Cell Fractionation: Isolation of Mitochondria from Cauliflower and Determination of Specific Enzyme Kinetics
Mitochondria is an organelle found in eukaryotic cells that play a role in biochemical processes such as respiration and energy production. Mitochondria even play an important role in apoptosis, or programmed cell death. This is achieved by disruption of electron transport, oxidative phosphorylation, and ATP production or even the release of proteins that trigger activation of caspase family proteases and alteration of cellular reduction-oxidation (redox) potential (Reed and Green, 1998). Succinate dehydrogenase (SDH) and malate dehydrogenase (MDH) are enzymes that play a role in the Citric Acid Cycle portion of cellular respiration and are investigated further in this experiment.
The purpose of this experiment was to prepare whole as well as broken preparation of mitochondria and compare the enzymatic activity of succinate dehydrogenase (SDH) and malate dehydrogenase (MDH). This experiment will answer the question of whether enzymatic activity will be greater in intact or broken mitochondria as well as in SDH or MDH. It is hypothesized that enzymatic activity will be higher in intact mitochondria because the intact mitochondria contains both SDH and MDH, where the broken mitochondria only contains MDH. Because the broken mitochondria only contain one enzyme complex, there should be less enzymatic activity. It is hypothesized that the MDH activity will be greater in broken mitochondria rather than intact because in the broken mitochondria, the membrane is removed. Because the membrane is removed, the MDH won’t have to diffuse through said membrane to reach the proteins, thus causing activity to occur faster. It is also hypothesized that SDH activity will be greater in the intact mitochondria rather than the broken mitochondria because the SDH is embedded in the membrane of the mitochondria. This would mean that there shouldn’t be any SDH in the broken mitochondria, thus making the SDH activity greater in the intact mitochondria. During this experiment, two tubes of mitochondria were isolated through filtering a suspension made of cauliflower, cold mannitol assay, and sand, centrifuging, and decanting the postmitochondrial supernatant. In two tubes, the mitochondrial pellet was resuspended, and then the pH was adjusted in order to rupture the mitochondria. After this, they were centrifuged. This is also done to the two other tubes, except the pH is not adjusted, therefore the mitochondria is still intact. After this was done, assay buffer, sodium azide, and DCIP were added to 24 test tubes. In six of those test tubes, succinate and the broken mitochondria were added. In another six, succinate and intact mitochondria were added. In another six, malate and broken mitochondria were added, while in the last six, malate and intact mitochondria were added. These test tubes were then placed in a spectrophotometer and the absorbance was read. From this, the enzymatic activity of the mitochondria could be compared. Results
The absorbance values were read multiple times over a thirty minute span and resulting data was plotted in four different plots in order to compare the enzymatic activity in broken and unbroken mitochondria with SDH and broken and unbroken mitochondria with MDH. The relative amount of enzymatic activity could be determined from the slope and r2 values of the four plots. A standard amount of protein was assumed in the plots. According to Figure 1,which represents SDH activity in broken mitochondria, the slope of the plot is -0.0002 and the r2 value is 0.497. With the slope being so low and the r2 value so far from 1, it can be determined that very little enzymatic activity occurred. Figure 2 represents SDH activity in intact mitochondria and has a slope of -0.0038 and an r2 value of 0.979. With an r2 value closer to one, it can be determined that a good amount of activity occurred....
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