Subcellular Fractionaion

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DeAndre Borders 26 October 2023 BIO 3810 Molecular Bio Subcellular Fractionation Mini Results This experiment allows for the use of biotechnological procedures to separate subcellular components within a cell. In this experiment, the organelles in mitochondria and the nuclei are the targeted organelles, and with isolation will be necessary. Isolation of organelles will be done by subcellular fractionation and incremental increases in centrifugation based on the densities of the organelles. In turn, the centrifugation will cause the production of samples of P1/P2 and Filtrate. The purpose of this lab is to take a sample of cauliflower and centrifuge the lysate to separate the cellular organelles, nuclei, and mitochondria. Once separated, the nuclei were stained using Azure C dye and observed via microscopy. Nevertheless, the mitochondria were observed under conditions tested by assaying succinate dehydrogenase (SDH). Succinate Dehydrogenase is an enzyme found in the inner membrane of mitochondria that reduces FAD+ to FADH2 and oxidizes succinate into fumarate. It was hypothesized that if sample P2 contains high amounts of SDH, P2 will contain the most mitochondria compared to the other samples. This experiment was conducted over a 3-week, which included the following controls of Dichlorophenolindophenol (DCIP), Malonate, Azide, and Succinate as independent controls. The measurement of DCIP velocity is the dependent control. In order to test to see if mitochondria activity was present, the previously stated controls severed to interfere with or manipulate SDH activity, thus reducing or increasing the speed at which DCIP is reduced. Utilization of competitive inhibitors affected different areas of aerobic respiration, such as the Citric Acid Cycle (TCA) or the Electron Transport Chain (ETC). The inclusion or exclusion of each of the enzymes affects the transport of electrons through aerobic respiration processing. Results: Figure 1, Filtrate Microscopy: The depicted Figure displays the cauliflower cell centrifuge with equal parts of mitochondria and nuclei, magnification of 40x. There is an abundance of both
Subcellular Fractionation Mini Results 2 organelles yet the focus for Figure 1 will be the amount the nuclei present within the figure. Though one nucleus is shown the surrounding surface area displays a multitude of nuclei. Figure 2, Sample P1 Microscopy: Figure 2m, depicts the nuclei in pellet form and observed under a microscope, magnification at 40x. However, in this figure the amount of mitochondria that is present cannot be ignored, the overall dominate organelle shown is nuclei. In order to separate this further the Mannitol Assay will need to be added to P1 and centrifuged. Figure 3, Sample S1 Microscopy: This figure depicts the supernatant of the cauliflower sample at a magnification of 100x. This liquid is mixed with cellular organelle content of nuclei and mitochondria. In this supernatant there are more nuclei than mitochondria. The focus of the microscopy is to focus on the presence of nuclei. This figure shows the subtle decreasing number of nuclei as the sample continues to undergo subcellular fractionation.
Subcellular Fractionation Mini Results 3 Figure 4, Sample S2 Microscopy: Figure illustrates the original sample after undergoing centrifugation, at a magnification of 400x. The number of nuclei has significantly reduced with the addition of Mannitol Assay. The cellular organelle that is dominant are the mitochondria, yet a small percentage of nuclei remain within the cell. Figure 5, Velocities of Subcellular Fractionation Bar Graphs: The velocities of each of the samples were measured after the SDH Assay, the utilization of DCIP served as a final electron acceptor within the ETC rather than oxygen. This allowed for the visual change to occur as DCIP was reduced, when oxidized in a solution it is blue and colorless when reduced. Since filtrate contained an equal part of mitochondria and nuclei its velocity is higher than S1,P1,S2. However, P2 displayed the highest velocity, showing the correlation between DCIP reduction and number of mitochondria as it relates to velocity and SDH presence. 1.428571429 0.825396825 0.666666667 0.126984127 4.53968254 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 Velcoities (μmole/min) Fractions Velocities of Fractions Filtrate S1 P1 S2 P2
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Subcellular Fractionation Mini Results 4 Figure 6, Control Velocity Bar Graphs : By adding the three controls—malonate, azide, and succinate control—the mitochondrial activity of the fractions F, S1, P1, S2, and P2 was measured. Their individual velocities were computed. The P2 and Malonate showed the highest velocity, while the S2 and Azide control showed the lowest. Succinate SDH activity was almost similar to Malonate control, indicating some SDH activity. Calculations: Velocity of DCIP reduction = ∆OD × 2mL 𝜀??𝐼𝑃 × 3?𝑖? 𝐹𝑖???𝑎?? = (0.575 − 0.620) × 2?? 21 ?? − 1?? − 1 × 3 = 0.001428 × 1000 = 1.428 𝑆𝑎???? 𝑆1 = (0.517 − 0.543) × 2 21 × 3 = 0.000825 × 1000 = .8250 𝑆𝑎???? 𝑃1 = (0.686 − 0.707) × 2 21 × 3 = 0.000667 × 1000 = .6667 𝑆𝑎???? 𝑆2 = (0.273 − 0.277) × 2 21 × 3 = 0.000127 × 1000 = .1270 0.349206349 1.492063492 0.158730159 1.365079365 4.53968254 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 Velocities (μmole/min) Fractions of Controls Velocities of Controls Positive Control Malonate Control Azide Control Succinate control P2
