6 Abstract: This experiment attempts to answer the question of whether an increase in a succinate concentration (a component of the Krebs cycle) will lead to an increased rate of cellular respiration within a cell. We measured the amount of electrons given off by the succinate to fumerate redox reaction by using DPIP. DPIP is an electron acceptor that takes the place of FAD by accepting the electrons and turning from its oxidized blue state to its reduced clear state.
We had three tubes with varying concentrations of succinate and measured the transmittance of each over a half hour period to determine whether more succinate led to more DPIP being reduced. The results showed that the tube with no succinate (no reaction) had an average increase in percent transmittance from 58. 13% to 59. 97%. The tube with the least succinate raised in transmittance from 59. 1% to 73. 27%. The tube with the highest succinate concentration raised from 58. 27% to 85. 03%. This data supports my hypothesis that an increased amount of succinate will lead to a higher rate of cellular respiration.
The tubes with more succinate gave off more electrons, which reduced more of the DPIP, raising the transmittance. Introduction: Cellular respiration is a biological process used by most organisms, enabling them to produce ATP in large amounts, which can be used to provide energy for cells (Campbell, 2008). It involves three processes: glycolysis, Krebs cycle and the electron transport chain. All of these three produce ATP in some way and work ideally under aerobic conditions (in the presence of oxygen). It can also function in anaerobic conditions (lacking oxygen), in a process called fermentation.
However, this is much less effective since it leaves out the greatest producer of ATP, the electron transport chain (Campbell, 2007). This experiment focuses specifically on one step in the Krebs cycle, a series of eight catalytic reactions (Wrischnik, 2010). It involves oxidation, the losing of electrons, and reduction, the gaining of electrons. In the cycle, a compound gives off electrons to an oxidized carrier (NAD+ and FAD) in order to make it reduced (NADH and FADH2). It can then transport then electrons to the next step and continue cellular respiration (Wrischnik, 2010).
The question that this lab attempts to answer is how much the concentration of one of these compounds in the cycle affects the rate of cellular respiration. In order to do this, we used one specific part of the cycle, the conversion of succinate to fumerate. This involves the compound succinate giving protons and electrons to FAD and being converted to fumerate as a result (Wrischnik, 2010). One way to determine the rate of cellular respiration is by measuring the amount of protons and electrons given off by succinate in the cycle.
However, since there is no easy way to measure the amount of FADH2 produced, we used a substance called DPIP (di-chlorophenol-indophenol). Oxidized DPIP is a deep blue color, but when it accepts electrons or protons, it turns colorless (Wrischnik, 2010). When placed in the presence of succinate, it will absorb all of the electrons meant for the electron carriers and become reduced and colorless. By measuring the absorbance of DPIP with either a high or low succinate concentration, we determined how many electrons were given off, and therefore how much of the reaction was occurring (Wrischnik, 2010).
If the amount of light transmitted through the DPIP is low, this means that the DPIP is absorbing a lot of light and the DPIP is fairly oxidized. In this case the reaction is not taking place in high abundance. On the other hand, if the transmittance is high, then the DPIP is absorbing little light and is in its clear, reduced state. This would mean that a great deal of the reaction is taking place (Wrischnik, 2010). The solution of DPIP and succinate will take place with a prepared mitochondrial suspension created from pulverized lima beans and added glucose.
This suspension will serve to provide an environment in which the Krebs cycle can take place as it normally would with glucose available for respiration. There will also be a buffer added to the solution to ensure that the pH remains relatively constant, preventing the possibility that any enzymes would be denatured. The main question of this experiment is whether or not a high concentration of succinate will increase the rate of the Krebs cycle. My hypothesis is that an increase in the substrate will result in an increase in the rate of reaction.
If we increase the substrate concentration, then ore DPIP will be reduced, turning the solution clear and increasing the transmittance. Materials and Methods: The first thing that we did is flip switch C to turn on the Bausch & Lomb Spectronic 20 five minutes before we started, in order to let it warm up and calibrate properly. We then set the wavelength to 600nm, the wavelength at which DPIP absorbs light. We then used the control knob to set the spectrophotometer by turning the transmittance level to 0%, with the cover closed and the chamber empty.
After labeling four different cuvettes B, 1, 2, 3 corresponding to each trial, we prepared the blank (B) solution. Using a micropipette, we measured 4. 6 ml buffer, 0. 3 ml mitochondrial suspension, and 0. 1 ml succinate into the B cuvette that contained no DPIP. Before placing the cuvette in the spectrophotometer, we wiped down the sides and aligned the mark on the cuvette with the line on the holder to ensure that the reading came from the clear side of the cuvette. After closing the cover, we adjusted the light control so that there was a 0% absorbance with our control in the machine.
