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Transcript
Running head: REACTION RATES OVER TIME The Reaction Rates of Enzyme-Catalyzed Reactions Over Time Trevor M. Dopp Madison High School 1 REACTION RATES OVER TIME 2 Abstract Enzymes are proteins that catalyze chemical reactions. Catalase catalyzes the process of breaking down hydrogen peroxide into water and oxygen. Enzyme-catalyzed reactions were created in order to measure the reaction rates over time. Catalase was added to hydrogen peroxide solutions and bubbles were formed showing that a reaction was occurring. Each solution was allowed to react for a different amount of time before being stopped by adding sulfuric acid. Each solution was then titrated using potassium promaganate to measure the amount of hydrogen peroxide used up in each solution. Using the equation y 2 y1 , the x 2 x1 reaction rates over each time interval were calculated. The data showed that the reaction rate of the catalyzed reaction was nearly constant at the beginning of the reaction but slowed down to almost a stop as the substrate was used up. Further study could be done to test the affects of temperature on enzyme-catalyzed reactions to show how body temp could inhibit the function of enzymes in the body. REACTION RATES OVER TIME 3 Introduction Enzymes are proteins that catalyze chemical reactions. They have active sites where substrates attach to the enzyme and a chemical reaction occurs in which a product is produced. Although a chemical reaction occurs, the enzyme remains unchanged. Salt concentration, pH, temperature, and competitive and noncompetitive inhibitors can inhibit the effectiveness of an enzyme (Ap biology lab, 2001). If these factors are not at their optimal levels the enzyme will become ineffective and may even denature. A change in pH, temperature, or salt concentration will cause the enzyme to denature. Competitive inhibitors will mimic the structure of the substrate and trick the enzyme into thinking that it is the substrate. Noncompetitive inhibitors will attach to the enzyme and alter the shape of its active site, not allowing for an induced fit with the substrate. “Biological systems utilize free energy and molecular building blocks to grow, to reproduce, and to maintain dynamic homeostasis” through the use of enzymes (Campbell, Reece, Urry, Cain, Wasserman, Minorsky & Jackson, 2008). Enzymes are proteins, which are macromolecules that have many functions including speeding up chemical reaction in the form of enzymes. An organism uses enzymes to catalyze chemical reactions in the body so the reactions can occur quickly in order to maintain homeostasis. This experiment was done in order to measure the reaction rates of enzyme-catalyzed reactions that occurred for different time lengths (Ap biology lab, 2001). It should show the change in reaction rate of an enzyme-catalyzed reaction over a specific period of time, which should be almost constant at first but slow down to almost zero over time (Ap biology lab, 2001). It should also show the amount of hydrogen peroxide that is converted to water and oxygen over specific periods of time due to enzyme catalysis. REACTION RATES OVER TIME 4 If an enzyme is added to its corresponding substrate solution then the reaction rate of the solution will increase at first but the reaction will slow down over time as the substrate is used up, because enzymes catalyze chemical reactions. Catalase, the enzyme used in this lab, contains four polypeptide chains each made up of over 500 amino acids (Ap biology lab, 2001). Catalase regulates hydrogen peroxide levels in the body, so adding it to a solution composed of its corresponding substrate, hydrogen peroxide, will speed up the chemical reaction of breaking down hydrogen peroxide into water and oxygen. The enzyme and substrate have an induced fit at the active site where they react. In order to stop the reaction after the desired period of time, sulfuric acid will be added to the solution, which will denature the enzyme and stop the reaction. Materials and Methods In order to conduct this lab I used 11 Dixie cups, a glass beaker, a burette, two syringes, a stopwatch, potassium promaganate, catalase, sulfuric acid, hydrogen peroxide latex gloves, goggles, a marker, tape, and my lab notebook. First I used the tape and marker to label each Dixie cup as follows: 0 sec, 10 sec, 30 sec, 60 sec, 90 sec, 120 sec, 180 sec, 360 sec, H2O2, catalase, and titration. I filled the H2O2 Dixie cup with hydrogen peroxide, the catalase Dixie cup with catalase, and the beaker with sulfuric acid. I then used a syringe to put 10mL of H2O2 in each of the Dixie cups labeled 0 sec, 10 sec, 30 sec, 60 sec, 90 sec, 120 sec, 180 sec, and 360 sec. I cleaned out the syringe I used for the hydrogen peroxide and filled it with 1mL of catalase and filled the other syringe with 10mL of sulfuric acid. I started with the Dixie cup labeled 10 sec and started the stopwatch at the same time I put the 1mL of catalase in the solution and stirred the solution for the designated time labeled on the Dixie cup. After the designated time I put the 10mL of sulfuric acid in the solution to stop the reaction. I REACTION RATES OVER TIME 5 repeated these steps with the other Dixie cups labeled with a specific time on them. When doing the 0 sec Dixie cup I did not add any catalase, but I did add the 10mL of sulfuric acid (this solution will be used to find the base line). In order to find the base line I filled my burette with potassium promaganate and recorded the initial reading of the burette in my lab notebook. 