Mitochondria and Cellular Respiration - Lab Report Example

Mitochondria and Cellular Respiration Introduction Mitochondria are double membrane and rods shaped organelles found in the cells of both animal and plants and are responsible for various metabolic functions within the cells. Mitochondria play a vital role in providing the necessary biological energy for the cell through enzymatic oxidation of the substrates of the Krebs’s cycle. Mitochondrion is made up of outer membrane, inner membrane, intermembrane space and matrix. The outer membrane consists of same amounts of proteins and phospholipids as well as large number of porins which are specialized proteins. The outer membrane is permeable to ions, nutrients and energy molecules such as ADP and ATP (Abdrakhimova et al, 2011). The inner membrane of the mitochondrion is complex and consists of a number of folds known as the cristae which are important since it increases the surface area within the organelle. The increased surface area within the organelle coupled by presence of various proteins within the inner membrane is crucial in aiding various enzymatic reactions including the production of ATP. However, unlike the outer membrane, the inner membrane is strictly permeable to ATP, Oxygen and movement of metabolites across the membrane (Jones, 2013). The space between the outer and the inner membranes is referred to as the intermembrane space and its composition is similar to that of the cytoplasm. The matrix which is the fourth component of the mitochondrion is made up of a complex mixture of enzymes and proteins which are important in the synthesis of ribosome, ATP, mitochondrial DNA and tRNAs (Miles et al, 2014). Succinate dehydrogenase is one of the enzyme complex bound to the inner membrane of the mitochondrion. Succinate dehydrogenase plays a vital role in Krebs’s cycle since it catalyzes the oxidation of Succinate to fumarate within the inner membrane (Gorbacheva et al, 2013). Objective In this experiment we examined the intracellular location of the Succinic dehydrogenase. As outlined above, succinic dehydrogenase are enzymes involved in redox reactions during cellular respiration by catalyzing the conversion of succinic acid to fomaric acid in the Kreb’s Cycle as shown below. Succinate dehydrogenase Succinate + FAD  fumarate + FADH2 Through measurements of FADH2 produced in the reaction, it is possible to determine the intracellular location of Succinic Dehydrogenase. Since measuring FADH2 directly is hard, this compound can be coupled with a second reaction that results into a colored product which can be measured using a spectrophotometer. Methods Fractionation of Cauliflower Cells The top 2-3 mm off the heads of fresh cauliflower were shoved to give 25g of tissue then blended for about 10 seconds in 20mL cold grinding buffer (0.4M sucrose; 6mM KH2PO4; 14mM K2HPO4, pH7.2. The slurry was then poured through 4 layers of muslin cloth and the filtrate collected in a tube. The blender was then rinsed with 10mL cold grinding buffer, filtered and added to the tube to form the homogenate. The homogenate was then centrifuged at 500 x g for 10 minutes at 4oC and the supernatant was then transferred to a clean tube, labeled S1 and kept on ice. The pellet was then re-suspended in 4mL cold suspension/assay buffer (0.4M sucrose, 6mM M KH2PO4; 14mM K2HPO4; 10mM KCl; 5mM MgCl2; pH 7.2), Labeled P1 and stored on ice. Tube S1 was then centrifuged at 20,000 x g for 30 minutes at 4oC then the supernatant transferred to a clean tube and labeled S2 and stored on ice. The pellet in 4mL was then re-suspended in cold suspension buffer, Labeled P2 and kept on ice. Assay for Succinate Dehydrogenase The solution from Tube 1 was transferred to a cuvette and used to zero the spectrophotometer at wavelength 620 nm. Using an autopipette, 0.4 mL 0.1% DCIP was added to Tubes 2 – 6. The tubes were then flickered to mix and each reaction solution were carefully transferred to a spectrophotometer cuvette. The absorption was then measured at 620 nm and the values recorded as the absorption at Time 0 for that tube. The curvette was then taken out of the spectrophotometer and placed on the bench and the absorbance