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Name:_______________________________ Biology Date:__________________ LAB: Investigating Enzyme Function (Part 1) Objectives: - What is the role of enzymes in chemical reactions? - Can enzymes be reused? - How can you demonstrate that enzymes can/cannot be reused? Materials: test tube rack 5 test tubes liver 3% hydrogen peroxide goggles Safety: Goggles must be worn. Wash hands after handling liver. Do not ingest solutions or eat liver. Background Information At any given moment, all of the work being done inside any cell is being done by enzymes. The purpose of an enzyme in a cell is to allow the cell to carry out chemical reactions very quickly. Enzymes are proteins that speed up the rate of reactions that would otherwise happen more slowly. These reactions allow the cell to build things or take things apart as needed. Enzymes are made from amino acids. When an enzyme is formed, it is made by stringing together between 100 and 1,000 amino acids in a very specific and unique order. The chain of amino acids then folds into a unique shape. That shape allows the enzyme to carry out specific chemical reactions -- an enzyme acts as a very efficient catalyst for a specific chemical reaction. For example, the sugar maltose is made from two glucose molecules bonded together. The enzyme maltase is shaped in such a way that it can break the bond and free the two glucose pieces. The only thing maltase can do is break maltose molecules, but it can do that very rapidly and efficiently. substrate In this lab, you will study an enzyme that is found in the cells of many living tissues. The name of the enzyme is catalase (KAT-uh-LAYSS); it speeds up a reaction which breaks down hydrogen peroxide, a toxic chemical, into 2 harmless substances--water and oxygen. Hydrogen peroxide is a product of cellular activities but needs to be broken down because it is poisonous to cells. The reaction is as follows: CATALASE 2H2O2 -----------------------> 2H2O + O2 Reactant Hydrogen peroxide Products Water + Oxygen Pre-Lab Questions 1. The reaction is catalase facilitating is : _______________________________________ 2. Why is this reaction necessary in the body? 3. The enzyme in this reaction is ________________ & the substrate is _______________ 4. The substrate fits into a location of the enzyme called the _______________________. 5. The reactants are _______________________ & the products are ________________. Pre-Lab Procedure and Discussion Points: 1. Place 2 pipettes of hydrogen peroxide (1 mL) in a small clean test tube and put the test tube in the rack. 2. Add a small piece of enzyme (liver) to the peroxide. 3. Record observations in Table 1 (New H2O2 + New Catalase) 4. Feel the bottom of the test tube and note any temperature change. What might be the source of the change? What are some possible explanations for the bubbles. Objectives: How do enzymes work and can enzymes be reused? Hypothesis: To explain this reaction, three hypotheses are possible. Choose ONE: a. Catalase and hydrogen peroxide together form one or more new products. b. The hydrogen peroxide is changed in the presence of the enzyme, but the enzyme does not change. c. The enzyme is changed in the presence of hydrogen peroxide, which does not change. Table 1. Predictions & Results: Reaction Prediction (bubbles or no bubbles?) New H2O2 + New Catalase Used H2O2 + New Catalase New H2O2 + Used Catalase New H2O2 + New, Cooked Catalase Observations Explanation Analysis: 1. What role does catalase play in a cell? Is this enzyme found only in the liver? (HINT: Hydrogen peroxide will bubble if placed on a cut finger. What does this tell you?) 2. Carbon dioxide is also produced by reactions in living cells. Does catalase cause the same reaction with carbon dioxide or carbonic acid as it does with hydrogen peroxide? (Carbonic Acid is formed when carbon dioxide dissolves in water.) Why or why not? (Consider how enzymes work). 3. If left open in the sunlight, a bottle of hydrogen peroxide will slowly change into water and oxygen. Under these conditions it takes a very long time for the reaction to happen. On the other hand, when catalase is placed in the hydrogen peroxide, the reaction takes place almost immediately. What does this tell you about the function of the enzyme regarding the breakdown of hydrogen peroxide in living systems? Conclusion: * Formal scientific writing has no personal pronouns and is written in the past tense. A Conclusion Paragraph usually contains a description of the purpose of the experiment, a discussion of your major findings, an explanation of your findings, and recommendations for further study. Usually the following questions are presented in paragraph form: 1. What was the purpose of the experiment? Format: The purpose of the experiment was to investigate (Insert Title.) Example: The purpose of the experiment was to investigate the effect of stress on the growth of bean plants by comparing the growth of bean plants subjected to stress for 15 days with a control (non-stressed plants.) 2. What were the major findings? Format: The major findings were (Insert Results Sentence.) Example: The major findings were that there was no significant difference existed between the mean height of stressed plants and non-stressed plants 30 days after transplanting. 3. Was the hypothesis supported by the data? Format: The hypothesis that (Insert Hypothesis) was (supported, partially supported, or not supported.) Example: The hypothesis that stressed plants would have a lower mean height was not supported. 4. How did your findings compare with those of researchers/lab groups? Example: In contrast, Japanese farmers found that hitting and pulling rice plants were beneficial to plant height. 5. What happened that you did not expect? How can you explain this? Format: This was unexpected. (Insert anomaly if there was one.) This can be explained by (insert explanation.) Example: The stressed bean plants were expected to have a lower height. The fact that they didn't and that Japanese rice farmers stress their points on purpose to achieve better growth means that something about stressing out plants makes them growth better. Perhaps some plants that are stressed release a chemical in response to the stress that promotes better growth and others don't release that chemical, such as rice vs. beans. Or perhaps there is a difference in reaction to stress between monocots and dicots. 