Introduction
Energy, the ability to do work, plays a key role in all living organisms by providing its cells with fuel to function properly. Because of this, metabolism also plays a key role in all living organisms. Metabolism measures all of an organism’s chemical reactions. One of the main functions of metabolism is to organize the energy of the organism which is done through metabolic pathways. The metabolic pathway begins with a specific molecule which is then altered down its pathway, each step catalyzed by an enzyme, and ending with a specific product (Urry, et al, 2017). An enzyme is a protein that acts as a catalyst in order to speed up a chemical reaction. An enzyme does this by lowering a reaction’s activation energy, which is the amount of energy needed to start a reaction. In a chemical reaction, the enzyme acts on a substrate, or the reactant. The substrate binds to a specific enzyme on the active site, the region where the substrate binds and is shaped specifically for a certain substrate. Together this complex is known as an enzyme-substrate complex. Without these enzymes, an organism’s metabolic pathways would be heavily affected and disorganized. Enzymes play an important role in the study of biochemistry and can also branch out into life science and medicine. Specifically, with metabolism and how humans and animals consume food in order to maintain the energy input their bodies require. The main purpose of food is to provide energy and nutrients to our bodies. The food that humans consume consists of biological molecules such as carbohydrates, lipids, and proteins. When enzymes break down these molecules, the chemical energy that is stored in these molecules will be released and used by cells (“Protein - Role of enzymes in metabolism | Britannica,” 2020). Through the following experiments, the importance of enzymes and their functions are highlighted.
The following experiments focuses on how the concentration of potato extract and the surrounding temperature affects the absorbance of benzoquinone by catechol oxidase. For the first experimental design, it is hypothesized that increasing the concentration of the enzyme extract solution will increase the amount of benzoquinone produced therefore increasing its absorbance. For the second experimental design, it is hypothesized that as the surrounding temperature increases, the amount of benzoquinone produced will increase therefore increasing its absorbance.
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Methods
The first experiment was designed to represent how the concentration of the potato extract enzyme affected the catalyzation of production of benzoquinone measured by its absorbance. There was a total of five experimental cuvettes prepared, with each going through two trials. Catechol oxidase was acquired by homogenizing potatoes in which produces an impurified version of catechol oxidase (Scott, et al, 2020). Furthermore, a buffer, a solution that maintains the pH levels during the reaction, was added. A spectrophotometer was used in order to measure the absorbance of benzoquinone. The wavelength on the spectrophotometer was set to 486 nm. First, for each treatment a blank was created. The first treatment blank consisted of 5.0 mL of pH 6 buffer and 0 mL of the potato enzyme extract. The second treatment blank consisted of 4.8 mL of pH 6 buffer and 0.2 mL of the potato enzyme extract. The third treatment blank consisted of 4.6 mL of pH 6 buffer and 0.4 mL of the potato enzyme extract. The fourth treatment blank consisted of 4.4 mL of pH 6 buffer and 0.6 mL of potato enzyme extract. Lastly, the fifth treatment blank consisted of 4.2 mL of pH 6 buffer and 0.8 mL of potato enzyme extract. For each treatment, the blank was inserted into the spectrophotometer and zeroed out. Next, the experimental cuvettes were created consisting of the same additions as the blank cuvettes except with 0.5 mL of catechol added as well as 0.5 mL less pH 6 buffer. Each experimental cuvette was tipped back and forth for approximately three minutes in order to incorporate oxygen into the solution. The experimental cuvettes were inserted into the spectrophotometer and their absorbances were measured. For this experimental design, the concentration of the potato enzyme extract served as the independent variable. This caused the amount of benzoquinone produced and its absorbance to be the dependent variables. Furthermore, the substrates, 0.5 mL of catechol and oxygen, served as the controlled variables.
