Cellular respiration is a process by which glucose is broken down in a complicated four step process to produce energy for cellular functions. Cellular respiration is vital for survival as it produces ATP which powers nearly all activities of all cells. Cellular respiration can be defined as “chemical mechanisms by which the cell converts the bound, radiant energy of the sun, stored in foodstuff molecules, to free utilizable biotic energy, thereby making possible cellular activity and even cellular existence” (Reid, 1940). Although this definition is dated it stands true to this day. Cellular respiration can be quantified; it “is normally determined by metabolic activity and the corresponding rate of ATP utilization (Chandel, Budinger, Choe, and Schumacker, 1997). With these things in mind cellular respiration can be separated into parts and those induvial parts can be quantified using various methods.
The first step of cellular respiration is glycolysis: in this step, glucose is broken down into two pyruvate molecules. Breaking down a six-carbon glucose into two three-carbon pyruvate molecules produces ATP (useable free energy) and NAD+ is converted to NADH. In the next step, Pyruvate enters the mitochondria and is converted into a two-carbon molecule called acetyl and is combined with an enzyme called Coenzyme A (CoA) to form Acetyl CoA. This process releases carbon dioxide and makes NADH. The third step of cellular respiration, the citric acid or Krebs Cycle, combines Acetyl CoA with a four-carbon molecule; this process produces ATP, FADH2, and NADH. The final step in cellular respiration oxidizes NADH and FADH2 to NAD+ and FAD. The oxidation process releases energy and the lost electrons travel down the electron transport chain and are pumped out of the cell which creates a concentration gradient. ATP synthase then pumped the electrons back into the cell which created ATP. At the end of the electron transport chains the uptake of electrons by oxygen to forms water.
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The Krebs cycle is a complicated process with the breakdown of many complex molecules. In this experiment, part of the Krebs cycle was observed as succinate converted to fumarate. Mitochondrial suspension extracted from lima beans was used as the enzyme to which succinate binds to be oxidized and loses electrons to FAD; however, FAD was replaced with DPIP in this experiment. DPIP was used because it is blue in its oxidized form and becomes colorless as it is reduced. By measuring the percent transmittance of the DPIP solution, the rate at which the Krebs cycle occurs can be quantified and analyzed.
A spectrophotometer was used to measure the percent transmittance of DPIP. A spectrophotometer can measure percent transmittance or percent absorbance by shooting light through a cuvette full of solution and comparing it to a blank (clear solution) to come up to a percent difference in the transmittance through the solution or absorbance of light by the solution. In the experiment, a spectrophotometer was used to find the percent transmittance of DPIP over a set amount of time to observe reaction rate of cellular respiration. The more substrate (succinate), the faster the rate of cellular respiration will be as DPIP goes from a blue to colorless solution. In this experiment, if the concentration of succinate increases then the rate of respiration will increase as shown by the change in percent transmittance.
Methods
This experiment highlighted cellular respiration through focusing on part of the Krebs cycle. The transformation of succinate to fumarate by loss of hydrogen ions and electrons was observed. DPIP was used in this experiment and acted as an electron acceptor. Different amounts of succinate (substrate) was added to three different solutions. The spectrophotometer measured percent transmittance of DPIP, as DPIP was reduced and changed from blue to clear, over a set interval of time. These percents measured determined the rate of cellular respiration in relation to the amount of substrate within the solution.
The spectrophotometer was set up first. The wavelength setting was adjusted to 600nm because that is the wavelength absorbed by DPIP. The spectrophotometer was set to read transmittance in order to compare color change of DPIP from dark blue to clear as it becomes reduced. The spectrophotometer was then calibrated to 0% transmittance (T) without a cuvette in the machine. A blank was then created with a ratio of 4.6mL of Buffer: 0.3mL of mitochondrial solution: 0.1mL of succinate and then was covered tightly using parafilm and inverted to mix the solution. The spectrophotometer was calibrated a second time. The blank was wiped with a Kimwipe to remove fingerprints and smudges. The blank was then placed in the spectrophotometer and the transmittance was adjusted to 100%T. The blank is used as 100%T to adjust for the mitochondrial solution. Therefore, by setting the spectrophotometer to 100%T for the blank solution, the spectrophotometer will read the transmittance with regards to the clarity of the mitochondrial solution.
