Aerobic Cellular Respiration in Isolated Mitochondria of Lima Bean Seeds - Lab Report Example

? Investigating the Aerobic Cellular Respiration in Isolated Mitochondria of Lima bean (Phaseolus lunatus) seeds during the Conversion of Succinate to Fumarate in Krebs cycle Introduction: A basic concept in cellular respiration is the transfer of chemical energy (electrons) from an electron donor (i.e. carbohydrate, proteins and fat from food molecules) to an electron acceptor. Most of the organisms use oxygen as the final electron acceptor although some organisms (eg. Yeast) use other alternatives. During aerobic respiration, oxygen in the air is used as the final electron receptor which subsequently gets reduced to water. Energy is generated during this process in the form of a high energy molecule, adenosine triphosphate (ATP). This is a complex process involving a series of reactions that use many chemicals and enzymes. Glucose is the most preferred source for cellular respiration and as reported by Rich (2003), it release high energy (29-30 ATP molecules per glucose molecule) during aerobic respiration. Aerobic respiration consists of 3 major steps as glycolysis, Krebs cycle (Citric acid cycle) and electron transport chain. During glycolysis, pyruvate is produced by glucose which is converted to a 2C molecule, acetyl-CoA. Acetyl-CoA combines with the 4C oxaloacetate (last product of the previous Krebs cycle) to produce citrate which is a very high energy source. During the Krebs cycle, citrate is consumed in an 8-step process to release this energy (electrons). Here, the coenzymes FAD (flavin adenine dinucleotide) and NAD+ (nicotinamide adenine dinucleotide) gets reduced to produce a small quantity of carbon dioxide and ATP. Therefore, hydrogen electrons coming from glucose will reduce FAD and NAD+ to FADH2 and NADH + H+ respectively. These electrons then enter the electron transport chain to get oxidized and produce ATP. Glycolysis occurs in the cell cytoplasm while Krebs cycle and electron transport chain occur in the mitochondria organelle of the cell. Hence mitochondria are a good source to study the aerobic respiration reactions. The rate of cellular respiration in aerobic organisms can be measured by monitoring enzyme activity in the Krebs cycle. One step of the Krebs cycle is the conversion of succinate to fumarate which takes place in mitochondria in the cytoplasm (Zaunmuller, Kelly, Glockner and Unden, 2006). This reaction is catalyzed by the enzyme succinic dehydrogenase using FAD as co-enzyme. In this reaction, 2 hydrogen atoms are removed from succinate and transfer to FAD thereby reducing it to FADH2. DPIP (2, 6-dichloro-phenol-indophenol) blue dye can act as a hydrogen molecule acceptor instead of FAD during this reaction. When DPIP receive hydrogen from succinate, blue color get decolorized. Thus the DPIP color change from blue to colorless is an indication of the level of enzyme activity in the mitochondria which can be measured and recorded with a spectrophotometer. The Krebs cycle is influenced by competitive and noncompetitive inhibitors. Competitive inhibitors compete with the substrate to bind to the active site of the enzyme and this can be overcome by providing more quantity of substrate molecules. Conversely, noncompetitive inhibitors such as metal ions (copper, Cu2+ and mercury, Hg2+) will deactivate the enzyme thereby making it impossible to return back to the reaction. Therefore, the reaction cannot be reactivated by incorporation of more substrate. In the succinate-to-fumarate reaction of the Krebs cycle, Malonate act as a competitive inhibitor on succinate molecule. Molecule shape of malonate is similar to succinate molecule and thus it obstructs the conversion reaction of succinate to fumarate by binding to active site of the enzyme succinate dehydrogenase. However, as described in Zeevalk, Derr-Yellin and Nicklas (1995) it will not react further and result in the termination of the reaction. Therefore FAD will not reduce to FADH2 and fumarate will not be produced, thus arresting the Krebs cycle. As malonate is a competitive inhibitor, the reaction can be regained by addition of more succinate to the reaction medium. Within this context, the objective of this study was to investigate aerobic cellular respiration in isolated mitochondria of lima bean seeds. Rate of cellular respiration will be monitored during the succinate to fumarate conversion in the Krebs cycle using the color change of DPIP which act as the electron acceptor. Null hypothesis of this experiment will be the addition of succinate will not improve the succinate-to-fumarate reaction while the alternate hypothesis suggests incorporation of succinate will improve the succinate-to-fumarate reaction in mitochondria. Materials and methods: A supernatant containing isolated mitochondria of Lima bean (Phaseolus lunatus) was prepared by soaking 50g of Lima bean seeds, followed by homogenization and centrifugation. One sample of this supernatant was boiled for 5 minutes. Reagents except succinate were mixed (as given in Table 1) in spectrophotometric test tubes (numbered from 1 to 6) to make a final volume of 5ml. Each tube was covered by a piece of Parafilm and the contents were mixed by inverting the tubes several times. A different piece of Parafilm was used for each tube as to prevent contamination. Then, succinate was added as given in Table 1 and after placing a Parafilm over the tube and inverting to mix, the tubes were immediately placed in the spectrophotometer (Spectronic 20). A ‘blank’ was set up without DPIP to ensure measuring decolorization of DPIP only and not absorbance of other materials in the solution. First, the spectrophotometer was set to 100% absorbance and 0% transmittance and inserted the blank. Next, the machine was set to 100% transmittance and 0% absorbance. This will permit the measuring of DPIP absorbance only in the test tubes. The absorbance of each tube was read at 600nm at 2 minute interval for 16 minutes and a graph was plotted by MS Office Excel (2007) software program. Table 1. Volume of reagents mixed in the tubes Tube no. Lima bean juice (ml) DPIP (ml) Phosphate buffer (ml) Malonate (ml) Succinate (ml) 1 0.3 0.3 4.3 - 0.1 2 0.3 0.3 4.4 - - 3 0.3 (boiled) 0.3 4.3 - 0.1 4 0.3 0.3 4.0 0.3 0.1 5 0.3 