.5mg/ml albumin, which kept the osmotic pressure from increasing in the mitochondria, to keep it from bursting. A blank, which included buffered sucrose, was used to zero out the spectrometer. The reactions were ran in sets of three. Group1 included crude homogenate, nuclear and soluble. Group 2 included the mitochondrial fractions. At time 0 minutes, 200ml of group1 was added to the tube and mixed thoroughly. The tube was placed in the spectrometer and the absorbency was read immediately. The tube was then placed in the water bath immediately afterwards. At time 3 minutes, the absorbency was read again, and the tube was placed in the water bath afterwards. At time 4 minutes Group2 was started the same way as Group1. At time 15 minutes, the absorbency was measured again and the tube was placed in the water bath afterwards. This procedure was repeated for the blank, homogenate, mitochondria, nuclear, and soluble fraction. The dilutions were made with the fraction and sucrose buffer as follows: for the crude homogenate, nuclear, and soluble there was a 1:2 dilution, 500ml of fraction and 500ml of sucrose buffer. For the mitochondria, three dilutions were made: 1:5, 200ml of fraction and 80ml of sucrose buffer, 1:10, 100ml of fraction and 900ml of sucrose buffer, 1:25, 40ml of fraction and 960ml of sucrose buffer. The time line for each group was as follows; Group1= 0-3-10-15-20: Group2 = 4-7-12-17-22. This data with the data from the above section was used to find the protein concentration, total proteins present, crude activity of the proteins, and specific activity for the proteins.
Results:
There was an error in the data gathered during the laboratory session, so data from another laboratory session was used for the results below.
The first time that the tubes were put in the spectrometer the readings were: