The Rate of Aerobic Respiration - Lab Report Example

This experiment aims at investigating aerobic process in the mitochondria. The experiment uses a suspension of mitochondria obtained from ground Lima beans. The experiment is based on the assumption that the mitochondria should continue functioning if they were intact cells. Succinate which is the substrate for the process of respiration is added to the suspension. The rate of aerobic respiration is measured by observing the reaction of a single enzyme found in one of the reactions taking place in the Kreb’s cycle. The enzyme is responsible for the conversion of succinate acid to fumarate. The hypothesis for the experiment is that if the substrate concentration of Succinate increases, then the rate of aerobic respiration increases was not confirmed. The conclusion of the experiment was discovered that succinate concentration and enzyme concentration does affect the rate of aerobic respiration. Introduction Aerobic respiration occurs in living things and is a process through which food substances are broken down to release energy in form of ATP. This process takes place in the mitochondria and requires energy. Unlike the process of fermentation where food substrate is broken down in absence of oxygen where only a lesser number of ATP are produced, aerobic respiration results in production more energy in form of ATP. The first part of aerobic respiration (glycolysis) is similar to that of fermentation and takes place in the cytoplasm. Pyruvic acid, a three carbon molecule is formed through a series of reactions on glucose molecules. Two molecules of ATP are released absence of oxygen as anaerobic respiration takes place (Apte et al 37). The pyruvic acid then proceeds to the next stage. C6H12O6 CH3COCOOH (Pyruvic acid) The second phase of aerobic reaction takes place in the matrix of the mitochondria. Pyruvic acid obtained from the first phase is broken down through a process of reactions controlled by enzymes in the Kreb’s cycle. The end products of this process are 38 molecules of ATP, carbon dioxide and water. C6H12O6 CH3COOH CO2+H2O+ ATP During the Kreb’s cycle, some of the reactions lead to the release of electrons which enter the electron transport chain. A high percentage of the ATP produced during this stage is produced by the electron transport system. Objectives To investigate the factors affecting the rate of aerobic respiration Hypothesis If the substrate concentration of Succinate increases, then the rate of aerobic respiration increases. Procedure The materials needed for this experiment includes test tubes, lima beans mitochondrial suspension, succinate, DCPIP, buffer and paraffin, In this experiment, four small test-tubes were obtained and labeled B, 1, 2, 3, 4 test tubes B was blank. In the blank test tube, 4.6 ml of the buffer, 0.1ml of succinate acid and 0.3ml of mitochondria suspension was placed. The tube was then covered with a film of paraffin and inverted to ensure that the contents mixed completely. A micropipette was then used to transfer 1ml of the mixture to a square corvette. The machine was then set to read transmittance. The wavelength of the machine was set at 600nm. The corvette was placed in an open spectrophotometer. The transmitter was set to 100 where the blank was used as the baseline. In tube 1, 2 and 3, DCPIP, mitochondria suspension and the buffer were placed in measurements indicated in the table below. Succinate was not added at first Reagents Tube 1 Tube 2 Tube 3 Buffer 4.4ml 4.3ml 4.2ml DCPIP 0.3ml 0.3ml 0.3ml Mitochondrial suspension 0.3ml 0.3ml 0.3ml Succinate 0ml 0.1ml 0.2ml Succinate was then quickly added to the test tubes which were then covered with paraffin and placed in the corvette. The corvette was wiped on the outside and then placed in the samples holder ensuring that the arrow pointed downwards. The lid of the machine was closed and the readings for each sample recorded at different times. The spectrometer was reset after every five minutes using the blank as the baseline. The content of the test tube were inverted to mix the mixture and their percentage transmittance recorded. The results were then recorded within intervals of five minutes for 30 minutes. Results Showing transmittance in % tube 0 min 5min 10min 15min 20min 25min 30 min 1 0.4 2.0 2.6 2.6 2.7 2.8 2.9 2 4.0 7.4 10.5 13.4 17.3 21.3 24.3 3 3.4 5.5 8.4 10.9 15.5 19.2 23.6 The transmittance in tube 1 rose a little bit and continued to be constant. This could be as a result that there was no substrate added to the test tube and the transmittance came from some substrate that was present on the lima beans extract. Test tube two had the highest amount of transmittance despite the fact that test tube three had the highest amount of substrate. This could be as a result of the amount of substrate in test tube three exceeding the amount of enzyme. It was observed that tube two with 0.1 ml of succinct, 4.3ml, 0.3ml of mitochondrial suspension and 0.3ml of DCPIP. In all the test tubes, transmittance increased as time progressed. Discussion The amount of electrons received in test tube 2 were the highest as it had the right amount of substrate and enzyme concentration. The results in substrate concentration without an increase in enzyme concentration doe not result in increase in respiration reaction. Lima Extract was used to provide the mitochondria which are the site for respiration. The substrate used in this reaction was succinate. Succinate is the substrate for the process of respiration and is converted to fumarate in the Kreb’s cycle. The buffer is used to ensure that the pH of the solution was enzymatic reactions are taking place is kept within a narrow range. This is because enzymes perform within a specific PH range. However, as the reactions proceeds, the products causes a change in PH and this has to be maintained with the range. Change in PH around the enzymes makes the charges within the enzymes to also change (Starr et al, 178). This causes internal repulsion which could denature the enzymes. The buffer provides both positive and negative charges to the solution. This offsets the effect the change in PH has on the enzymes protecting them from being denatured. Different amounts of the buffer were placed in test tube 1, 2 and 3 because the three tubes have different amounts of substrate which affects the amount of product produced. DCPIP act as an indicator of enzymatic reactions taking place and change in color indicate that some electrons are being released in the Kreb’s cycle. DCPIP is used in this experiment as a sign of enzymatic actions. DCPIP is blue is color and absorbs the electrons when they are released in the Kreb’s cycle before they enter the electronic chain. When this happens, DCPIP changes color from blue to colorless. In this reaction where DCPIP was used, when the solutions in the test tubes turned colorless, it was a sign of enzymatic reactions resulting in the release of electron. The amount of product has different effects on the PH of the solution. For a balance in PH to be maintained, then different amount of buffer should be used for different amount of substrate (Starr et al. 134). The right amount of buffer should be used. Failure to do so would lead to there being excess charges. Test tube one was used as a negative control since there was no substrate in it. Succinate was added to the test tube last to ensure that all reactions with the substrate are accounted for as it is placed on the spectrometer for observation. Tube 1 has the highest amount of the buffer and this produced a large amounts of both positive and negative charges to offset the effect of the electrons released in the process of respiration. Conclusion It can be concluded that increase in the rate of aerobic reaction is affected by both substrate and enzyme concentration. Increase in the concentration of succinate would only result in increase in the rate of the reaction if the enzymatic concentration is also increased. The limitation to this study was that it is possible there was more than one substrate used in this experiment. This is because the mitochondrial suspension could have contained other substrates which were not accounted for in the reaction. Work Cited Apte, Shireesh P, and Rangaprasad Sarangarajan. Cellular Respiration and Carcinogenesis. New York: Springer, 2008. Print. Starr, Cecie, Christine A. Evers, and Lisa Starr. Biology: Concepts and Applications. Australia: Brooks/Cole, Cengage Learning, 2011. Print. Starr, Cecie, Christine A. Evers, and Lisa Starr. Biology: Today and Tomorrow : with Physiology. Australia: Brooks/Cole Cengage Learning, 2010. Print. Transmittance Tube 2 tube 3 Tube1 Time in Minutes Read More