0.3 4.1 - 0.3 6 0.3 0.3 3.8 0.3 0.3 Blank 0.3 - 4.6 - 0.3 Results: All 6 tubes showed a reduction of absorbance during the tested time period (figure 1). Figure 1. Absorbance level of Lima bean samples over the tested time period Tube 1, which contained the lima bean sample with 0.1ml succinate and the sample without any added succinate (tube 2) had higher absorbance level in the beginning but both showed reduced absorbance over time. The sample with 0.3ml succinate (tube 5) showed a lower absorbance than tubes 1 or 2 up to the fourteenth minute. Thereafter it was similar to the level of tube 2. Tube 3 which contained boiled lima bean supernatant and 0.1ml of succinate gave lower recordings than other samples except tube 6. The absorbance reduction pattern of tube 4 which contained 0.1ml succinate and 0.3ml inhibitor malonate was similar to the pattern of tube 5 which had a higher level (0.3ml) of succinate. A remarkably lower absorbance level was observed with tube 6 which contained equal high volumes (0.3ml) of succinate and malonate. The total absorbance reduction levels (difference between zero and the sixteenth minute) was highest in tube 2 followed by tubes 3, 6, 5, 1 and 4. Discussion: Indicator dye DPIP absorb maximum amount of light in the 600nm wavelength range. When a sample is placed in the pathway of light, the spectrophotometer can show the percent of light absorbed by the sample and the percent of light transmitted through. When the enzymatic action of the Krebs cycle is faster, DPIP receives more and more hydrogen molecules (electrons) which get reduced rapidly. This causes the solution to become colorless quickly resulting a reduced absorption and a higher transmittance. Hence, if the tested sample exhibit lower and lower readings over time, it indicates that the DPIP is receiving electrons and getting decolorized. This proves that respiration is occurring in the tested solutions. Results in the present experiment which contained lima bean mitochondria showed that all 6 tubes had reducing absorbance over the time. This indicates that respiration reactions are happening in mitochondria in the tubes and thus is in agreement with Zaunmuller, Kelly, Glockner and Unden (2006). However, some of the results obtained are somewhat contradictory to the theoretical explanations. For example, tube number 3 had a boiled sample of lima bean supernatant. Therefore, the enzymes in mitochondria in the supernatant of that sample should be destroyed due to high heat and should not be able to reduce DPIP. This should result in higher absorbance. However, as shown in figure 1, the boiled sample has a lower absorbance than other samples (except tube 6) and it continued to reduce over the time indicating the occurrence of respiration reactions in that sample. Similarly, lima bean sample with no succinate (tube 2) had lower absorbance, thus faster reaction, than the sample with succinate (tube 1). Actually, presence of succinate should enhance the reaction and thus produce lower absorbance level. Sample with a high level of succinate (tube 5) had lower absorbance than the sample with the lower level of succinate (tube 1). This is in agreement with the explanation that the presence of more succinate enhances the respiration reaction. The competitive inhibitory effect of malonate can be well explained in this experiment with tubes 4 and 6. Both samples contained equal volume of malonate which inhibit succinate to fumarate reaction and cause lesser DPIP reduction. Therefore the absorbance levels of these samples should be higher. However, when higher volume of succinate was incorporated in to the sample (tube 6) the inhibition reaction was terminated as the presence of more succinate molecules could compete with malonate and DPIP reducing action was restored. Thus the absorbance level was reduced as explained by Zeevalk, Derr-Yellin and Nicklas (1995). Therefore more fumarate is produced in tube 6 than in tube 4. It is again confusing that the sample with high level of succinate but no malonate (tube 5) expressed lower reaction than the sample with equal volume of succinate but with high volume of malonate (tube 6). Here, the succinate-only sample should have expressed faster reaction, thus lower absorbance than the sample containing the malonate inhibitor. Some of the results in the experiment are not in accordance with the theoretical explanations. It may be possible that errors in tube numbering, errors in measuring reagents and errors in sample preparation might have caused this. Another factor which is needed to consider is the correct operation of the spectrophotometer. One or several of these reasons may have produced erroneous readings. According to these results, following conclusions can be drawn from this study. Conclusions: 1. All tested samples showed reduced absorbance in the given time period indicating that they are alive and succinate to fumarate reaction occurred in the mitochondria samples. 2. Presence of malonate inhibited the reaction but its inhibitory effect could be controlled with addition of more succinate. 3. DPIP functioned as the hydrogen molecule receptor and continued the reaction even without addition of succinate (as in tube 2). 4. The results in general support the alternate hypothesis, i.e. addition of succinate improves the conversion reaction to produce fumarate from succinate in mitochondria. However, since there are certain confusions with the results of the present study, it is advisable to carefully plan and repeat this experiment to clarify the contradictory issues before accepting/rejecting the hypotheses. References: Rich, P.R., 2003. The molecular machinery of Keilin's respiratory chain. Biochemical Society Transactions, 31, pp.1095–1105. Zaunmuller, T., Kelly, D. J., Glockner, F. O. and Unden, G. 2006. Succinate dehydrogenase functioning by a reverse redox loop mechanism and fumarate reductase in sulphate-reducing bacteria. Microbiology, 152(2), pp.2443 – 2453. Zeevalk, G. D., Derr-Yellin, E. and Nicklas, W. J. 1995. Relative vulnerability of dopamine and GABA neurons in mesencephalic culture to inhibition of succinate dehydrogenase by malonate and 3-nitropropionic acid and protection by NMDA blockade. Journal of Pharmacological Experimental Theory, 275(3), pp. 1124 – 1130. Read More