Introduction

Enzymes are proteins that speed up reactions by lowering the amount of activation energy needed for the reactions. This lab will be a series of four tests relating to the actions of the enzyme catalase, which is found in all aerobic organisms. We will see how catalase reacts to temperature (boiling) and how it affects decomposition of H2O2.

Hypothesis

Activity A The process of boiling will denature most enzymes. Because of this, I predict that the boiling of the catalase will destroy it, so there will be no reaction in the H2O2. This will be measured by observing if the catalase still causes a reaction in H2O2. Activity D Since catalase is an enzyme, I predict that it will speed up the decomposition of H2O2. This will be measured by adding catalase to H2O2 after an increasing period of time.

Materials and Methods

Materials Activity A
H2O2 Catalase on ice 60 mL cups Test tube with holder Test tube rack Syringe for H2O2 Transfer pipets Glass rod for stirring Scalpel Piece of potato or liver Boiling water bath Activity B H2O2 H2SO4 KMnO4

Cup labeled Baseline Cup labeled Titration Syringe for H2O2 Syringe for H2SO4 Syringe for Transfer Transfer pipet Titration Syringe Distilled water in cup labeled dH20

Activity C H2O2 H2SO4 KMnO4 Cup labeled Uncatalyzed Decomposition Cup labeled Titration Syringe for H2O2 Syringe for H2SO4 Syringe for Transfer Transfer pipet Titration Syringe Distilled water in cup labeled dH20 Cup labeled H2O2 Overnight Activity D H2O2 H2SO4 KMnO4 Catalase on ice Cup labeled Titration Syringe for H2O2 Syringe for H2SO4 Syringe for Transfer Transfer pipet Titration Syringe Cups labeled 10 Second, 30 Second, 60 Second, 120 Second, and 180 Second

Methods

Activity A Testing for enzyme activity 1. Use the labeled H2O2 syringe to transfer 10 mL of H2O2 into the unlabeled 60 mL cup. Use a transfer pipet to add 1 mL of catalase solution to the unlabeled cup. Observe for 30 to 60 seconds. Do you observe anything that indicates a chemical change is taking place? If so, explain. 2. On the basis of your observation of the reactions and your knowledge of chemistry, write a balanced equation for the reaction. 3. Give the following for this reaction: a. Substrate b. Enzyme

c. Intermediate complex that formed (not observed) d. Products The effect of boiling on enzyme activity 1. Use the same transfer pipet to transfer 3-5 mL of catalase to a test tube. Place the test tube in a boiling water bath for five minutes. 2. While waiting, rinse your unlabeled cup. Use the H2O2 syringe to transfer 10 mL of H2O2 into the rinsed, unlabeled cup. Use a clean, unused transfer pipet to add 1 Ml of the boiled catalase to the unlabeled cup. Observe the results. What is the effect of boiling the catalase? Explain how boiling affects catalase activity. 3. List at least three other factors that could affect the activity of the catalase and explain why they would have an effect. Testing for catalase in living tissue 1. Rinse your unlabeled 60 mL cup. Use a scalpel to cut a cube about one cm on a side from a piece of potato or liver. Place the cube in the unlabeled cup and macerate with a glass rod. 2. Use the H2O2 syringe to add 10 mL of H2O2 to the cup of tissue, and then observe. Does the tissue contain catalase? Explain your answer. Activity B Preparation of baseline sample 1. Use the syringe labeled H2O2 to put 10 mL of H2O2 in the 60 mL plastic cup labeled Baseline. 2. Use the plastic transfer pipet to add 1 mL of distilled water from the dH20 cup to the cup labeled Baseline. 3. Use the syringe labeled H2SO4 to add 10 mL of 1 M H2SO4 from the H2SO4 cup to the cup labeled Baseline. 4. Gently swirl the Baseline cup to mix the contents. 5. Use the 5 or 6 mL syringe labeled Transfer to remove 5 mL of solution from the cup labeled Baseline and put it in the cup labeled Titration. Rinse the Transfer syringe. 6. Titrate your 5 mL sample (refer to the Titration Protocol) to determine the baseline amount of H2O2. Record your data in Table 1. 7. You will use the value of change in volume from Table 1 as the baseline amount of H2O2 present in solution. Record the baseline. Activity C 1. Use the syringe labeled H2O2 to transfer 10 mL of H2O2 from the H2O2 Overnight cup to the 60 mL plastic cup labeled Uncatalyzed Decomposition. 2. Use the transfer pipet to add 1 mL of distilled H2O from the dH2O cup to the cup labeled Uncatalyzed Decomposition. 3. Use the syringe labeled H2SO4 to add 10 mL of 1 M H2SO4 from the H2SO4 cup to the cup labeled Uncatalyzed Decomposition. 4. Gently swirl the Uncatalyzed Decomposition cup to mix the contents. 5. Use the 5 or 6 mL syringe labeled Transfer to remove 5 mL of solution from the cup labeled Uncatalyzed Decomposition and put it in the cup labeled Titration. Rinse the Transfer syringe. 6. Titrate your 5 mL sample (refer to the Titration Protocol) to determine the amount of H2O2 left in the solution after 24 hours. Record your data in Table 2. Activity D Preparing the cups 1. Line up the 60 mL plastic cups with the labels 10 Seconds, 30 Seconds, 60 Seconds, 120 Seconds, and 180 Seconds.