This ensured that our experimental samples were only showing readings for the light absorbed by the DPIP and not by the mitochondrial suspension as well. Tube 1 consisted of 4. 4 ml buffer, 0. 3 ml DPIP, 0. 3 ml mitochondrial suspension, and 0 ml of succinate. This served as a control because there was no succinate to give off electrons and reduce the DPIP. Tube 2 consisted of 4. 3 ml buffer, 0. 3 ml DPIP, , 0. 3 ml mitochondrial suspension and 0. 1 ml succinate. This was the trial with a low concentration of substrate. Tube 3 had 4. 2 ml buffer, 0. 3 ml DPIP, 0. 3 ml mitochondrial suspension and 0. 2 ml succinate.
This final tube was used to notice the effect of increased substrate on the amount of electrons given off, or the amount of DPIP reduced. We also made sure to add succinate to each tube last, so that the succinate to fumerate reaction would not occur before the DPIP was there to accept the electrons. Immediately after creating each cuvette, we covered it in parafilm to minimize contamination and placed it directly in the spectrophotometer to get an accurate reading. We recorded the absorbance of cuvettes 1, 2, and 3 every 5 minutes for the next thirty minutes and recorded the absorbances in a table.
We also made sure to properly mix the contents of the cuvettes thoroughly before getting a reading in order to best distribute the DPIP throughout the solution. Results: Transmittance of Experimental Cuvettes 1, 2, and 3 TABLE 1:| | Time| | | | | | | Cuvette 1| Group| 0| 5| 10| 15| 20| 25| 30| | 1| 59. 6| 58. 2| 60. 5| 61. 5| 61. 5| 61. 6| 61. 2| | 2| 55. 2| 51. 3| 51. 2| 52. 3| 51. 5| 52. 2| 50. 7| | 3| 59. 6| 64. 8| 65. 7| 63. 7| 64. 8| 66. 1| 68| | Average| 58. 13333333| 58. 1| 59. 133333333| 59. 16666667| 59. 26666667| 59. 96666667| 59. 96666667| TABLE 2:| | Time| | | | | | |
Cuvette 2| Group| 0| 5| 10| 15| 20| 25| 30| | 1| 58. 9| 63. 8| 68. 3| 70. 2| 72. 3| 72. 9| 73. 3| | 2| 59. 3| 60. 5| 62. 9| 67| 66. 6| 68. 5| 70| | 3| 59. 1| 65. 3| 69. 2| 71. 4| 73. 4| 74. 2| 76. 5| | Average| 59. 1| 63. 2| 66. 8| 69. 53333333| 70. 76666667| 71. 86666667| 73. 26666667| Caption: Contains 4. 3 ml buffer, 0. 3 ml DPIP, and 0. 3 ml mitochondrial suspension. Control. Caption: Cuvette 2 contains 4. 3 ml buffer, 0. 3 ml DPIP, 0. 3 ml mitochondrial suspension, and 0. 1 ml succinate. Purpose is an experimental sample with low concentration of succinate. TABLE 3:| | Time| | | | | | |
Cuvette 3| Group| 0| 5| 10| 15| 20| 25| 30| | 1| 58. 9| 73. 5| 78. 2| 79. 6| 82| 82. 6| 83. 1| | 2| 58. 2| 69. 2| 71. 5| 74. 5| 75. 5| 77. 1| 81| | 3| 57. 7| 73. 2| 79. 4| 82. 5| 79. 9| 85. 2| 91| | Average| 58. 26666667| 71. 96666667| 76. 36666667| 78. 86666667| 79. 13333333| 81. 63333333| 85. 03333333| Caption: Cuvette 3 contains 4. 3 ml buffer, 0. 3 ml DPIP, 0. 3 ml mitochondrial suspension, and 0. 1 ml succinate. Purpose is an experimental sample with low concentration of succinate. The three cuvettes each represent a different amount of succinate, which will affect how much DPIP is reduced.
