bio lab report-formative (7)

P301442, FC303 Pubudo Biology Lab Report Effect of pH on the rate of enzyme activity Word count: P number: P301442 Name: Janah alQallaf I confirm that this assignment is my work. Where I have referred to academic sources, I have provided in-text citations and included the sources in the final reference list

The effect of pH on the rate of breakdown of hydrogen peroxide by catalase Introduction: The rate of hydrolysis is responsible for converting hydrogen peroxide to oxygen and water (Kaushal et al, 2018). Enzymes are needed to break down nutrients in the body so that they can be absorbed. Enzymes are extremely important proteins that impact an organism’s life tremendously, their main purpose is to increase the rate of reaction including the breakdown of hydrogen peroxide. The pH can have a huge impact on enzyme activity, for example, if the environment of the enzyme happens to have a ph. level of 1-5 meaning it is too acidic which can lead to the enzyme stopping working. This is also true if the pH level was between 8-14 meaning it is too basic; this information is valid if it was compared to the natural environment of an enzyme. (UKEssays. November 2018.). Temperature is one factor that affects the rate of enzyme activity, and pH and the presence of inhibitors are factors as well. Catalase is an enzyme that is made by cells to break down hydrogen peroxide, which is a waste product of the cellular activity, and it acts as a poison to cells. The thing that speeds up the decomposition of hydrogen peroxide into water (H2O) and oxygen (O) is catalase. The rate of enzyme activity can be measured by the number of bubbles produced which is what the aim of this experiment is. There are several theories that potentially explain how pH affects how quickly the catalase enzyme breaks down hydrogen peroxide. Some of the primary hypotheses are as follows: The hypothesis of competitive inhibition: According to this idea, pH fluctuations may have an impact on the ability of certain molecules, such as inhibitors or stimulators, to bind to the peroxide enzyme's active site, which may have an impact on the enzyme's enzymatic performance (Tao et al., 2009). The theory of ionization: This idea states that the ionization state of the amino acid residues in the catalase enzyme's active region can be influenced by the pH of the buffer solution. Some of the amino acid residues may become ionized if the pH is too high or too low, changing the structure of the enzyme and lowering its catalytic activity. According to the protonation theory, pH fluctuations may have an impact on the hydrogen peroxide substrate's protonation state, which may then have an impact on the substrate's capacity to attach to the catalase enzyme's active site. Finally, the catalase enzyme's overall shape or conformation may vary in response to pH variations, which may impair the enzyme's capacity to bind to and catalyze the breakdown of hydrogen peroxide, according to conformational theory (Tao et al., 2009). By considering these theories, researchers may create experiments that alter the buffer solution's pH and track variations in how quickly catalase breaks down hydrogen peroxide (Tao et al., 2009). Researchers can comprehend the variables that affect the catalase enzyme's activity and how it might be enhanced for a variety of industrial and medicinal uses by analyzing the results considering these hypotheses. Materials: You would need the following materials to study how pH affects how quickly catalase breaks down hydrogen peroxide: 1. Three Boling tubes 2. Graduated cylinders /Gilson pipette: Use these to measure and transfer the correct volume of reactants and buffer solutions. 3. Connecting tube ( Test tubes or cuvettes ): Use these to hold the reaction mixtures. 4. Stopwatch/ timer : Use to time the reaction 5. Distilled Water: Use to make the buffer solutions and dilute the hydrogen peroxide solution. 6. Catalase enzyme: Obtain a pure sample of catalase enzyme. 7. Buffer solutions: Prepare a series of buffer solutions with different pH values ranging from acidic to alkaline. You can use a buffer system, such as the phosphate buffer system, to maintain a constant pH.

