Subcellular Fractionation Results and Discussion

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Subcellular Fractionation Results and Discussion Subcellular Fractionation is a technique used to separate the cellular components based on the density of the molecules. The technique specifically used differential centrifugation, which involves incrementally centrifuging a filtrate at different speeds. At slower speeds, the pellet formed will contain components with smaller densities. Then, at higher speeds, molecules with higher densities will centrifuge down. The purpose of this particular lab was to separate the mitochondria and nuclei of cauliflower cells. The presence of nuclei was tested using microscopy, and the presence of mitochondria was tested using an SDH assay. If a filtrate is centrifuged twice at a lower and higher speed, then there will be more nuclei present in the first pellet, than the first supernatant, and there will be more mitochondria present in the second pellet and first supernatant. Results To observe the effects of differential centrifugation, microscopy was done on the filtrate, supernatant 1, and pellet 1, to determine which sample would have more nuclei. The filtrate has the greatest number of nuclei present, while S1 and P1 have similar amounts (Figure 1). Filtrate 10x S1 10x P1 10x Figure 1. Microscopy of Nuclei. Images taken of microscopy for Filtrate, S1, and P1, after differential centrifugation. In order to observe the SDH activity within each sample, the absorbance of each sample was measured at 600nm immediately after the addition of the subcellular fraction sample, and then again after 3 minutes. The difference between the ODs was calculated, and that number was used to determine the velocity of the reaction. The first graph displays SDH activity for the subcellular fractions. The first pellet had the highest amount of SDH activity, followed by S1, Filtrate, P2, and S2, having the least amount of SDH activity. The second graph displays the control activity. The exclusion of succinate displayed the highest reaction rate, followed by malonate, the P2, then finally azide. (Figure 2 and Figure 3).

Filtrate S1 P1 S2 P2 SDH Activity Fraction Velocity (µmoles/min) Figure 2. SDH Activity Bar Graph of Fraction Velocities. Bar graph of SDH activity velocities for each fraction. P2 malonate azide succinate Control Activity Fraction Velocity µmoles/min Figure 3. Control Activity Bar Graph of Fraction Velocities Bar graph of velocities of reactions for the controls that were excluded from the cuvettes during the measurement of absorbance. Discussion If a filtrate of cauliflower cells is centrifuged using a differential centrifugation technique, there will be more nuclei present in the filtrate and pellet than in the supernatant. Based on Figure 1, the microscopy of filtrate showed a more substantial number of nuclei. Although it is hard to determine a large difference between the P1 and S1 microscopies, it can be concluded that S1 has more nuclei. Because of this result, we can determine that nuclei are denser than mitochondria, causing them to be more prevalent in the pellet. We are able to determine that

the cellular components being seen are nuclei because of the usage of azure C dye. This dye particularly stains nuclei making them visible under the microscope. Since there are similar amounts of nuclei in the S1 and P1 samples, there must have been errors along the way. These could have come about from improper centrifugation technique such as not properly balancing the centrifuge or centrifuging at a gravity number too small or too high. If the cellular components of cauliflower are separated using subcellular fractionation, then the filtrate, supernatant 1 and pellet 2 will have more mitochondria present than supernatant 2 and pellet 1. If malonate is added to, or succinate or azide is excluded from the P2 fraction, there will be less SDH activity. The results expected from Figure 2 was that P2 would have the highest velocity of reaction because of a higher amount of mitochondria present. The actual results determine that P1 had the highest number of mitochondria. The results expected from the control graph was a decrease in velocity of reactions for the controls in comparison to the P2 sample. The addition of malonate would decrease SDH activity, and the removal of sodium azide and succinate would cause little to no SDH to be reduced. According to figure 3, there was a decrease in SDH activity for the malonate and azide controls, however there was a significant increase for the succinate control rate of reaction. These controls are expected to reduce SDH activity because of their roles the oxidation of succinate to fumarate. Adding sodium azide to the reaction blocks the flow of electrons through the electron transport chain. This allows for an artificial electron acceptor to accept electrons in place of SDH. DCIP was used as the artificial electron acceptor because when it is oxidized, it is a dark blue color, but when it is reduced, it is colorless. This allows for the visualization and experimental testing of the affects that the controls have on the reaction. Malonate is a competitive inhibitor that binds to the active site of SDH. This prevents succinate from binding which causes a decrease in SDH activity. Sodium azide is an inhibitor to the electron transport chain. It increases the amount of FADH 2 to be oxidized by DCIP. Therefore, when it is removed the amount of FADH 2 decreases which decreases DCIP reduction. Finally, the removal of succinate means that fumarate cannot be formed which results in little to no SDH activity. The calculations of the velocity of the reactions from the measured absorbances are shown below.

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Based on the results of this experiment, it can be concluded that mitochondria and nuclei were not successfully separated from the cauliflower cells. This is because of an excess amount of mitochondria present in the first pellet which was expected to contain little to no mitochondria. In addition, there was not a significant difference in number of nuclei visible in the microscopies of the first pellet and supernatant. Subcellular fractionation is important because it can allow cellular components to be separated without disrupting their functions. This allows us to study them in greater detail which can help find cures for diseases or mutations of particular molecules.

Works Cited Paulo, J. A., Gaun, A., Kadiyala, V., Ghoulidi, A., Banks, P. A., Conwell, D. L., & Steen, H. (2013). Subcellular fractionation enhances proteome coverage of pancreatic duct cells. Biochimica et biophysica acta , 1834 (4), 791–797. https://doi.org/10.1016/j.bbapap.2013.01.011

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