5 mL of the 0 sec solution was placed in the Dixie cup labeled “titration” using a clean syringe. After placing the solution under the burette I added the potassium promaganate to the solution drop by drop until the solution changed color. I recorded the final reading of the burette in my lab notebook. This is the base line assay. I repeated the previous steps for each of the solutions I created in order to find the amount of hydrogen peroxide used in each reaction. Because I thought there may have been an error in titrating the 90sec solution, I redid the titration for that solution. The control in this lab is the 0 sec Dixie cup solution because no catalase was added to it. The independent variable is the time each enzyme-catalyzed reaction occurred for, while the dependent variable is the amount of hydrogen peroxide that is used up in the reaction. In order to calculate the amount of potassium promaganate used in each solution the following formula was used: final reading of the burette minus the initial reading of the burette. The answer to the previous formula was then subtracted from the base line assay to get the amount of hydrogen peroxide that was used up in each reaction. These formulas should show that the longer the enzyme-catalyzed reaction occurred, the more hydrogen peroxide was used. The amount of hydrogen peroxide used should always be less than the base line assay as well. The reaction rate is found by using the equation y 2 y1 . x 2 x1 REACTION RATES OVER TIME 6 Results When titrating each sample of the hydrogen peroxide solution, a logistic trend was found. The longer the catalase reacted with the hydrogen peroxide solution, the more hydrogen peroxide was used up. It was found that the rate of reaction of the catalase and hydrogen peroxide was almost constant at first, but slowed down over time. While the 10sec solution consumed 2.5mL of potassium promaganate, the 360sec solution only consumed 0.01mL of potassium promaganate. Titration of H2O2 Table 1 Reaction Rate of Enzyme-Catalyzed Reaction Table 2 REACTION RATES OVER TIME 7 Figure 1 Discussion Enzymes have an induced fit with their corresponding substrate that allows them to efficiently react with the substrate. If an enzyme is added to its corresponding substrate solution then the reaction rate of the solution will increase at first but slow down over time as the substrate is used up, because enzymes catalyze chemical reactions. The data collected supports this hypothesis. The enzyme used was catalase and its corresponding substrate is hydrogen peroxide. It was observed that when 1mL of catalase was added to 10mL of hydrogen peroxide, bubbles formed showing that a chemical reaction was occurring. It was expected that the reaction rate of the enzyme-catalyzed reaction would slow down to close to zero as more hydrogen peroxide was used up. This was also supported with the data. The titration showed that the reaction rate from time 0 seconds to 10 seconds was 0.05 mL/sec. As more hydrogen peroxide was used up over time, the reaction rate slowed to 0.0005 mL/sec REACTION RATES OVER TIME 8 from time 180 seconds to 360 seconds. An error that could have occurred with the titration of the solutions was a time constraint. Doing the titration a day after catalyzing the hydrogen peroxide solution could affect the results because the hydrogen peroxide may have broken down even more within a day. It would be beneficial to test the affects of temperature on the enzyme-catalyzed solution in order to see how body temperature affects enzyme function and show the affects that long-term fevers can have on enzymes. This could be done by repeating the same procedure done in this lab but making a few changes. Instead of catalyzing each hydrogen peroxide solution for different amounts of time, each solution would be held at a different temperature. Each solution could be catalyzed for 10 seconds but the temperature of each solution would vary. For example the solutions could be set at 0oC, 20 oC, 40 oC, 60 oC, 80 oC, and 100 oC using a hot plate and ice bath. The titration should show that the enzyme works best at or around body temperature, which is 37oC. If the enzyme is too cold, it will not function and when the enzyme becomes too hot, it will denature and become ineffective. Conclusion This lab was conducted to measure the reaction rate of an enzyme-catalyzed reaction over time. The results of the lab show that enzyme-catalyzed reactions are almost constant at first, but slow down over time as the substrate is used up. The idea that sulfuric acid denatures enzymes and stops chemical reactions was also supported by the observations. It was also shown that catalase does indeed catalyze the process of breaking down hydrogen peroxide into water and oxygen. REACTION RATES OVER TIME 9 References (2001). Ap biology lab manual for students. (lab 2 enzyme catalysis). College Entrance Examination Board. Campbell, N., Reece, J., Urry, L., Cain, M., Wasserman, S., Minorsky, P., & Jackson, R. (2008). Ap edition biology. (Eighth ed.). San Francisco, CA: Benjamin Cummings.