Question and Hypothesis
Experiment I
Eukaryotic cells contain several membrane-bound organelles. Plant cells are a specific type of eukaryote that are both aerobic and photosynthetic. Because of this, they contain mitochondria that serve a vital role in aerobic respiration that follows photosynthesis. The enzyme succinate dehydrogenase (SDH) is an enzyme associated with the Citric Acid Cycle that takes place in the mitochondrial matrix. SDH serves as a catalyst for a reaction in which succinate and FAD become fumarate and FADH2. The SDH-FADH2 complex then reduces Ubiquinone in the electron transport chain. SDH can be indirectly measured using the artificial electron acceptor DCIP that is blue in its oxidized state and colorless in its reduced …show more content…
This change in color can then be related to the concentration of SDH , which catalyzes the reaction in mitochondria. To begin, bean sprout homogenate was made. The homogenate was then centrifuged three different times at increasing speeds. Pellet two, pellet three, and supernatant three were then saved. From here, solutions containing assay buffer mixed with with the electron inhibitor sodium azide and the artificial electron acceptor DCIP, and SDH’s substrate, succinate, were prepared. When the saved cell fractionates stored on ice were added to the prepared room temperature solutions, the color change of the solution due to the reduction of DCIP was immediately measured using a spectrometer. Measurements were then taken two more time in seven minute increments.
The amount of reduction of DCIP as measured by the decreased absorbance by the Spec 20 over time can be viewed in Figure 1. The total change in absorbance can be seen in Figure 2. The line representing no cell fraction was a negative control in which little to no change was expected. The independent variable was the cell fraction added to solution, and the dependent fraction was the resulting absorbance decrease. The solution with supernatant 3 has the smallest change in absorption besides the control, and the solution containing pellet 3 had the largest …show more content…
The Spec 20 measurements showed the greatest decrease in absorbance for the solution with pellet three. This decrease in absorbance is due to reduction of DCIP by the SDH-FAD2 complex. Because the enzyme SDH is only found in the mitochondria, it can be concluded that pellet three contained the most mitochondria of the cell fractions. Repeating the same experiment but varying the final centrifugation speed would be a way to maximize the amount of mitochondria in pellet 3 which could then be used for further experiments.
Experiment II Our hypothesis that if the cell fraction with mitochondria is exposed to an environment either too hot or too cold, it will change the structure of the enzyme enough that it will not function properly since protein confirmation is sensitive to temperature was supported by the data collected in experiment II. The SDH warmed to 37C showed the largest reduction in absorbance. In other words, the enzyme SDH was most effective at 37C. However, when the enzyme was exposed to temperatures hotter and colder than 37C, the absorption decreased significantly less showing that SDH was not as effective in transferring electrons to DCIP for those temperatures. It is also notable that when the enzyme was incubated at 65C, the absorbance increased very slightly, which would technically correspond to DCIP oxidation. However, his seems unlikely
Experiment I
Eukaryotic cells contain several membrane-bound organelles. Plant cells are a specific type of eukaryote that are both aerobic and photosynthetic. Because of this, they contain mitochondria that serve a vital role in aerobic respiration that follows photosynthesis. The enzyme succinate dehydrogenase (SDH) is an enzyme associated with the Citric Acid Cycle that takes place in the mitochondrial matrix. SDH serves as a catalyst for a reaction in which succinate and FAD become fumarate and FADH2. The SDH-FADH2 complex then reduces Ubiquinone in the electron transport chain. SDH can be indirectly measured using the artificial electron acceptor DCIP that is blue in its oxidized state and colorless in its reduced …show more content…
This change in color can then be related to the concentration of SDH , which catalyzes the reaction in mitochondria. To begin, bean sprout homogenate was made. The homogenate was then centrifuged three different times at increasing speeds. Pellet two, pellet three, and supernatant three were then saved. From here, solutions containing assay buffer mixed with with the electron inhibitor sodium azide and the artificial electron acceptor DCIP, and SDH’s substrate, succinate, were prepared. When the saved cell fractionates stored on ice were added to the prepared room temperature solutions, the color change of the solution due to the reduction of DCIP was immediately measured using a spectrometer. Measurements were then taken two more time in seven minute increments.
The amount of reduction of DCIP as measured by the decreased absorbance by the Spec 20 over time can be viewed in Figure 1. The total change in absorbance can be seen in Figure 2. The line representing no cell fraction was a negative control in which little to no change was expected. The independent variable was the cell fraction added to solution, and the dependent fraction was the resulting absorbance decrease. The solution with supernatant 3 has the smallest change in absorption besides the control, and the solution containing pellet 3 had the largest …show more content…
The Spec 20 measurements showed the greatest decrease in absorbance for the solution with pellet three. This decrease in absorbance is due to reduction of DCIP by the SDH-FAD2 complex. Because the enzyme SDH is only found in the mitochondria, it can be concluded that pellet three contained the most mitochondria of the cell fractions. Repeating the same experiment but varying the final centrifugation speed would be a way to maximize the amount of mitochondria in pellet 3 which could then be used for further experiments.
Experiment II Our hypothesis that if the cell fraction with mitochondria is exposed to an environment either too hot or too cold, it will change the structure of the enzyme enough that it will not function properly since protein confirmation is sensitive to temperature was supported by the data collected in experiment II. The SDH warmed to 37C showed the largest reduction in absorbance. In other words, the enzyme SDH was most effective at 37C. However, when the enzyme was exposed to temperatures hotter and colder than 37C, the absorption decreased significantly less showing that SDH was not as effective in transferring electrons to DCIP for those temperatures. It is also notable that when the enzyme was incubated at 65C, the absorbance increased very slightly, which would technically correspond to DCIP oxidation. However, his seems unlikely