LAB ASSIGNMENT ONE: Isolation of Mitochondria
Succinate dehydrogenase (SDH) is bound to the inner mitochondrial membrane, and is crucial in the citric acid cycle. SDH catalyses the oxidation of succinate into fumarate (succinate + FAD fumarate + FADH2), and can be used as a marker enzyme during the isolation of mitochondria through differential centrifugation. Once the mitochondria have been separated, an azide reagent is used to block the normal electron transport system in the cell sample. An artificial electron acceptor (2,6-dichlorophenolindphenol, DCIP) is then used to receive the two electrons that would normally be accepted by an SDH-FAD prosthetic group.
The oxidized DCIP is reduced when is receives these electrons, and a colour change from blue to colourless is visible. Spectrophotometry at the 600nm range can then be used to quantify this colour change, and give an indication of the mitochondrial content of a given sample.
RESULTS AND DISCUSSION
The spectrophotometry results obtained by our group (3A), as well as the class averages, are presented in Table 1, and shown graphically in Figure 1. The greatest absorbance reading was obtained for Tube 6 (1.068), which is in good agreement with the average class findings. Tube 6 served as a negative control, and did not contain any cellular suspension. The lack of mitochondria in this sample means there was no biochemical reaction between the marker enzyme (SDH-FADH2) and the DCIPoxid, which remained blue in colour. The second highest reading was found for Tube 2 (0.987), which was also in concordance with the class results. This sample contained the heaviest constituents of the cell (mostly nuclei), as well as any unbroken whole cells that may have remained after the mechanical grinding and initial centrifugation at 600x.
We found Tube 8 to have the third highest absorbance reading (0.626) and Tube 4 with the lowest (0.483). However, the sample from Tube 8 should have had a lower...