Subcellular Fractionation Mini Results 5 𝑆𝑎???? 𝑃2 = (0.992 − 0.849) × 2 21 × 3 = 0.004540 × 1000 = 4.544 𝑃??𝑖?𝑖?? ??????? = (0.267 − 0.278) × 2 21 × 3 = 0.000349 × 1000 = 0.349 0 ??????? − ?𝑎???𝑎?? = (0.400 − 0.447) × 2 21 × 3 = 0.000349 × 1000 = 1.4920 ??????? − 𝐴𝑧𝑖? = (0.274 − 0.279) × 2 21 × 3 = 0.000158 × 1000 = 0.158 ??????? − 𝑆???𝑖?𝑎?? = (0.115 − 0.158) × 2 21 × 3 = 0.00136 × 1000 = 1.365 Discussion: The goal of this experiment was to separate the cauliflower's mitochondria and nuclei using the subcellular fractionation technique via differential centrifugation. In addition, when observing the experiment, the verification of the hypothesis that if sample P2 contains high amounts of SDH, then P2 will contain the most mitochondria compared to the other samples was either supported by the data or refuted. The various densities displayed by the mitochondria and nucleus dictated the differentiable centrifugation of the fractions F, S1, P1, S2, and P2. Due to mitochondria's more significant density, the organelle will produce a larger pellet than a nucleus. Following the centrifugation of each sample, the presence of nuclei was assessed using microscopy at different magnifications. The enzyme succinate dehydrogenase is present in the mitochondria in the TCA Cycle during photosynthesis; an SDH assay was utilized to identify and validate the mitochondria present in the fractions. The enzyme succinate dehydrogenase's purpose is to catalyze the oxidative reaction, causing the conversion of succinate to fumarate. The first phase of this experiment allowed for the observation of the nuclei isolated from the cauliflower. The expectation was that as the samples underwent centrifugation, the number of nuclei would decrease in each sample, specifically in samples S2 and P2. Filtrate, S1, and were expected to have the most significant number of nuclei when observed under microscopy. This expectation was verified and supported. After dying the solutions with Azure dye, the organelles can be identified clearly. Figure 1 filtrate depicts equal amounts of mitochondria and nuclei. Compared to Figure 4, there is some presence of nuclei, but minimal when compared to Figure 1 or 2. In Figure 4, the large amounts of mitochondria can be identified in a dark purple pigmentation. In order to accurately measure the presence of mitochondria, the enzyme SDH was used as an indicator. As succinate is oxidized to fumarate, the reaction is coupled with the reduction of FAD+ to FADH2, which the ETC then uses. Succinate from the TCA cycle links to the ETC to transfer electrons from FAD+ to ubiquinone. (Jones, 2013). In order to completely replicate the ETC, DCIP was used as the electron acceptor. The expected results of the SDH Assay were that sample P2 would more than likely have the highest velocity due to the high volume of mitochondria presence and its ability to oxidize DCIP at a faster rate than the other samples that contained smaller amounts of mitochondria activity. Figure 5 supports the
Subcellular Fractionation Mini Results 6 expectation with sample P2 velocity at 4.5mM/min. P2's velocity also indicates that the solution for P2 was clear if DCIP was completely reduced without interference. Surprisingly, S2 was the lowest at .12mM/min; the expectation was that this would be higher, assuming that the S2 was mostly mitochondria like P2. However, S2 data indicates little presence of mitochondria and more nuclei. In order to observe the effects of aerobic respiration regarding the transferring of electrons to produce ATP, controls were added and removed from the solution. Each solution was then processed through a spectrophotometer at 600nm to measure the amount of SDH activity. Malonate was implemented as a competitive inhibitor and control, effectively binding to the substrate instead of succinate dehydrogenase. (Anastacio et al, 2013) The expected result was that P2 would have the most SDH activity and succinate would have the most minor activity. Interestingly, Figure 6 indicates that even though Malonate is an inhibitor, DCIP could still be reduced and display mitochondrial activity. P2 displayed the highest velocity of DCIP being reduced, which was expected due to the number of mitochondrial organelles in the sample. Sodium Azide provided little to no SDH activity, yet when this is removed from the ETC, more FADH2 can be oxidized and, therefore, should have SDH activity since electrons are moving through the chain. Sodium Azide, in this case should have shown higher mitochondrial SDH activity. Compared to the succinate negative control, succinate had more mitochondrial activity than Sodium Azide. The removal of succinate will effectively halt the ETC because there are no electrons to pass through the chain. Figure 6 displays the complete opposite and indicates SDH activity. This opposite result was caused by possible contamination of the samples or ineffective removal or omission of succinate and sodium Azide. In summary, Subcellular fractionation is helpful for drug research when attempting to see how organelles are affected by the drugs and the overall response of the body. Figures 4 and 5 show that mitochondria are active in P2, the most abundant sample. Some patterns, meanwhile, were not anticipated. Although P1 was more prevalent in nuclei, its velocity was just 0.153 μmol/min slower than S1's; this could have been caused by mistakes made during separating P1 and S1. It is possible that some samples were contaminated during the transfer, or the centrifuge tube was not clean. However, the hypothesis was mainly supported, claiming that the P2 would have the most SDH activity and, thus, more mitochondria to reduce DCIP faster. When repeated in this experiment, contamination and timing must be monitored closely.
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Subcellular Fractionation Mini Results 7 References Anastacio, M. M., Kanter, E. M., Keith, A. D., Schuessler, R. B., Nichols, C. G., & Lawton, J. S. (2013, June). Inhibition of succinate dehydrogenase by diazoxide is independent of the ATP-sensitive potassium channel subunit sulfonylurea type 1 receptor . Journal of the American College of Surgeons. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3660462/ Jones, A. J. Y., & Hirst, J. (2013, July 22). A spectrophotometric coupled enzyme assay to measure the activity of succinate dehydrogenase . Analytical Biochemistry. https://www.sciencedirect.com/science/article/pii/S0003269713003369?via%3Dihub

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