In cuvette 1 with no succinate, the transmittance level raised from 58. 13% to 59. 97%, only raising a little under 2%. In cuvette 2, with 0. 1 ml succinate, the transmittance raised from 59. 1% to 73. 27%, a rise of 14%. In cuvette 3, with 0. 2 ml succinate, the transmittance raised from 58. 27% to 85. 03%, a rise of a little under 27%. The graph (Figure 1), on the following page, shows the trend in transmittance over time for each different solution, based on three sets of data for each cuvette. The time is the independent variable and the transmittance is the dependent variable.
Caption: This graph displays the trends and the line of best fit slope equations Discussion: The original question that my research attempted to answer was whether an increase in succinate, a substance present in the Krebs cycle, would accelerate the rate of cellular respiration. I hypothesized that an increase in succinate concentration would lead to an increase in the amount of the reaction taking place, and I predicted that if the quantity of succinate is increased, then the amount of amount of DPIP reduced would also increase.
Based on the evidence collected, it is fair to conclude that an increased amount of succinate does in fact lead to a faster rate of cellular respiration. Since DPIP turns colorless when it accepts electrons and is reduced, less light will be bouncing off of the blue pigment in it, and instead will be transmitted through. Therefore, the tubes with the highest transmittance have the most DPIP being reduced during the conversion of succinate to fumerate. Table 3 shows the tubes with the highest concentration of succinate went up the largest amount in percent transmittance.
On the other hand, Table 1 shows that a solution with no succinate, and therefore no reaction, increases very little. As expected Table 2 shows that a solution with a low succinate concentration has a transmittance in between the two extremes. The data supports my hypothesis because the transmittance increase for tube 1 is 1. 84%, 14. 17% for tube 2, and 26. 77% for tube 3. This shows that the tubes with more succinate became clearer than those with less. The trend lines in Figure 1 also support this fact, showing the steepest upward slope for tube 3, followed by tube 2 and then tube 1.
The graph also shows that the first few readings contain the most significant jump in transmittance. This is because the concentration of succinate is highest, and it is being converted in abundance until it begins to become scarce, providing fewer electrons for DPIP to become reduced. In this experiment, the amount of electrons produced was used as an indicator of cellular respiration rate, and the reduction of DPIP was used as a tool to measure this indicator. Based on this, the data collected definitely supports my hypothesis that the succinate increase leads to more cellular respiration taking place.
My prediction was also supported because raising the succinate concentration did indeed lead to the transmittance raising. While the experiment was effective in producing the results that I expected, errors were made and there was room for improvement. One possible source of error is differing times in between adding the succinate and measuring the transmittance between trials. Ideally, we would’ve placed the completed solution in the spectrophotometer immediately after introducing the succinate. However, this was not possible and the times between adding the succinate and taking the first reading differed among trials.
The effect of this is simply that some reactions will have started before others, and some may be farther along when the first and subsequent readings are taken. Another possible source of error is insufficient mixing of the tubes before transmittance readings. Not mixing the tubes properly may allow some of the important elements of the substance to settle at the bottom, giving the tube an inaccurate reading. Improvements on the experiment include more trials for each tube. An increase in trials would enhance the reliability and credibility of the data.
Another possible improvement would be to include more succinate concentrations. While, this would require more work, having more tubes with higher concentrations of succinate could only further help support the conclusions being made. Further research that could be done on a similar topic would be to analyze the effect of temperature or pH on the rate of cellular respiration. While a different method than the one used in this experiment would need to be used, there is a lot of potential for discovering more about what affects cellular respiration.
Examining the rates of cellular respiration is important, mainly because it plays such an important role in our lives and there are so many factors that can influence it. For example, a study by Geeraerts and Vigue studied the effects of temperature on cellular respiration in the brain. Traumatic brain injury in people can sometimes lead to exceeding brain temperature, which can later lead to cerebral dysfunction (Geeraerts and Vigue, 2009). By analyzing how cellular respiration is affected by different factors can go a long way towards helping people and solving a lot of unanswered questions.
My conclusions from this experiment are that the rate of cellular respiration is increased with higher levels of succinate. Literature Cited: Campbell, N. , Reese, J. , Urry, L. , Cain, M. , Minorsky, P. , Wasserman, S. , and Jackson, R. 2008. Biology. 8th ed. San Francisco: Benjamin Cummings. 162-175. Geeraerts, T. , and Vigue, B. 2009. Cellular metabolism, temperature and brain injury. Annales francaises d’anesthesie et de reanimation. 28: 339-334. Morgan, J. G. , and Carter, M. E. B. 2010. Symbiosis: the pearson custom library for the biological sciences. Special Edition. San Francisco: Pearson Custom Publishing. 97-102.