The second experiment was designed to represent how the temperature of each solution affected the production of benzoquinone measured by its absorbance. This experimental design consists of the same substrates and products as the previous experimental design. First, five blanks were created by filling five test tubes with 5 mL of pH 6 buffer and 0.5 mL of potato enzyme extract solution. Each blank was placed in either room temperature water, 33°C water, ice water, 55°C water, or boiling water. Next, 10 experimental test tubes were filled with 4.5 mL of pH 6 buffer and 0.5 mL of potato enzyme extract solution. In pairs, the experimental test tubes would be placed in differing temperatures, have 0.5 mL of the potato extract enzyme added, be parafilmed then slightly mixed, and placed back into the water for 3 minutes. The temperatures would be one of five including: 3°C (an ice bath), 20°C (room temperature), 37°C, 55°C, or 63°C (boiling water). The experimental test tube placed in boiling water (63°C) was not parafilmed due to high temperature. Finally, the blank test tube and experimental test tubes would be placed into the spectrophotometer where its absorbance would be recorded. For this experiment, temperature served as the independent variable. This causes the production of benzoquinone and its absorbance to be the dependent variables. Similar to the first experimental design, the controlled variables would be the amount of substrates (catechol and oxygen) as well as the pH 6 buffer solution.
Discussion
The results for the experimental design dealing with the concentration of potato extract supports the hypothesis which states that increasing the concentration of the enzyme extract solution will increase the amount of benzoquinone produced therefore increasing its absorbance. According to the results, as the volume of the potato enzyme extract solution increased, the absorbance of benzoquinone increased. This experiment included a blank that served as the negative control. The blank consists of the enzyme extract and the buffer. Since the blank does not include catechol, there is no reaction occurring. The cuvette with no potato extract enzyme had 0 absorbance. It’s possible that this cuvette can serve as a negative control as well since the chemical reaction did not occur fast enough for results to be seen. The following cuvettes serve as the positive control since a chemical reaction did occur and there was an absorbance of benzoquinone that was measured. The absorbance of benzoquinone started at 0.087 for both trials when 0.2 mL of potato extract enzyme was added to the experimental test tube. The next cuvette consisted of 0.4 mL of potato extract enzyme and for both trials the absorbance of benzoquinone increased, both trials falling in close proximity with each other. The next cuvette consisted of 0.6 mL of potato extract enzyme and for both trials the absorbance of benzoquinone increased. However, the results of each trial do not fall in close proximity with each other with the difference between the trials being 0.108 au. This could have been due to a systematical error where, possibly, the cuvette was not properly placed in the spectrophotometer. The final cuvette consisted of 0.8 mL of potato extract enzyme and the absorbance of benzoquinone increased, both trials falling in close proximity with each other. The absorbance of benzoquinone increased as the volume of potato extract enzyme increased because the amount of benzoquinone produced increased. Absorbance is related to the amount of benzoquinone produced because of the color of the experimental solution once taken out of the spectrophotometer. As the volume of enzyme increases, the experimental solution turns a darker brown which would cause measurements of absorption to increase (Scott, et al, 2020). In a chemical reaction, the role of an enzyme is to speed up or catalyze the reaction by decreasing the amount of activation energy used to start the chemical reaction (Urry, et al, 2017). More specifically, the substrates present in the solution, which in this case is catechol, are more likely to collide with the enzyme present in the solution. Because the volume of the potato extract enzyme increased, the chances of the substrate colliding with the enzyme increases which then increases the amount benzoquinone produced (Scott, et al, 2020). This experiment showcases the biological importance of an enzyme’s impact on a chemical reaction. Without the presence of an enzyme, a chemical reaction is still likely to occur, just at a slower rate. However, certain reactions could take days, months, or years to completely occur which can be inefficient. This serves especially true when it comes to how cells use enzymes to facilitate its metabolic pathways and maintaining homeostasis (Cooper, 2016). For this experiment specifically, the enzyme catechol oxidase serves an important role when it comes to its use in plants. In plants, catechol oxidase is released when the cells in the plant are injured, in which the plant produces benzoquinone (Scott, et al, 2020). Benzoquinone has insecticidal and bactericidal properties that help protect the plant when it is injured (Scott, et al, 2020). Without the enzyme, the plant would not be able to protect itself and could potentially die. Overall, depending on the amount of potato extract enzyme that is present in the solution, the amount of benzoquinone produced will either increase or decrease due to their direct relationship.
