Isolation Of Mitochondria
Using Differential Fractionation
by Amanda Ellison
© 2006 Amanda Ellison
Originally Submitted for Biology 350: Cell Biology
Professor John Egan
The mitochondria is an important organelle found in large numbers in Eukaryotic cells of both plants and animals. It is responsible for a multitude of chemical reactions in the production of energy in the form of ATP as a result of oxidative phosphorylation. It plays important roles in catabolic and anabolic reactions and is also important in programmed cell death (1). Mitochondria, unlike most other organelles, contain their own genome, known as mtDNA, which encodes components involved in oxidative phosphorylation (3). Various damaging diseases have arisen from mutations in the mtDNA (3). Plant mitochondria also serve other functions that are not incorporated in animal cell mitochondria (1).
In order to carry out the complex chemical reactions, many enzymes are located in the mitochondria to catalyze the reactions. One such enzyme is succinate dehydrogenase (SDh). SDh catalyzes the oxidation of succinate to fumarate in the tricarboxylic acid cycle in order to use succinate as a source of energy. This is the first step in a process involving an electron transport chain that allows for aerobic respiration (2). One important aspect of succinate dehydrogenase, especially in its use in mitochondria identification, is that it is bound to the inner membrane of the mitochondria (2).
In this study, we isolated mitochondria from cauliflower florets then confirmed the presence of the mitochondria tissue by using DCIP to detect the presence of succinate dehydrogenase activity. Finally, we adjusted our findings of enzyme activity based on the portions of each solution used to determine which portion contain more specific enzyme activity. Since we expected the pellet isolated to contain mitochondria tissue, and therefore the enzyme succinate dehydrogenase, we expected that the mitochondrial pellet solution would reduce DCIP rapidly while the solution of supernatant would not. What we found was that both solutions significantly decreased DCIP concentrations at similar rates. Using specific enzyme activity, we found that the supernatant, rather than the pellet, contained more activity but diluted to a greater extent.
MATERIAL AND METHODS
Isolation of Mitochondria. 25 g of florets were cut from the head of the cauliflower. The tissue was placed in a cold ceramic mortar along with 20ml isolation buffer and 6 g sand and ground until the mixture was a smooth paste. Another 20 ml isolation buffer was then added and the tissue was ground for a few more minutes. Using a funnel made of four layers of cheesecloth, the mixture was strained into a 50ml centrifuge tube. The cheesecloth was squeezed to remove the remaining liquid and the solid portions caught in the cloth were discarded. The homogenate was then centrifuged at 600g for 10 minutes at 4°C. Once centrifuged, the supernatant was decanted off the top into an Oak Ridge tube and the pellet material on the bottom was discarded. The supernatant was then centrifuged at 12,000g for 30 minutes at 4°C. Once centrifuged, 5ml of the supernatant was placed in a 15ml centrifuge tube labeled “supernatant”. The remaining supernatant liquid was discarded. 5ml assay buffer was added to the mitochondrial pellet at the bottom of the tube and the pellet was re-suspended. The mixture was transferred to a 15ml centrifuge tube labeled “pellet”. Both mixtures were then stored on ice.
Assay of Mitochondrial Enzyme Activity – Construction of a DCIP standard curve. 6 test tubes were prepared with varying concentrations of DCIP, from 0mM to 10mM in 2mM increments. Each tube was covered and mixed thoroughly. The absorbance of each tube at 600nm was found, using the tube with no DCIP as the blank. A standard curve was constructed and an equation for the line was determined using Excel (Figure 1).
Assay of Mitochondrial Activity. 6 test tubes were prepared with assay buffer, 0.5ml sodium azide, and 0.2M succinate. 0.5ml DCIP was added to tubes 2, 4, and 6 only. 0.5ml mitochondrial suspension was added to tubes 1 and 2 and the time was recorded. The tubes were mixed then the absorbance of tube 2 at 600nm was recorded, using tube 1 as a blank. Both tubes were set aside at room temperature. Next, 0.5ml supernatant was added to tubes 3 and 4. They were mixed thoroughly and the absorbance of tube 4 at 600nm was recorded, using tube 3 as a blank. The absorbance of tube 6 was found using tube 5 as a blank. At 5 minute intervals, the absorbance of tubes 2 and 4 were found using tube 1 as a blank for tube 2 and tube 3 as a blank for tube 4 for each measurement. Readings were recorded for 30 minutes (Table 2). The absorbance of tube 6 was found at 30min to confirm that it remained constant. Using the equation for the concentration of DCIP found previously, the concentration of DCIP in each tube was calculated and plots of concentration versus time were constructed for the supernatant and the mitochondrial suspension (Figure 2).
Enzyme Specific Activity. To determine enzyme activity, a plot of DCIP concentration in mitochondrial solution and the supernatant were plotted over time. A regression line was found and the slope of the line was recorded, representing the activity of the enzyme by the decrease in concentration of DCIP per minute. To determine the enzyme activity in the solution assayed, the slope of the line was divided by the volume of each solution assayed, which was 0.5ml for each solution. To find the activity in the entire fraction, we multiplied the activity found by the total volume of the original fraction. 5ml of the mitochondria had been used versus 35ml of the supernatant (Table 2).
