Enzyme Pre-Lab. Using the Enzyme worksheet and Enzyme lab handout answer the Pre-Lab questions the pre-lab must be complete before beginning the lab.

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1 Enzyme Pre-Lab Using the Enzyme worksheet and Enzyme lab handout answer the Pre-Lab questions the pre-lab must be complete before beginning the lab. Background: In this investigation, you will study several factors that effect the activity of enzymes. Enzymes are made up of amino acids and have optimal working conditions. The enzyme you will use is catalase, which is present in most cells and found in high concentrations in liver and blood cells. You will use liver as the source of catalase. Catalase promotes the decomposition of hydrogen peroxide (H 2 O 2 ) in the following reaction: 2H 2 O 2 2H 2 O + O 2 Hydrogen peroxide is formed as a by-product of chemical reactions in cells. It is toxic and would kill cells if not immediately removed or broken down. Read the entire lab before answering the questions. USE COMPLETE SENTENCES!!! 1. In the reaction above, what is the substrate? What are the products? 2. What is the purpose of an enzyme? 3. Define the following: a) Active site: b) Substrate: c) Enzyme-substrate complex: 4. Describe the Lock and Key model. (Use a picture and descriptions) 5. Based on the Lock and Key model, do enzymes get used-up? 6. What are enzymes made of? 7. What are the safety precautions for this lab? 8. Why do we need to use CLEAN test tubes for each part? 9. Briefly describe the purpose of each part of the Enzyme lab. 10. Describe activation energy and how enzymes affect it.

2 Materials: Raw liver Enzyme solution on ice Dropper Graduated cylinder Glass rod Straw Enzyme Lab Boiling water Warm water bath Ice bath 0.1 M sodium hydroxide 0.1 M hydrochloric acid 6 test tubes 13 x 100 mm 3% hydrogen peroxide ph paper forceps test tube rack hot plate test tube holder Safety: Safety goggles Keep lab area CLEAN and ORGANIZED Procedure: RECORD DETAILED LAB RESULTS (temperature change, color change, if bubbles were produced, measure the height of the bubbles, time) Complete each part entirely (including the questions) before continuing PART 1: Mixing an enzyme and substrate 1. Obtain supplies: 2 test tubes and test tube rack. 2. Put 20 drops (1ml) of hydrogen peroxide in one test tube (you will use the other test tube in part 2). (CLEAN ALL SPILLS) 3. Add 5 drops of stock enzyme solution to the peroxide. (BE SURE TO KEEP THE ENZYME SOLUTION ON ICE) Note what happens inside the test tube. 4. Feel the bottom of the test tube and note your observation. 5. Dump solution in the waste container (NOT down the sink) and CLEAN the test tube (use test tube brushes). 6. Answer questions. PART 2: Is an enzyme or substrate changed ( used up ) in reaction? 1. Obtain supplies: Glass stirring rod (make sure it does not roll off the table). 2. Label the test tubes A and B. 3. Put a piece of liver (the size of a rice grain) into the bottom of test tube A, using a glass stirring rod to push it to the bottom of the test tube. 4. Put the tube in a rack and add 40 drops (2 ml) of hydrogen peroxide. Note what happens and record in the table in part Allow the reaction to continue until it stops; stir gently. 6. Pour only the reaction liquid into test tube B, do not transfer over the left over liver pieces use the glass rod to assist you. 7. Put another small piece of liver (the size of a rice grain) into test tube B. Note whether or not a reaction occurs when using the used peroxide (Is it bubbling?). Record your results in the table in part Pour another 2ml (40 drops) of hydrogen peroxide in test tube A to see if the used liver is still reactive. Record your results in table in part 2.

3 9. Dump solution in down the sink, but put the piece of liver in the large trash. CLEAN the test tube (use test tube brushes) 10. Using the results, answer questions. PART 3: Is an enzyme specific for only one substrate? 1. Obtain supplies: straw 2. Put 3 ml of distilled water in a test tube and carefully blow into the water with a straw for 1 minute. Carbon dioxide (CO 2 ) is produced in reactions in living cells. Blowing into the tube will add carbon dioxide to the water which will form carbonic acid. 3. Add a small piece of liver to the test tube to see what effects the enzyme catalase (from the liver) has on carbon dioxide. Record your results in the table in part Dump solution in the waster container (NOT down the sink) and CLEAN the test tube (use test tube brushes) 5. Answer questions. Part 4: Effects of ph on enzyme activity 1. Obtain a total of 6 test tubes, label test tubes A, B, C. Do not label the other 3 2. Add 2 ml of distilled water to test tube A. Gently swirl the test tube. 3. Add 2 ml of hydrochloric acid (HCl) to test tube B. Gently swirl the test tube 4. Add 2 ml of sodium hydroxide (NaOH) to test tube C. Gently swirl the test tube. 5. Add 1 ml of stock enzyme solution to test tubes A, B, C. 6. CLEAN-UP ALL SPILLS. Be sure not to splash any chemicals in your face. 7. Use a small piece of ph paper and tweezers, tilt the test tube slightly to test the ph of test tube A, B and C (use a new piece for each test tube). Record results. 8. To each of the three unmarked tubes add 1 ml of hydrogen peroxide. 9. AT THE SAME TIME, pour the contents of the unmarked tubes into each of the labeled test tubes. 10. Record your results. Note the relative rates of each individual reaction. 11. Dump solution in the waster container (NOT down the sink) and CLEAN the test tube (use test tube brushes) 12. Answer questions. Part 5: Effects of temperature on enzyme activity 1. Using your cleaned test tubes, label three A, B, C. Do not mark the other three. 2. To each of the labeled tubes, add 1 ml of stock enzyme solution (liver catalase). 3. AT THE SAME TIME: put tube A in boiling water bath (100 ºC) USE TEST TUBE HOLDER put tube B in warm water bath (about 37 ºC) USE TEST TUBE HOLDER put tube C in an ice bath (0 ºC) 4. Wait 4 minutes. While you wait, add 1 ml of hydrogen peroxide solution to each of the three unmarked test tubes. 5. After 4 minutes, remove the tubes from their baths and put them in a test tube rack. 6. AT THE SAME TIME, add the contents of the unmarked tubes to each tube. A, B, C. 7. Observe the results and BE SURE TO NOTE THE RATE OF EACH REACTION! Record your results. 8. CLEAN AND RETURN ALL SUPPLIES 9. Answer questions.

