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Using pH, temperatures, and substrate concentration to determine enzyme activity levels....

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Effects of Temperature, pH, and Substrate Concentration on Catalase Activity

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University of South Carolina Biology 101 Laboratory Section 046 Nikita Kirkise Effects of Temperature, pH, and Substrate Concentration on Catalase Activity Submitted by Ivana Daniels October 1, 2020

Effects of Temperature, pH, and Substrate Concentration on Catalase Activity

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Abstract Enzymes are biological catalysts that bring about a specific biochemical reaction. Enzymes are used to initiate a reaction or speed up a reaction. Many factors occur in the body that could prohibit or induce enzyme activity, determined through a series of experiments. Specifically, in this lab, the factors that tested included: temperature, pH, and substrate concentration. In terms of temperature, the hypothesis was that catalysis would occur faster as the temperature increased, and upon reaching the optimum temperature, the activity of catalysis would cease. In terms of pH, the hypothesis was, an excess of H+ or OH- ions would result in the distortion of the active site; therefore, the pH should be neutral. In terms of substrate concentration, the hypothesis was that an increase in substrate concentration would increase the chemical reaction rate until a sufficient amount was present to saturate all present enzyme molecules. Each factor had an experiment where the two remaining factors were the constants, and the one factor tested for changed. The optimum temperature, pH, and substrate concentration determine maximum enzyme activity.

Introduction The human body possesses many processes carried out by an enzyme's work; a biological molecule produced by a living organism. (Worthington et al., 1972) It acts as a catalyst to bring about a specific biochemical reaction. The function of an enzyme is specific to its location, shape, and many other factors. In general, an enzyme's primary function is to speed up or initiate chemical reactions by breaking down or constructing specific molecules. Without enzymes, many of the systems set up throughout the human body would not function accordingly.

Effects of Temperature, pH, and Substrate Concentration on Catalase Activity

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There are many factors needed to carry out an enzymatic reaction, one of which is the substrate. The substrate in an enzymatic reaction is what the enzyme is acting on. The molecule or molecules created after the reaction are called the products. The rate at which a chemical reaction occurs does increase as the concentration of substrate increases. However, factors such as temperature or pH can also affect chemical reaction rates. The enzyme used throughout all three experiments was catalase. Most living cells contain the catalase enzyme because a plethora of cellular reactions results in hydrogen peroxide production. (Goodsell, 2004) Hydrogen peroxide in the body is dangerously reactive, and catalase substantially accelerates the decomposition thereof. (William, 1928) This reaction produces water, oxygen, and heat, as shown in the following equation: 2H2O2 + catalase (catalyst) à 2H2O + O2 + heat Specifically, there is a very high concentration of catalase in mammals, the catalase source used throughout the lab. The oxygen gas produced was used as the evaluation factor in the experiment. To evaluate, answer the following questions: Oxygen production in mL? Production speed? The most effective temperature, pH, or substrate concentration was the optimum choice out of all the other choices.

Materials and Methods As previously stated, three separate experiments provided the answer to one question: how does temperature, pH, and substrate concentration affect catalase activity? The first experiment determined the optimum temperature for catalase activity. A constant temperature bath was taken from the instrument shelf and set to 10 ºC. A small test tube was then taken from the containers

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shelf and placed in the constant temperature bath. Five mL of 3% hydrogen peroxide was taken from the materials shelf and added to the test tube. Next, five mL of distilled water was added to the same test tube. A thermometer monitored temperature. The hydrogen peroxide and water solution temperature had to stabilize at 10 ºC before continuing the experiment. Once the temperature stabilized, one mL of catalase solution was added to the test tube. After adding the catalase solution, the gas syringe was added to the test tube, and a rubber stopper was placed on it. The initial gas volume was recorded before proceeding. A fifteen-second timer began, and the rubber stopper was removed from the test-tube. After the conclusion of the fifteen seconds, the final gas volume was recorded. Both the thermometer and gas syringe were put back in the instruments shelf, and the test tube was emptied and placed in the sink. This same procedure was repeated four more times using temperatures of 21.5, 40, 60, and 80 ºC instead of 10 ºC. The optimum temperature for catalase activity based on the results was recorded and used in the next experiment. In the second experiment, substrate concentration was tested. A constant temperature bath was set to the optimum temperature for catalase activity, determined in the first experiment. Four test tubes were taken from the container shelf and labeled 1-4. The first test-tube contained eight mL of water and two mL of 3% Hydrogen Peroxide. The second test tube contained five mL of water and five mL of 3% Hydrogen Peroxide. The third test tube contained two mL of water and eight mL of 3% Hydrogen Peroxide. The fourth test tube contained no water and ten mL of 3% Hydrogen Peroxide. After ensuring each test-tube had a specific concentration, the experiment began. The first step involved placing the first test tube into the constant temperature bath. A thermometer was placed inside the test tube. Once the temperature stabilized and the test tube reached the same temperature as the bath, one mL of catalase solution was added to the first test

