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Mitochondrial Enzyme Lab Report

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Testing for the Activity of a Mitochondrial Enzyme
BIOL: 1411: 0A09

Jordyn Kuehl
October 3, 2017

Partners: Lexi Zocher, Steve Coutteau

I. Question & Hypothesis
In experiment I and II we attempted to take cell fractions of cauliflower, created through a series of differential centrifugations, and ultimately determine which cell fraction contained the greatest number of mitochondria. The Citric Acid Enzyme succinate dehydrogenase (SDH) is a biochemical marker that allows us to indirectly asses the presence of mitochondria. SDH is an enzyme found in the inner mitochondrial membrane and is responsible for catalyzing the oxidation of succinate into fumarate. The mitochondria can be isolated through differential centrifugation and then treated …show more content…

We were only able to take 3 readings (at 0 min, 7 min, and 14 min) of the test tubes (E1-E4). The greatest change in absorbance was obtained for test tube E4 (Supernatant 3) at 1.8 nm. This tells us that supernatant 3 contained the largest number of mitochondria relative to the other cell fractions. E3 (pellet 3), contained the lowest absorbance reading, indicating that it had the lowest number of mitochondria present. This was shown experimentally when the solution of E3 (Pellet 3) remained the initial shade of blue, and did not become lighter over time. Without the SDH enzymes found in the inner mitochondrial membrane the DCIP does not get reduced, therefore there is no color change in the solution. The dependent variable of this experiment was the change in absorbance at 600 nm, as the differing number of mitochondria in the cell fractions is what causes the change in absorbance. The independent variable was the number of mitochondria in the cell fractions, as the mitochondria levels were manipulated, the values of reduction of DCIP varied. The control was the solution E1 (None), which contained no cauliflower cell fraction. This provided data that guaranteed a solution without any mitochondria present, that tubes E2-E4 could be compared to.

Figure 3. Effect of Temperature on Enzyme Activity Over Time. This experiment involved taking four identical test tubes and performing …show more content…

Generally, when the temperature is greater the decrease or change in absorbance is greater. The highest readings are for the solutions carried out at 3.7 C and 65.0 C. This is to be expected as they are the two highest temperatures used in experiment III. Although the solution carried out at 65.0 C was expected to have a greater change in absorbance than the solution carried out at 37.0 C, this could be due to the enzymes denaturing in the higher temperature of 65.0 C. The two lowest reading were also identical despite their significant temperature

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