re-read every 2 minutes for 20 minutes. The readings were then recorded in a table. The change in absorbance for each tube over each time interval were calculated by subtracting the absorbance at 2 min. from the absorbance at 0 min, etc and recorded. Results Table 1: Change in Absorbance at 620 nm Change in Absorbance (OD) at 620 nm for each period interval Time (min) Tube 2 Tube 3 Tube 4 Tube 5 Tube 6 T0 0 0 0 0 0 2 0 -0.002 -0.002 0.002 0.009 4 -0.002 0.000 0.012 -0.008 -0.004 6 0.001 -0.003 -0.004 0.005 -0.004 8 -0.004 0.000 -0.003 0.000 0.001 10 -0.001 0.004 0.002 -0.013 -0.003 12 0.001 0.001 -0.001 0.005 -0.011 14 -0.004 -0.001 0.001 0.000 -0.002 16 -0.005 0.006 -0.002 -0.008 -0.002 18 0.003 -0.004 0.001 0.006 -0.001 20 -0.006 -0.002 -0.002 -0.006 0.002 Figure 1: Change in Absorbance for each tube over time interval Discussion The objective of this investigation was to determine the intracellular location of Succinic Dehydrogenase by measuring its activity. The Succinic acid is oxidized by succinic dehydrogenase and the enzyme accepts the resultant free hydrogen atoms and binds it to FAD to form FADH2. FADH2 then conveys energy in the electron transport chain that result into ATP and oxygen is the receptor in the electron transport chain (Keshavarzian et al, 2013). However in our experiment, we used DCIP (2, 6-dichlorophenolindophenol) dye to act as electron acceptor. This results into the change of color of DCIP (2, 6-dichlorophenolindophenol) dye from blue to colorless. The change in color as demonstrated by the spectrophotometer absorbance rates shows that Succinic dehydrogenase is active. Succinic dehydrogenase enzyme activity, just like all other enzymes are affected by several factors including temperature, enzyme concentration, concentration of the substrate and the pH. This is the main reason why the prepared homogenate was stored on ice. This is to keep temperatures low and prevent oxidation. Since enzyme activity is also affected by changes in temperature, it was important to use a buffer to maintain the pH of the solution. Tubes 1, 2, and 3 were used as controls mainly for comparisons and generalizations about the outcome of the experiment. In these tubes, changes in absorbance are very little because there is no oxidation taking place and thus succinic dehydrogenase is inactive. The results shows that tubes 4, 5 and 6 contain active succinic dehydrogenase as exhibited by absorbance as shown in figure 1. However, there is high oxidation in tube 5 which is an indication of highly active Succinic Dehydrogenase. Conclusion This experiment shows that Succinic dehydrogenase is actively involved in the redox reaction in cellular respiration. This has been confirmed by the absence of active oxidation in control tubes while tube 5 which had the homogenate showed high changes in absorbance, an indication of oxidation. Cellular respiration is therefore highly affected by the activity of enzyme Succinic dehydrogenase. References Abdrakhimova, Y., Andreev, I., & Shugaev, A. (2011). Involvement of the energy-dissipating systems in modulating the energetic efficiency of respiration in mitochondria from etiolated winter wheat seedlings. Russian Journal Of Plant Physiology, 58(4), 567-574. Gorbacheva, T., Syromyatnicov, M., Popov, V., Lopatin, A., Eprintsev, A., & Fedorin, D. (2013). Characteristics of functioning of succinate dehydrogenase from flight muscles of the bumblebee Bombus terrestris (L.). Biology Bulletin, 40(5), 429-434 Jones, Aj; Hirst, J. A spectrophotometric coupled enzyme assay to measure the activity of succinate dehydrogenase. Analytical Biochemistry. 442, 19-23, Nov. 1, 2013. Keshavarzian, M., Gerivani, Z., Sadeghipour, H. R., Aghdasi, M., & Azimmohseni, M. (2013). Suppression of mitochondrial dehydrogenases accompanying post-glyoxylate cycle activation of gluconeogenesis and reduced lipid peroxidation events during dormancy breakage of walnut kernels by moist chilling. Scientia Horticulturae, 161314-323. Miles, T. D., Miles, L. A., Fairchild, K. L., & Wharton, P. S. (2014). Screening and characterization of resistance to succinate dehydrogenase inhibitors in Alternaria solani. Plant Pathology, 63(1), 155-164 Read More