Firstly, three Boling tubes were labeled as acid, base, and pH7 these are tube 1 in figure 1. Secondly, 3 ml of potato catalase was placed into the acid tube, this was done using a 5ml Gilson pipette. Thirdly, 3 drops of 0.5m HCL were added to the acid tube then, the acid test tube was left for 5 minutes. Meanwhile, test tube 2 was filled with tap water halfway, after that 3ml of hydrogen peroxide solution was added to the acid tube using a 5 ml Gilson pipette. After doing so, the two contents were mixed by gently swirling them around. Next, the bung was replaced, and the delivery tube connected to the bung was placed into test tube 2 as shown in the diagram above. After a wait of 10 seconds, the bubbles that evolved throughout 1 minute were counted and recorded in a table. Moreover, the contents of the acid tube were discarded in the sink and rinsed thoroughly under the tap. Furthermore, the procedure was repeated precisely as before using the rinsed tube and fresh potato catalase filtrate to obtain a second count of the number of bubbles that evolved per minute. The second count was recorded as well, in addition, the procedure was replicated a third time to gain a third count of the number of bubbles produced. Results 1mol/dm-3 concentration of hydrogen peroxide 0.5 HCL pH0.3 pH 7 0.5M NaOH pH 13.7 1 st count of bubbles per min 1 10 10 2 nd count of bubbles per min 7 12 10 3 rd count of bubbles per min 11 15 11 The average count of bubbles per min 6 12 10 Figure 1.2 shows the result 0 2 4 6 8 10 12 14 16 Shows The Line Graph On The Effect Of Ph On The Enzyme Catalase 0.5 HCL pH0.3 pH 7 0.5M NaOH pH 13.7 Figure 1.3 shows the line graph on the effect of pH on the enzyme catalase Results The results table was used to record the relative rate of reaction which was the number of bubbles produced per minute at different pH levels, it was also used to plot the line graph above. There were 3 pH levels, 0.3 which is acidic/ acid form, 7 which is neutral, and 13.7 which is basic/ base form. The average rate was calculated by adding together the number of bubbles at each count and dividing it by three which is the number of times the experiment was repeated. At the pH level of 0.3, the number of bubbles was at its lowest averaging at 6 bubbles per minute, this indicates that the lower the pH level the

fewer bubbles were produced. At a pH level of 7, it was seen that most bubbles were formed specifying that pH 7 was the optimum value of the reaction. At a pH level of 13.7, the bubbles averaged at 10 bubbles meaning that it is still less than the optimum value of pH 7 so the result was accurate. Overall, the table showed that most bubbles produced were at a pH level of 7 and lower or higher pH levels will result in fewer bubbles evolving. Discussion : This lab aims to show the breakdown of hydrogen peroxide by the enzyme catalase, this was done to show evidence of how catalase can do that at different pH levels by producing bubbles because of the breakdown (oxygen being the bubbles formed). Hydrogen peroxide is poisonous, so cells need to break it down to get rid of any toxic materials, they do that by using the enzyme catalase. All aerobic cells produce catalase, and one enzyme can work on over 35 million molecules of hydrogen peroxide per second. (Ellen Genovese, Laura Blinderman, Petrik Natale, 2019) When hydrogen peroxide is broken down it turns into 2 nontoxic subcomponents of water (H2O) and oxygen (O2) which are in their gaseous state therefore bubbles can be seen. Catalase is an enzyme that speeds up chemical reactions, therefore it was used in this experiment to increase the rate at which hydrogen peroxide was being broken down. Enzyme pH levels affect the rate of enzyme activity and can change the rate of their active site, each enzyme has its specific optimal rate at which it performs best for catalase it was pH 7. When the pH was higher or lower than 7 it was denatured and lost its structure. (Claire Gillespie, 2018) The rate of enzyme activity was measured by the number of bubbles produced in each reaction at different pH levels which were 0.3, 7, and 13.7. The goal of the lab was reached when pH 7 was where the most bubbles were produced because it is the optimum level this indicated that the experiment was made correctly. This lab was accurate because the results were what we expected. Errors: An error had occurred because the bung was removed too quickly leading to gas escaping and bubbles not being for a pH level of 0.3, however, the results needed were still obtained. There were a few safety precautions that were taken before starting the experiment which were washing hands thoroughly before wearing gloves, as well as never taking the lab goggles off to avoid any toxic or hazardous substances reaching the eyes. In addition, Lab coats were buttoned down throughout then the whole experiment following the laboratory rules. Once the experiments were done the gloves were carefully disposed of into a trash can and hands were washed multiple times. Conclusion: To conclude, each pH level had a different effect on the breakdown of hydrogen peroxide by catalase., the higher or lower the pH the more chance it would be desaturated. The aim of the experiment was reached by analyzing and discussing the results throughout the lab report. There was a clear link between the pH level and the rate of reaction, as the pH varies the number of bubbles differs, and a higher rate of enzyme activity resulted in a faster pace of reaction. Catalase is most active at pH 7.