In order to accurately compare the relationship of the independent variable (amount of succinate) and the dependent variable (percent transmittance) controlled variables were established. These variables included the total volume of the solutions in each cuvette, time, the amount of DPIP, and the amount of mitochondrial suspension.
Three other solutions were measured into three different cuvettes. Each cuvette contained 0.3mL of DPIP and 0.3mL of mitochondrial suspension. Each cuvette also contained varying amounts of buffer and succinate. Cuvette one contained 4.4mL of buffer and 0mL of Succinate, Cuvette two 4.3mL of buffer and 0.1mL of succinate, and Cuvette three contained 4.2mL of buffer and 0.2mL of succinate. The succinate was not added until all other elements of the solutions were combined into the appropriate cuvettes and the experiment was ready to begin. The proper amounts of contents for the solutions in each cuvette can also be found below in Table 1. Cuvette one acts as the control for the experiment. Cuvette one had all elements of the solution aside from succinate and therefore had no main reactant.
Table 1. Contents of Cuvettes for Cellular Respiration Experiment. Each tube contains 0.3mL or DPIP and mitochondrial suspension (controlled variables) and varying amounts of succinate (independent variable) and buffer. The buffer amount varied in order to keep the total volume of all solutions consistent; even though the buffer amounts varied it kept the total volume consistent and is therefore apart of the controlled variable. Cuvette one was the experimental control as it did not contain succinate.
Once the succinate was added, the cuvettes were immediately covered with Parafilm and inverted. Cuvette one was wiped with a Kimwipe and placed in the spectrophotometer. The percent transmittance was recorded in Table 2 in the Results section. The results recorded in Table 2 were then used to make a line graph (Figure 1 in the Results section). After this step was completed, the exact procedure was performed on Cuvette two and Three. Every five minutes of the thirty-minute experiment, the cuvettes were inverted and wiped down with Kimwipes, and the percent transmittance was recorded in Table 2 for all three cuvettes. In between each set of readings, the blank was placed in the spectrophotometer and recalibrated to 100%T to ensure each transmittance reading of Cuvettes One, Two, and Three are accurately corrected for mitochondrial suspension.
Discussion
This experiment tested whether the amount of succinate in solution affected the rate at which cellular respiration occurs in the Krebs cycle. The hypothesis and prediction for this experiment was supported. Increased levels of succinate in solution did increase the rate of cellular respiration. This general trend of increased concentration of succinate increased the rate of cellular respiration is show in Figure 1 in the results section. Notice that the purple line, which represented Cuvette three had the most succinate (0.3mL) and had the greatest change in percent transmittance over the thirty-minute interval (9.4%T). This means the solution in Cuvette three had the fastest rate of change. Cuvette one containing no succinate did react; however, it had the lowest change in percent transmittance, and therefore, had the slowest reaction rate. Cuvette two, containing 0.1mL of succinate had a change in percent transmittance in between Cuvette one and Three. This means Cuvette two had a reaction rate that also fell in between Cuvette one and Three.
There was, however, an outlier in the data. Cuvette two had a starting amount of 11.0%T which was greater than Cuvette three’s starting amount of 10.4%T. This was an outlier because Cuvette three was supposed to have a greater percent transmittance than both cuvettes throughout the entire experiment. This was most likely due to an increased wait time before the percent transmittance was recorded for Cuvette two; meaning, the succinate had more time to react before the reading was taken. As the experiment continued, the percent transmittance followed the predicted trend and the outlier was negligible.
Experimental conclusions are pertinent to the field of biology in order to fully understand the mechanics of life. Conclusions help to summarize experiments and can be compared to replicates of the same experiment and/or other similar experiments. These comparisons analyze the experiments performed and therefore verify the data. This verified data can then be recognized as true. In this experiment the conclusions can be compared to other similar experiments or replicates to verify the data and results. The conclusion drawn express the importance the amount of succinate as it is oxidized in cellular respiration.
Suggestions for future research would be to run the experiment at a longer time interval and add cuvettes with more succinate to compare to the three cuvettes already being tested. By increasing the time interval, the reaction could be observed from start to total completion. By adding more cuvettes with larger amounts of succinate, a reaction can be observed where the amount of reactants (succinate) will not change the percent transmittance of DPIP. Future research allows for more conclusions to be drawn and more comparisons to be made. This further of data therefore furthers the understanding of the research taking place.
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