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tube. A rubber stopper was added, using a gas syringe, the initial gas volume was recorded. A fifteen-second timer was set, and the rubber stopper was removed. After the fifteen seconds elapsed, the final gas volume was recorded. The same procedure was conducted for the second, third, and fourth test tube. The hydrogen peroxide concentration that produced the most gas was recorded. The third and final experiment tested the effect of pH on catalase activity. The constant temperature bath remained at the optimum temperature determined in experiment 1. A small test tube from the containers shelf was placed in the constant temperature bath. The thermometer inside the test-tube helped monitor the temperature. Five mL of 3% Hydrogen Peroxide from the materials shelf was added to the test tube. Next, five mL of pH 2 buffer was added to the test tube. Once the test tube's temperature was the same as the constant temperature bath, one mL of the catalase solution was added. A rubber stopper was added to the gas syringe, and the initial gas volume was recorded. A fifteen-second timer was set, and the rubber stopper was removed. After the conclusion of the fifteen seconds, the final gas volume was recorded. The same procedure was used two more times, using the pH 6 buffer and pH 10 buffer instead of the pH 2 buffer. Based on the results, the optimum pH was determined. All materials, containers, and instruments were cleaned and returned.

Results

Effects of Temperature, pH, and Substrate Concentration on Catalase Activity

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Figure 1. The data table above shows the amount of gas (oxygen) produced at different substrate concentrations. At the conclusion of all trials, the averages of the data points were taken and graphed in Figure 2.

Figure 2. The scatter plot above illustrates the volume of gas produced in mL at different substrate concentrations. The data shows how substrate concentration can affect catalase mediated breakdown of hydrogen peroxide.

Figure 3. The data table above shows how much gas (oxygen) was produced in 15 seconds at five different temperatures (Celsius). The averages of these values are graphed in Figure 4.

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Effects of Temperature, pH, and Substrate Concentration on Catalase Activity

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Figure 4. The bar graph shown above shows the average production of oxygen at each temperature. The graph illustrates how temperature affects catalase mediated breakdown of hydrogen peroxide.

Figure 5. The chart above shows the p-values at each temperature against 40°C. These values were determined using paired t-tests.

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Discussion and Conclusions The point of this lab was to determine how temperature, substrate concentration, and pH impact catalase activity. There were three different experiments which provided three different hypotheses. Those hypotheses are as follows: In terms of temperature, the hypothesis was that catalysis would occur faster as the temperature increased, and upon reaching the optimum temperature, the activity of catalysis would cease. In terms of pH, the hypothesis was, an excess of H+ or OH- ions would result in the distortion of the active site; therefore, the pH should be neutral. In terms of substrate concentration, the hypothesis was that an increase in substrate concentration would increase the chemical reaction rate until a sufficient amount was present to saturate all present enzyme molecules. In figure 4, the average production of gas (oxygen) at each temperature used in the experiment within 15 seconds is shown. The temperature with the greatest production was 40°C, which makes it the optimum temperature for maximum catalase activity. The hypothesis for temperature mentioned, once the optimum temperature was achieved, the activity of catalase would cease and at 60°C and 80°C, catalase activity decreased to 0.04 mL of gas (oxygen) produced. In figure 5, the p-values are shown for each temperature (10°C, 21.5°C, 60°C, and 80°C) against the optimum temperature of 40°C. All values were under 0.05 which means there is a statistically significant difference between the two temperatures. This completely rules out the null hypothesis of temperature not having an impact on catalase activity. In figure 2, showed how substrate concentration impacted catalase activity. The scatter plot showed the maximum catalase activity was achieved with the maximum substrate concentration which was 3.00%. The four different concentrations have a linear relationship, meaning, there is a direct relationship between the substrate concentration and the production of gas (oxygen). As the substrate concentration increased, the about of gas produced increased. The standard deviations are not large enough to raise any concerns as to experimental errors or incorrect interpretations thereof. There are so many studies involving the study of enzymes and how they work inside mammals. Every mammalian species has biological processes that require enzymes, catalase is one them. Knowing how enzymes work without invading a species is important, which is how an

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experiment like this was formulated. The experiment was successful because of all the research and preparation that went into it. Without the research or prep, the methodology associated with the experiment wouldn’t have been where it needed to be to generate useful data. Figures 1-5 provide the data needed to support the three hypotheses wanting to be tested. The experiment could be taken further by conducting experiment 1, testing temperature, to a more detailed level. 40°C is the optimum temperature, but is there a more specific temperature that would provide an even greater production of gas? The same question can be applied to ph. There is always a more precise value that would make the data even more accurate.

Effects of Temperature, pH, and Substrate Concentration on Catalase Activity

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References Goodsell, D. G. (2004). PDB101: Molecule of the Month: Catalase. RCSB: PDB-101. https://pdb101.rcsb.org/motm/57 Williams J. (1928). THE DECOMPOSITION OF HYDROGEN PEROXIDE BY LIVER CATALASE. The Journal of general physiology, 11(4), 309–337. https://doi.org/10.1085/jgp.11.4.309 Worthington, C. C. W., Worthington, V. W., & Worthington, A. W. (1972). Factors Affecting Enzyme Activity (Introduction to Enzymes). Worthington Biochemical Corporation. http://www.worthington-biochem.com/introbiochem/factors.html...