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Bio In this laboratory you will use a chemical titration to measure and then calculate the rate of conversion of hydrogen peroxide (HO) to water and oxygen using the enzyme catalase. Before doing this exercise, you should understand: Ų the general functions and activities of enzymes Ų the relationship between the structure and function of enzymes Ų the concept of initial reaction rates of enzymes Ų how the concept of free energy relates to enzyme activity Ų that changes in temperature, pH, enzyme concentration, and substrate concentration can affect the initial reaction rates of enzyme-catalyzed reactions. After doing the exercise, you should be able to: ü measure the effects of changes of temperature, pH, enzyme concentration, and substrate concentration on rates of an enzyme-catalyzed reaction in a controlled experiment. ü explain how environmental factors affect the rate of enzyme-catalyzed reactions. Part A: Test of Catalase Activity 1. a) Transfer 10 mL of 1.5% (0.44 M) hydrogen peroxide (H2O2) into a 50-mL glass beaker. b) Add 1 mL of freshly made catalase solution. 2. a) Fill a glass beaker with 300 mL of water. b) Place beaker on a burner and allow it to boil. c) Transfer 5 mL of purified catalase extract to a test tube. d) Put the test tube with the catalase extract in the beaker on the burner and leave it for 5 minutes. e) Allow the catalase to cool. f) Transfer 10 mL of 1.5% hydrogen peroxide (H2O2) g) Add 1 mL of the cooled, boiled catalase solution. Part B: The Establishment of a Baseline *These procedures must be performed without adding catalase (enzyme) to the reaction mixture. 1. Accumulate 10 mL of 1.5% hydrogen peroxide (H2O2), two 50-mL glass beakers, 10 ml of 1M sulfuric acid (H2SO4), approximately 6 mL of 2% potassium permanganate (KMnO4) 1 mL of water (H2O), two appropriately labeled syringes (for contamination prevention), white paper, a pipette, aprons, 2. Put on aprons, protective goggles, and latex gloves. 3. a) Put 10 mL of 1.5% hydrogen peroxide (H2O2) into a clean b) Add 1 mL of water (H2O), in place of the enzyme solution. c) Add 10 mL of 1M sulfuric acid (H2SO4), USING EXTREME CAUTION WHEN HANDLING 4. Using a syringe remove a 5 mL sample from the resulting 5. Place this 5 mL sample in another beaker. 5. a) Assay, or examine, for the amount of hydrogen peroxide b) Place the beaker containing the sample over a white paper. c) Use another syringe with a capacity of 5 mL to add potassium permanganate (KMnO4) a drop at a time until a persistent pink or brown color is obtained while gently shaking the Part C: This portion of the lab was not able to be executed. Part D: An Enzyme-Catalyzed Rate of H2O2 Decomposition 1. Gather 50 mL of 1M sulfuric acid (H2SO4), 50 mL of 1.5% hydrogen peroxide (H2O2), 5 mL of catalase, 10 50-mL glass beakers, 30 mL of potassium permanganate, 8 syringes, a white 2. Use syringe to put 10 mL of 1.5% hydrogen peroxide (H2O2) 4. At 10 seconds add 10 mL of sulfuric acid. MAKE CERTAIN TO WEAR LATEX GLOVES AND PROTECTIVE GOGGLES WHEN HANDLING DANGEROUS 6. Remove a 5 mL sample and use syringe to drop potassium permanganate (KMnO4) into solution. Once the sample turns pink or brown calculate the amount of hydrogen peroxide in the sample. 7. Repeat steps 1 through 5 above, making sure to change the 10 seconds to 30 seconds, the 30 seconds to 60 seconds, 60 seconds to 120 seconds, 120 seconds to 180 seconds, and 180 seconds to 360 seconds. Exercise 2A: Test of Catalase Activity 1) To observe the reaction to be studied, use the labeled syringe to transfer 10mL of 1.5% (0.44-MH2O2) into your unlabeled 60-mL plastic cup. Use a plastic transfer pipet to add 1mL of the catalase solution. The bubbles coming from the reaction mixture are the O2 resulting from the breakdown of H2O2 by catalase. How could you show that the gas evolved is O2? § The enzyme in this reaction is the catalase. The substrate in this reaction is the hydrogen peroxide. The products in this reaction are water (H2O) and oxygen (O2). The way you show that the gas evolved is O2 is by the way the reaction caused bubbles to emerge from the solution. 