Bean Sprout Homogenate

Question: Which cell fraction from the prepared bean sprout homogenate, after multiple different intensity centrifuges, will contain the most present amount of mitochondria represented by the biochemical marker succinate dehydrogenase (SDH) in the cell fraction? The initial cell fraction, Pellet 1 (P1), consisted of 20 g of bean sprouts grinded down with 5 g of purified sand and Grinding Buffer chilled for a total amount of 40 mL. This (P1) was then centrifuged for 2 min at 600 x g to get the denser solid Pellet 1 at the bottom of the tube and its liquid supernatant at the top of the tube. The supernatant from P1 was then taken and centrifuged in a different tube for 5 minutes at 2000 x g to get Pellet 2 (P2) at the bottom and its liquid supernatant
at the top, like done to P1. This process was then repeated again using the supernatant of P2 centrifuged for 15 minutes at 21,000 x g. Hypothesis: Since we discovered that most the enzymes of the Citric Acid Cycle and the electron transport chain are in the mitochondria (Leicht and McAllister, 59).
Also, that when electrons from SDH-FADH2 are transferred to DCIP a change in color occurs from blue to colorless. This color change is proportion to the amount of SDH which represents the mitochondria in the cell fraction (Leicht and McAllister, 71). We developed the hypothesis that the less dense mitochondria will continue to be separated from other denser cell organelles after each centrifuge until the least dense mitochondria will be left in Supernatant 3, a higher fraction that is more colorless than the rest due to the SDH
enzyme.
Experiment Two Question: What effect does the presence of malonate in the reaction have on the activity of SDH?
We chose to test the mitochondria under the presence of malonate because from research we discovered that malonate is the specific inhibitor for succinate dehydrogenase (C. A. Baumann and F. J. Stare). The inhibitor in this case will slow down or stop the catalysis action of the SDH enzyme causing the cuvette tubes to become more colorless. Hypothesis: Since unexpectedly our P3 had the greatest amount of SDH activity, which corresponds to greatest amount of mitochondria in a cell fraction, we used this in continuation to Part III. We expect that the cuvette with the most amount of malonate (0.1 mL for cuvette 3) will be the least absorbent at 600 nm of DCIP compared to the other two cuvettes which we will react with a lesser amount of malonate (0.01 mL for cuvette 1 and 0.05 ml for cuvette 2). Due to the malonate slowing down the action of the SDH enzyme to form DCIP, the cuvette with the most malonate will be more colorless than the rest signaling the most mitochondria
present.