2. Use the syringe labeled H2O2 to add 10 mL of H2O2 to each cup. 3. Before beginning each test, prepare 10 mL of H2SO4 in the H2SO4 syringe, so you can stop the reaction precisely when required. Doing the time trials 1. Use the transfer pipet to add 1 mL of catalase extract to the 10 Second cup. 2. Gently swirl the 10 Second cup to mix the contents. 3. At 10 seconds, add 10 mL of H2SO4 to the cup. 4. Repeat steps 1-3 for each time trial, allowing each reaction to occur for the allotted amount of time. Titrating the time trials 1. Use the rinsed syringe to remove 5 mL of solution from the 10 Second reaction cup and place it in the plastic cup labeled Titration. 2. Rinse the Transfer syringe. 3. Titrate according to the Titration Protocol. 4. Record your results in Table 3. 5. Repeat steps 1-4 for each assigned time trial.

Data

Activity A 1. Do you observe anything that indicates a chemical change is taking place? If so, explain. Yes. The water is bubbling, which means that O2 is being produced. 2. On the basis of your observation of the reactions and your knowledge of chemistry, write a balanced equation for the reaction. 2H2O2 + catalase = 2H20 + O2 3. Give the following for this reaction: e. Substrate: H2O2 f. Enzyme: Catalase g. Intermediate complex that formed (not observed): H3O+ h. Products: H20 + O2 4. What is the effect of boiling the catalase? Explain how boiling affects catalase activity. When the catalase is boiled, it doesnt react in the H2O2. Boiling causes the catalase to denature. 5. List at least three other factors that could affect the activity of the catalase and explain why they would have an effect. pH levelcatalase requires a neutral pH level to function. temperatureif the catalase is too cold or hot, it will not function. Amount of catalaseif there is more catalase, the reaction will occur more quickly than if there was less catalase. 6. Use the H2O2 syringe to add 10 mL of H2O2 to the cup of tissue, and then observe. Does the tissue contain catalase? Explain your answer. Yes. There is a chemical reaction (bubbling). Activity B Table 1 Initial Volume 10 mL Final Volume 4 mL Change in Volume 6 mL

Baseline = 6 mL Activity C Table 2 Initial Volume 10 mL Final Volume 7 mL Change in Volume 3 mL The amount of H2O2 that spontaneously decomposed at room temperature was 3 mL/24 hours. Activity D Table 3 Initial Volume Final Volume Change in Volume H2O2 Decomposed 10 Seconds 10 mL 8 mL 2 mL 4 mL 30 Seconds 10 mL 8 mL 2 mL 4 mL 60 Seconds 10 mL 9 mL 1 mL 5 mL 120 Seconds 10 mL 9.5 mL .5 mL 5.5 mL 180 Seconds 10 mL 7 mL 3 mL 3 mL

1. Why was it necessary to determine a baseline for the H2O2? It was needed to compare how much H2O2 decomposed normally. 2. Why did the addition of H2SO4 stop the reaction? H2SO4 is an acid. It raised the level of the pH in the catalase, which stopped the reaction. 3. Graph the following data from Table 3 for H2O2 decomposed by catalase. Title the graph and supply the following information a. The independent variable is the time b. The dependent variable is the amount of H2O2 decomposed

Catalyzed Rate of Decomposition

H2O2 Decomposed, in mL 6 5 4 3 2 1 0 10 30 60 120 Time, in seconds 180

4. What does the line on your graph represent? The correlation between how much time passed and the change in volume. 5. What does your graph tell you about the rate of the reaction over time? Over time, the catalase decomposes H2O2 at a faster rate.

6. Calculate the results for your data. Record your results in a Data Table that you construct below. 0-10 Seconds .4 mL/sec 10-30 Seconds .4 mL/sec 30-60 Seconds .5 mL/sec 60-120 Seconds .55 mL/sec 120-180 Seconds .3 mL/sec 7. On the basis of your results, what is the initial rate of the catalase/H2O2 reaction? .4 mL/sec

Experimental Error

There were many possible errors in the experiments. The boiling of the catalase may have gone on for too long (or not long enough), which would affect the data. The baseline of the H2O2 mightve had an errorthe measurements werent exact. In Activity D, the 180 second test seemed to have an error because it didnt fit with the rest of the data.

Conclusion