1. Enzyme Function

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1 1 Enzyme Function National Science Standards Science as Inquiry: Content Standard A: As a result of activities in grades 9-12, all students should develop: Abilities necessary to do scientific inquiry. Fundamental abilities and concepts that underlie this standard include: Designing and conducting a scientific investigation Life Science: Content Standard C: As a result of their activities in grades 9-12, all students should develop an understanding of the cell. Fundamental concepts and principles that underlie this standard include: Most cell functions involve chemical reactions. Food molecules taken into cells react to provide the chemical constituents needed to synthesize other molecules. Both breakdown and synthesis are made possible by a large set of protein catalysts, called enzymes. California State Standards for Biology: Cell Biology #1b: Students know enzymes are proteins that catalyze biochemical reactions without altering the reaction equilibrium, and the activities of enzymes depend on the temperature, ionic conditions, and the ph of the surroundings. Investigation and Experimentation #1. Scientific progress is made by asking meaningful questions and conducting careful investigations. As a basis for understanding this concept and addressing the content in the other four strands, students should develop their own questions and perform investigations. Students will: #1a. Select and use appropriate tools and technology (such as computer-linked probes, spreadsheets, and graphing calculators) to perform tests, collect data, analyze relationships, and display data.

2 2 What is Catalase? Pre-Lab Readings Beef liver catalase is an iron-containing enzyme of molecular weight M = 240, which is responsible for the elimination of hydrogen peroxide (H ) without formation of free radicals. The protein contains four heme groups and four NADPH molecules, each of which consists of 506 known amino acid residues. Tzu-Ping Ko, John Day, Alexander J. Malkin and Alexander McPherson, Structure of orthorhombic crystals of beef liver catalase. Acta Crystallographica Neonatal oxidative liver metabolism: effects of hydrogen peroxide, a putative mediator of septic damage. Romeo C, Eaton S, Quant PA, Spitz L, Pierro A. Institute of Child Health and Great Ormond Street Hospital for Children, London, England. BACKGROUND/PURPOSE: Surgical neonates are at risk for sepsis and liver dysfunction. These complications are more common in preterm neonates and in those who receive total parenteral nutrition. Elevated levels of reactive oxygen species (eg, hydrogen peroxide) have been reported in these "at-risk" patients and may be the mediators of liver impairment via their effect on oxidative energy metabolism.

3 3 Comparing Catalysts Introduction Hydrogen peroxide is a very reactive compound that can be used for a variety of reactions including bleaching and disinfecting minor wounds. Acting as an oxidizing agent, it is also toxic to cells, hence its value as a disinfecting agent that disrupts the metabolism of bacteria. Our body cells and those of many other animals contain an enzyme called catalase that accelerates the conversion of toxic hydrogen peroxide to water and oxygen gas. 2H 2 O 2 2 H 2 O + 2O 2 Enzymes and Temperature Temperature can directly affect the rate of an enzymatic reaction. Firstly, all chemical reactions are affected by temperature, according to the laws of thermodynamics. The increased molecular motion that occurs as a result of increased temperature, makes collisions between the enzyme and substrate more likely, and therefore the reaction will occur at a greater rate. So generally, as temperature increases so does the rate of reaction. However one must also bear in mind that high temperatures can cause thermal denaturation of the enzyme and freezing may also damage an enzyme. Denaturation is a change in the tertiary structure of an enzyme. An enzyme's function is related to its 3-dimensional (tertiary) structure. This structure can be altered by heat, thus causing the enzyme to lose function.

4 4 Catalase Isolation Lab During this initial lab catalase will be isolated from potatoes. The catalase will be used in the next lab when each pair designs experiments to test enzymes under several conditions. Materials Potato Centrifuge Graduated Cylinder Funnel Blender Ice Bucket Conical Tubes with lids Beakers Scalpel Centrifuge tubes Coffee Filters Ice Procedure 1. Peel the potato. 2. Cut into small pieces. 3. Weigh 50 g of potato pieces. 4. Put the 50 g of potato into a blender with: 50 ml cold water 50 ml of crushed ice 5. Blend the potato mix for 30 seconds: pulse 3 times for 10 seconds each. 6. Filter mixture using a coffee filter in a funnel over a beaker. 7. Pour the filtrate (liquid that came out of the filter) in equal volumes into centrifuge tubes for 10 minutes at 1300 X g. 8. While waiting, put ice in the Styrofoam ice container. 9. Carefully decant (pour slowly) the supernatant (the clear liquid above the solid after centrifuging) into clean tubes. Put on ice immediately. Don t disturb the pellet (solid material) at the bottom of the centrifuge tube. Catalase is in the supernatant. It will be active for several hours if kept on ice. 10. Add 50 ml of water to the supernatant. Note: This enzyme may be frozen for use during another class period.

5 5 Lab: Proteins as Enzymes 1. Design a quick test for catalase activity using the isolated catalase and hydrogen peroxide. Describe your test: Is the enzyme used up at the end of your test? Are you sure? Design another simple test to find out. Explain the test and the results. 2. Ask 3 questions regarding hydrogen peroxide and catalase one each from: a. Pre-Lab readings b. State Standard Cell Biology, 1b c. The test for catalase activity that you designed in A. These questions will be the basis for designing an experiment. Read through the experiment at the following website for ideas.

6 Choose one question, suggest a hypothesis, and design an experiment using the equipment provided to test your hypothesis, then do the experiment. Your teacher will assign you to a group: Groups A & B: Vary the ph Group C: Vary the ionic conditions Group D & E: Vary the temperature Use Probes in your experiment. Question Hypothesis Experiment Materials Protocol a. b. c. d. e. f. g.

7 Analyze your results. What did you observe? Discuss these results with your group and write down their comments. Ask 3 questions about your results. a. b. c. These questions will lead to slight modifications in your experiment not a completely new experiment. Choose one question (circle the letter). Suggest a hypothesis. Make experimental design changes and perform the experiment again.

8 Analyze your results. What did you observe? Discuss these results with your group and write down their comments. 6. Discuss your results with the whole class and collect information that they have found about their experiments. Complete the following note-taking guide during the discussion. a. The influence of temperature on enzyme function; b. The influence of ph on enzyme function: c. The influence of ionic conditions on enzyme function:

9 Using your experimental results and the above Pre-Lab Readings type a 500 word discussion of State Standard Cell Biology, 1b. Comment on why it is important to understand the action of catalase. Include how what you did during your experiments relates to the diagram of Scientific Inquiry below. 8. Find out what HEME is, what it does and what it looks like. You may attach a picture from the Internet but reference your source. 9. Find out what NADPH is, what it does and what it looks like. You may attach a picture from the Internet but reference your source. 10. What structural conformation do you think catalase is in when it is functional? (primary, secondary, tertiary, quaternary) Hint: View the power point lecture for this week. (You can also see some structures of catalase at:

10 10. Communicating With Others Observing Defining Problem Reflecting On the Findings Questions Forming Question Examining The Results Investigating the known Carrying Out the Study Articulating The Expectation Harwood, William S A New Model for Inquiry in Journal of College Science Teaching. Vol. XXXII, #7.