6. What recommendations do you have for improving this experiment? Example Improved experimental design techniques including a larger sample and a longer growing period would benefit a similar study. 7. What recommendations do you have for further study? (This is above and beyond this experiment.) Example: Additional investigations using various sources of stress at more frequent intervals would be a good additional experiment. another idea would be to use different types, such as a monocot and a dicot. If further research were done, perhaps scientists have isolated a chemical released by plants during stress. It would be interesting to investigate the amounts of this chemical released during stress. Name:_______________________________ Biology Date:__________________ LAB: Determining the Optimal Conditions for the Enzyme Catalase (Part 2) Objectives: What are the optimal temperature and pH conditions for catalase function? What conditions can cause a decrease in catalase function and why? Hypothesis: Write a hypothesis for each experimental (independent) variable in “If/then/prediction of observed result” format. Temperature Hypothesis: pH Hypothesis Materials Liver HCl solution 3% hydrogen peroxide NaOH solution 6 test tubes & rack Goggles 3 beakers Indicator (pH paper) ice Hot hands/ clamps Thermo-meter Safety: Goggles must be worn with glassware; Wash hands after handling liver. Gloves must be worn to handle acidic and basic solutions. Procedure Determining Optimal Temperature Conditions for Catalase Room Temperature: Make a “Baseline Observation” for rate of reaction at room temperature. 1. Place 2 ml of the 3% hydrogen peroxide solution into a clean test tube. 2. Add a small piece of liver to one test tube. Observe the bubbles. What gas is being released? _______________________________________________ 3. Throughout this investigation you will estimate the rate of the reaction (how rapidly the solution bubbles) on a scale of 0-5 (0=no reaction, 1=slow,...., 5= very fast). Assume that the reaction in step 2 proceeded at a rate of "4" and record the speed in the DATA TABLE as the rate at room temperature. Boiling (100 C) 4. Put a piece of liver into the bottom of a clean test tube and cover it with a small amount of distilled water. Place this test tube in a boiling water bath for 5 minutes. What effect do you think boiling will have on the enzyme? Provide a reason for your answer. ___________________________________________________________________________________________ _____________________________________________________________________________ 5. Remove the test tube from the hot water bath, allow it to air cool, then pour out the water. Add 2 ml of hydrogen peroxide. CAUTION: Use a test-tube holder when handling the hot test tubes. What is happening in the test tube? ___________________________________________ Record the reaction rate (0-5) the DATA TABLE. Ice Bath (0 C) and Warm water bath (37 C) 6. Put equal quantities of liver into 2 clean test tubes and 1 ml H2O2 into 2 other test tubes. Put one test tube of liver and one of H2O2 into each of the following water baths: Ice bath (0 deg.C) and Warm water bath (37 deg.C) 7. After 3 minutes, pour each tube of H2O2 into the corresponding tube of liver and observe the reaction. Record the reaction rates (0-5) in the DATA TABLE. o What is the "optimum" temperature for catalase? (This is the temperature at which the reaction proceeds fastest.) ____________________________________________________________________________ o How was the rate of reaction affected by cooling to 0 degrees C? Provide an explanation for your observation. _____________________________________________________________________________________ _____________________________________________________________________________________ _____________________________________________________________________________________ _____________________________________________________________________________________ How was the rate of reaction affected by heating to 100 degrees C? Provide an explanation for your observation. _____________________________________________________________________________________ _____________________________________________________________________________________ _____________________________________________________________________________________ _____________________________________________________________________________________ o Determining the optimal pH for catalase activity: Teacher Station 1. Add 2 ml hydrogen peroxide to each of 3 clean test tubes. Treat each tube as follows: Tube 1--add a drop of 1molar HCl (acid) at a time until pH 3. Tube 2--add a drop of 1molar NaOH (base) at a time until pH 10. Tube 3--adjust the pH to 7 by adding single drops of either 1molar HCl or 1molar NaOH as needed. CAUTION: Do not let acids or bases contact your skin or clothing. Swirl each test tube after adding each drop and measure the pH of each solution with pH paper. To do this, remove a drop or two of solution from a test tube using a clean glass stirring rod. Rinse your stirring rod and wipe dry before you dip it into each test tube. Place the drop on pH paper. Record the pH of each solution in DATA TABLE 1. 2. Next, add a small piece of liver to each test tube. Estimate the reaction rates (0-5) and record in DATA TABLE 1. Does there appear to be a pH "optimum"? At what pH? Provide evidence to support your answer. _______________________________________________________________________________ _______________________________________________________________________________ Data Table: Catalase Activity at different Temperatures and pH Temperature Ice bath (0 C) Room Temp. (22 C) Warm water (37 C) Boiling Water (100 C) Enzyme Activity (0 none 5 very fast) 0 1 2 3 4 5 0 1 2 3 4 5 0 1 2 3 4 5 0 1 2 3 4 5 0 1 2 3 4 5 pH 3 (acidic) 7 (neutral) 10 (basic) Enzyme Activity (0 none 5 very fast) 0 1 2 3 4 5 0 1 2 3 4 5 0 1 2 3 4 5 Lab Write Up Your lab write up should have the following sections (in this order). Please use your Experimental Design Reference packet (and its checklists) as you prepare your lab report. Title Hypothesis (Make sure you address temperature and pH conditions.) Procedure Data Table Graph Results sentence Summary Conclusion