The results for the experimental design dealing with the surrounding temperature partially supports the hypothesis which states that increasing surrounding temperature will increase the amount of benzoquinone produced therefore increasing its absorbance. According to the results, as the temperature of the water bath surrounding the test tube increases, the absorbance of benzoquinone increases. This experiment included a blank that served as the negative control and consists of catechol and oxygen. Because no chemical reaction occurred due to the absence of catechol, it served as the negative control. The following test tubes served as the positive controls because catechol is present in the solution in which a chemical reaction would occur. Different pairs of test tubes are placed in different temperatures of water including 3°C (an ice bath), 20°C (room temperature), 37°C, 55°C, and 63°C (boiling water). Within the two trials, the absorbances of benzoquinone at 55°C falls in close proximity with each other being 0.230 and 0.217 au. With the other temperatures, the absorbances of benzoquinone do not fall in close proximity with each other, mainly with a difference in around 0.70-0.100 au. This could be due to a systematical error with the spectrophotometer. It could have been possible that the cuvette was not placed in the instrument properly, therefore affecting the amount of absorbance measured. However, at 63°C (boiling water) the absorbance of benzoquinone drops drastically to 0.000 au. Similar to the previous experiment, absorbance is related to the amount of benzoquinone produced because of the color of the experimental solution once taken out of the spectrophotometer. As the surrounding temperature increases, the amount of benzoquinone produced increases because heat has the ability to speed up reactions. As temperature increases, the substrates have a higher chance of colliding with an enzyme due to the increase in motion (Urry, et al, 2017). However, high temperatures can also lead to the denaturation of an enzyme, which is a protein, which causes the speed of the enzymatic reaction to drop drastically (Urry, et al 2017). This occurs because the enzyme has gone over its optimal temperature where its performance is at its greatest. Seeing how the absorbance of benzoquinone dropped to 0.000 au when the surrounding temperature reached 63°C, it can be assumed that 63°C is above the potato extract enzyme’s optimal temperature. Therefore, the hypothesis for this experiment is not fully supported since enzymes have optimal temperatures. An example of the negative effects of high temperature on enzymes is with fevers when body temperatures usually reach 38°C or greater. When the body reaches high temperatures, enzymes are also affected and could potentially become denatured if temperatures reach high levels (“Enzyme Function Dependent On Temperature - Wilson’s Syndrome,” 2020). This also applies to lower temperatures when experiencing hypothermia. If enzymes become denatured, it has the effect to negatively impact the body’s functions and overall homeostasis. Overall, depending on the surrounding temperature, the amount of benzoquinone that is produced will either increase or decrease due to their direct relationship.
Conclusion
Ultimately, both experiments conducted above were designed to showcase the effects of enzyme concentration and surrounding temperature on the production of benzoquinone and its absorption. The results for the enzyme concentration experiment support the hypothesis which states that as the concentration of potato extract enzyme increases, the amount of benzoquinone that is produced increases. Furthermore, the results for the temperature experiment partially supports the hypothesis which states that as the surrounding temperature increases, the amount of benzoquinone produced increases. The hypothesis is only true to a certain extent until the enzyme reaches its optimal temperature. Although two different variables were used to calculate the amount of benzoquinone produced, both variables have a direct relationship to the amount and rate of production of benzoquinone in a chemical reaction.
Reference List
- Cooper, G. M. (2016). The Central Role of Enzymes as Biological Catalysts. Retrieved November 9, 2020, from Nih.gov website: https://www.ncbi.nlm.nih.gov/books/NBK9921/#:~:text=A%20fundamental%20task%20of%20proteins,reactions%20are%20catalyzed%20by%20proteins.
- Enzyme Function Dependent On Temperature - Wilson’s Syndrome. (2020). Retrieved November 9, 2020, from Wilson’s Syndrome website: https://www.wilsonssyndrome.com/ebook/body-function-dependent-on-body-temperature/enzyme-function-dependent-on-temperature/
- Protein - Role of enzymes in metabolism | Britannica. (2020). In Encyclopædia Britannica. Retrieved from https://www.britannica.com/science/protein/Role-of-enzymes-in-metabolism
- Scott, S. M., Deneke, C., & Neff, E. (2020). Principles of Biology Lab Manual. Sacramento, CA: Cosumnes River College.
- Urry, L. A., Cain, M. L., Wasserman, S. A., Minorsky, P. V., Reece, J. B., & Campbell, N. A. (2017). Campbell biology. New York, NY: Pearson Education
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