The absorbance values collected for known concentrations of DCIP were plotted to form a standard curve (Figure 1). The line obtained in this section has an r-squared value of 0.998 which relates how closely the data points follow the line. Clearly, the absorbance increases linearly with an increase in concentration of DCIP. The equation for the line found from the graph of these points can then be used to find the concentration of DCIP in an unknown sample.
When DCIP was added to the mitochondrial pellet solution and the supernatant, it was reduced by the products of succinate dehydrogenase activity. The DCIP, normally a blue color, turned clear as it was reduced. As the experiment progressed, more of the DCIP was reduced and the absorbance of the solution continually decreased for both solutions, as recorded in Table 1.
Based on the equation for the line of the standard curve found for DCIP, the concentration of DCIP in the mitochondria pellet solution and the supernatant was found based on the absorbance recorded in Table 1. Then, the concentration of DCIP versus time for both solutions was plotted on the same graph (Figure 2). Figure 2 shows the change in DCIP concentration as it is reduced as a result of succinate dehydrogenase activity. The slope of the lines formed from the data conveys the average amount of DCIP that is reduced per minute. Both solutions caused a substantial reduction in DCIP concentration, suggesting the presence of succinate dehydrogenase in both solutions. Based on the slope found, the mitochondrial pellet solution reduced slightly more DCIP then the supernatant.
From the slope of the lines found, the enzyme activity in each solution was calculated. Dividing the slope by the volume assayed gives the amount of enzyme activity in the portion of solution that was assayed. This value was then multiplied by the total volume initially isolated. Following the isolation process, 35ml of supernatant was obtained and the mitochondrial pellet was re-suspended in 5ml of buffer. By multiplying by the initial volume, the enzyme activity in the total fraction is found. Even though the slope for both the mitochondrial pellet solution and the supernatant were similar, there was substantially more enzyme activity in the supernatant; it had simply been diluted to a greater extent.
Since there was a significant reduction in DCIP concentrations in our solutions, we did isolate some mitochondrial tissue. Contrary to what we expected, both the mitochondrial pellet solution and the supernatant reduced DCIP at approximately the same rate. Since both solutions caused a decrease in DCIP concentrations, there must be succinate dehydrogenase, and therefore mitochondrial tissue, in both the mitochondrial pellet solution and the supernatant. We found, however, that there was substantially more enzyme activity in the supernatant than in the mitochondrial pellet. Calculating the enzyme activity in the original fraction isolated showed that the enzyme was significantly diluted in the supernatant since it was in 35ml while it was concentrated in the mitochondrial pellet solution, since it was re-suspended in only 5ml. From this lab, it is clear that differential fractionation may be a useful method of dividing cells into their component parts.
1) Brugiere, S. et al. 2004. The hydrophobic proteome of mitochondrial membranes from Arabidopsis cell suspensions. Pytochemistry. 65(12): 1696-1701.
2) Mason, P., E. Matheson, A. Hall, and R. Lightowlers. 2003. Mismatch repair activity in mammalian mitochondria. Nucleic Acids Res. 13(3): 1052-1058.
3) Westenberg, D. and M. Guerinot. 1999. Succinate Dehydrogenase (Sdh) from Bradyrhizobium japonicum Is Closely Related to Mitochondrial Sdh. J of Bacteriology. 181(15): 4676-4679.
Figure 1. DCIP standard curve. The absorbance at 600nm of a solution of DCIP was found at different concentrations and plotted to find a regression line. The equation for the line is important in order to determine the concentration of DCIP in a sample by finding the absorbance.
Table1. Absorbance of Mitochondrial pellet solution and Supernatant solution over time.
a - DCIP
concentration decreases over time due to the activity of the
enzyme succinate dehydrogenase, indicating the presence of
b – control absorbance was recorded only at the start and end of the experiment to confirm that there was little change since succinate dehydrogenase is not present
Figure 2. Concentration of DCIP over time. Using the equation for the line in the DCIP standard curve, the concentration of DCIP in each sample was determined based on its absorbency and which was recorded every 5 minutes. The slope of the regression lines shows the average decrease in DCIP over time, which indicates the presence of succinate dehydrogenase. The equation for the line from the mitochondrial pellet is at the top of the graph while the equation on the bottom represents the supernatant data. Both the mitochondria pellet and the supernatant have similar slopes indicating that both solutions contained succinate dehydrogenase.
Table 2. Specific Enzyme activity of the Mitochondrial pellet and the Supernatant.
|Total ml a||Slope b (mmoles/min)||volume assayed (mL)||Activity c (mmoles/ min · mL)||activity in the entire fraction d (mmoles/min)|
a – the original volume obtained during the isolation process
b – average decrease in DCIP concentration found from the graph of DCIP concentration versus time
c – found by dividing the slope by the volume assayed
d – found by multiplying the activity by the total volume initially obtained
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