During this experiment, mitochondria were isolated from 20.2 grams of cauliflower using extraction buffer, filtration through Miracloth, and centrifusion. Twelve samples containing various volumes of mitochondrial suspension, assay buffer, DCIP, sodium azide, and citric acid cycle intermediates were prepared to be read by a spectrophotometer. The inclusion of the dye DCIP allowed for the absorbance of the reactions between the mitochondrial suspension and the TCA cycle intermediates succinate, malonate, and oxalate to be measured, as DCIP turns from blue to colorless as the activity of succinate dehydrogenase increases. Experimental Findings Increasing the number of mitochondria in the reaction did increase the reduction of DCIP relative to the amount of mitochondrial suspension present. Although the overall absorbance increases as more milliliters of mitochondrial suspension is added to a mixture of 0.25 mL of 0.5 mM DCIP, 0.5 mL of 50 mM sodium azide, various volumes of assay buffer (20 mM potassium …show more content…
While the absolute value of slope of the graph for the solution containing only 0.5 mL mitochondrial suspension was 4 x 10-4, the slope of the graph for the solution containing 0.5 mL of mitochondrial suspension, 0.5 mL of 100 mM succinate, and 0.5 mL of 100 mM malonate was 7 x 10-4. Although this change is not large, it does demonstrate that the addition of TCA cycle intermediates has an impact on reaction rate. The decrease in the rate of reaction of the sample containing 0.5 mL of mitochondrial suspension, 0.5 mL of 100 mM succinate, and 0.5 mL of 100 mM malonate as compared to the sample with only 0.5 mL of mitochondrial suspension and 0.5 mL of 100 mM succinate shows that the addition of malonate inhibits the reduction of