4 Name: Period: Enzyme Lab Results Sheet RECORD DETAILED LAB RESULTS (temperature change, color change, if bubbles were produced, measure the height of the bubbles, time) PART 1: Mixing an enzyme and substrate Observation notes: 1. What was the source of the temperature change? 2. What is a possible explanation for the bubbling during the reaction? (What chemical might be being released?) 3. The stock enzyme solution was made by crushing a piece of liver in a small amount of water. This released many molecules of enzyme (catalase) into the water. The reaction mixture in your test tube contained hydrogen peroxide (the substrate for the reaction) and this enzyme catalase. Below are three possible explanations: a) The enzyme, catalase, and the substrate, hydrogen peroxide, react together to form a new product and both changed during the reaction. b) The substrate, hydrogen peroxide, is changed during the reaction, but the enzyme, catalase, is unchanged. c) The enzyme, catalase, is changed during the reaction, but the substrate, hydrogen peroxide, is unchanged. Which of the above explanations do you think is correct? Explain why. PART 2: Is an enzyme or substrate changed ( used up ) in reaction? Step # Tube Enzyme Source Substrate Source Results 4 A Fresh piece of liver 2 ml of peroxide 6 B Fresh piece of liver Used peroxide (A) (reaction liquid from A) 7 A Used liver (A) 2 ml fresh peroxide

5 1. Does the peroxide substrate (used peroxide) change after the reaction? How do you know, use evidence from the lab? 2. Does the enzyme (used liver) change after the reaction? How do you know, use evidence from the lab? 3. If left open in the sunlight, a bottle of hydrogen peroxide would slowly break down into water and oxygen. Under these conditions, it takes a long time for the reaction to occur. On the other hand, when catalase is present, this reaction takes place almost immediately. What does this tell you about the function of an enzyme? PART 3: Is an enzyme specific for only one substrate? Step # Tube Enzyme Source Substrate Source Results 3 - Small piece of liver Carbonic acid Using your data collected in parts 1-3 answer the questions below: 1. Write out the formula for the breakdown of hydrogen peroxide in the cell. 2. What role does catalase play in keeping the cell in a healthy environment? 3. Is this enzyme found only in the liver? (Hint: if you put hydrogen peroxide on a cut finger, it bubbles) 4. Compare your results for the action of the liver catalase on the two substrates (hydrogen peroxide in part 2 and carbonic acid) you have tested so far. Did the enzyme catalase have an effect on the carbonic acid (formed when you blew bubbles into the water)? Based on your results, is an enzyme substrate specific? Explain.

6 Part 4: Effects of ph on enzyme activity Tube Contents ph Results A Distilled water + 1 ml enzyme B HCl + 1 ml enzyme C NaOH + 1 ml enzyme 1. Write out an explanation about the possible effects of ph on enzyme activity. 2. What effect does a high ph (base) have on the enzyme activity? Use evidence to support your answer. 3. What effect does a low ph (acid) have on the enzyme activity? Use evidence to support your answer. 4. What effect does a neutral ph have on the activity of the enzyme? Use evidence to support your answer. 5. Which ph is optimal for this reaction to occur? How does this relate to what we know about the body s normal ph level? Use evidence to support your answer. 6. Graph ph versus enzyme activity rate (slow, medium, fast). Label the acid, base and neutral ph ranges and use an arrow to show the optimum rate and ph level for this reaction. fast Rate of Reaction medium slow ph

7 Part 5: Effects of temperature on enzyme activity Tube Contents Temp Results A 1 ml enzyme + 1 ml 100 ºC hydrogen peroxide B 1 ml enzyme + 1 ml 37 ºC hydrogen peroxide C 1 ml enzyme + 1 ml hydrogen peroxide 0 ºC 1. At which temperature was the optimal for enzyme activity? Use evidence to support your answer. 2. Graph the enzyme activity rate (slow, medium, fast) versus temperature. Use an arrow to point out the area of the graph showing the optimal rate and temperature at which the reaction occurred. fast Rate of Reaction medium slow Temperature ºC 3. What is the effect of the temperature on enzyme activity and how does this relate to the normal body temperature of mammals? Use evidence to support your answer.