2) To demonstrate the effect of boiling on enzymatic activity, use the same transfer pipet (not the H2O2 syringe) to transfer 5mL of purified catalase extract to a test tube. Set the transfer pipet aside. Place the test tube containing catalase in a boiling water bath for 5 minutes. While the catalase boils, rinse out the unlabeled plastic cup you used in step 1 for re-use. Transfer 10mL of 1.5% H2O2 into the unlabeled 60mL plastic cup. Use a fresh plastic transfer pipet to add 1mL of the boiled catalase solution. How does the reaction compare to the one using the unboiled catalase? § The reaction compares to the one using the unboiled catalase in the way that the reaction proceeds much slower, due to the boiled catalase being denatured by the heat of the burner when boiled in the test tube. 3) To demonstrate the presence of catalase in living tissue, cut 1cm³ of potato or liver and macerate it. Rinse your unlabeled 60mL plastic cup again, and add 10mL fresh 1.5% H2O2. Add the potato or liver tissue to the cup. What do you observe? What do you think would happen if the potato or liver was boiled before being added to the H2O2? § The results would be a bubble reaction. If the potato or liver were to be boiled before being added to H2O2 their functions would slow down due to being denatured by the heat of the boiling. Procedure for Establishing Baseline: q Left alone, H2O2 spontaneously decomposes to water and oxygen at a slow rate. Therefore a solution that contained 1.5% H2O2 at the time of preparation does not necessarily contain 1.5% H2O2 after storage. In addition, the preparation may not have been perfectly precise. To determine the amount of H2O2 initially present in your nominally 1.% solution, you need to perform all the steps of the procedure without adding catalase (enzyme) to the reaction mixture. This amount is known as the baseline and is an index of the initial concentration of H O in solution. In any series of experiments, a baseline should be established first. 1. Using the 10-12mL syringe labeled H2O2, put 10mL of 1.5% H2O2 in the 60mL plastic cup labeled Baseline. 2. Use the plastic transfer pipet to add 1mL of distilled H2O2 (instead of enzyme solution) from the dH2O cup. 3. Using the 10-12mL syringe labeled H2SO4 , add 10mL of 1.0 M H2SO4 from the labeled beaker. Use Extreme Care In Handling Acids. Your teacher will instruct you about the proper safety procedures for handling hazardous materials. 5. Using the 5mL syringe labeled Transfer, remove 5mL of the reaction mixture and put it in the cup labeled Titration and assay for the amount of H2O2 present. Rinse the Transfer syringe by drawing up some water from the Wash Water cup and expelling it into the Waste cup. q If you are using a titration syringe (the syringe with the piece of Tygon tubing attached), draw in about 0.2mL air, then about 5mL of KMnO4 solution and record the initial volume reading. If you are using a burette, fill it and record the initial reading. Use the syringe or burette to add KMnO4 one drop at a time to the solution after adding each drop. When you get the persistent pink or brown color, record the final volume of KMnO4 below. Check to be sure that you understand the calibration on the syringe or burette. The Uncatalyzed Rate of H2O2 Decomposition 1. To determine the rate of spontaneous conversion of H2O2 to H2O and O2 in an uncatalyzed reaction, transfer approximately 15mL of 1.5% H2O2 to the 60mL plastic cup labeled H2O2 overnight. Store it uncovered at room temperature for approximately 24 hours. 2. Use the syringe labeled H2O2 to transfer 10mLs of H2O2 from the H2O2 overnight cup (from step 1) to the 60mL plastic cup labeled Uncatalyzed Decomposition. 3. Use the plastic transfer pipet to add 1mL of distilled H2O (instead of enzyme solution) from the dH2O cup. 4. Using the 10-12mL syringe labeled H2SO4, add 10mL of 1.0 M H2SO4 from the labeled beaker. Use Extreme Care In Handling Acids. Your teacher will instruct you about the proper safety procedures for handling hazardous materials. 6. Using the 5mL syringe labeled Transfer, remove 5mL of the reaction mixture and put it in the cup labeled Titration and assay for the amount of H2O2 present. Rinse the Transfer syringe by drawing up some water from the Wash water cup and expelling it into the Waste cup. 7. Titrate with KMnO4 as you did in Exercise 2B. Amount used after 24 hours 74.9 mL KMnO4 Amount of H2O2 spontaneously decomposed (mL baseline-mL 24 hours) 2.2-74.9= -72.9 What percent of the H O spontaneously decomposed in 24 hours? [(mL baseline-mL 24 hours)/mL baseline]x100= [(2.2-74.9)/2.2]x100=[-72.9/2.2]x100=-33.136x100=-3.3136% An Enzyme-Catalyzed Rate of H O Decomposition v In this experiment, you will determine the rate at which a 1.5% H2O2 solution decomposes when the reaction is catalyzed by catalase. To do this, you should determine how much H2O2 has been consumed after 10, 30, 60, 120, and 180 seconds. If a day or more has passed since you did Exercise 2B, you must reestablish the baseline by determining the amount of H2O2 present in your 1.5% solution. Repeat the assay (steps 1 through 5) and record the results below. Baseline (initial-final) 48 mL KMnO4 Procedure for a Time-Course Determination: v Set up the 60mL plastic cups with the labels 10 sec., 30 sec., 60 sec., 120 sec., and 180 sec. Use the 10-12mL syringe labeled H2O2 to add 10mls H2O2 to each cup. Do one time point assay at a time. One student should add reagents and a different student should keep time. Read the directions thoroughly before beginning. For the 10 sec. and 30 sec. tests, you will need to have the H2SO4 measured and ready to add before beginning the assay. 3. At 10 seconds, add 10mL of H2SO4 (1.0 M). 3. At 30 seconds, add 10mL of H2SO4 (1.0 M). Each time, repeat steps 1 through 4, as above, except allow the reactions to proceed for 60, 120, and 180 seconds, respectively. v Use the rinsed Transfer syringe to remove 5mL from the 10 sec. reaction cup and place it in the plastic cup labeled Titration. Rinse the transfer syringe by drawing in water from the Wash water cup and expelling it into the Waste cup. Assay for the amount of H2O2 remaining in the reaction cup by titrating with KMnO4. As you did in the Baseline determination, use a syringe or burette to add KMnO4 a drop at a time until the solution remains pink or brown. Gently swirl the Titration cup after each addition. Should the end point be overshot, there is sufficient sample left to repeat the titration. Record your results in Table 2.1-on the next page. KMnO4 (mL) 10 sec. 30 sec. 60 sec. 120 sec. 180 sec. B. Initial Reading 8.6 10.2 11.2 11.5 11.6 C. Final Reading 6.1 8.6 10.2 11.2 11.6 D. Amount of KMnO KMnO4(B-C) 2.5 1.6 1.0 0.3 0.1 E. Amount of H2O2(A-D) 0.9 1.8 2.4 3.1 3.3 0 10 20 30 40 50 60 70 80 90 100 110 120 2. From the formula described earlier, determine the initial rate of the reaction and the rates between each of the time points. Initial (0-10) 10-30 30-60 60-120 120-180 3. When is the rate the highest? Why? § The rate is at its highest at the beginning because the concentration of substrate at hand for which the catalase can react with is at its greatest quantity. 4. When is the rate the lowest? For what reasons is the rate low? § The rate commences to decrease after the first interval, continuing to decrease until the last interval due to the small quantity of substrate left to react with after each passing interval. 5. Based on facts related to enzyme structure and chemistry, explain the inhibiting effect of sulfuric acid on the function of catalase. § The inhibiting effect that sulfuric acid takes on catalase is that when introduced it incredibly lowers the pH level of the catalase. Therefore, the sulfuric acid does not allow the enzyme to function properly when it comes to attaching itself to the substrate due to the difficulty of forming the needed hydrogen bonds as well as disulfide “bridges”. 6. Explain the effect of lowering the temperature on the rates of enzyme activity. § The effect of lowering the temperature on the rates of enzyme activity is a slower reaction rate because the lowering of the temperature would slow down the particles. Bibliography:

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