Catalase enzyme lab ap biology, Schemes and Mind Maps of Biology

Download Catalase enzyme lab ap biology and more Schemes and Mind Maps Biology in PDF only on Docsity! AP BIO ENZYME ACTIVITY This activity is an alternative to the titration proposed for Enzyme Catalysis (AP Bio Lab #2, Biology Lab Manual). There are numerous alternative lab activities that measure the rate of enzyme activity (i.e. Gen Nelson, Catalase Lab, http://www.accessexcellence.org). This one is Erol Altug’s, which has been modified from the one developed by Tom Carroll of St. Alban’s School. The introduction and some questions are from Biology Lab Manual. Introduction Enzymes are biological catalysts that carry out thousands of chemical reactions, which occur in living cells. They are generally large proteins made up of several hundred amino acids, and often contain a non-proteinaceous group that is important in the actual catalysts. In an enzyme-catalyzed reaction, the substance to be acted upon, the substrate, binds in the active site of the enzyme. The enzyme and substrate are held together in an enzyme-substrate complex by hydrophobic bonds, hydrogen bonds, and ionic bonds. The enzyme then converts the substrate to the reaction products in a process that often requires several chemical steps, and may involve covalent bonds. Finally, the products are released into solution and the enzyme is ready to form another enzyme-substrate complex. As is true of any catalyst, the enzyme is not used up as it carries out the reaction, but it recycled over and over. One enzyme molecule can carry out thousands of reaction cycles every minute. Each enzyme is specific for a certain reaction because its amino acid sequence is unique and causes it to have a unique three-dimensional structure. The “business” end of the enzyme molecule, the active site, also is specific so that only one or a few of the thousands of compounds present in a cell can interact with it. If there is a prosthetic group on the enzyme, it will form part of the active site. Any substance that blocks or changes the shape of the active site will interfere with the activity and efficiency of the enzyme. If these changes are large enough, the enzyme can no longer act at all, and has become denatured. 1 There are several factors that are especially important in determining the enzyme’s shape, and these are closely regulated both in the living organism and in laboratory experiments to give the optimum or most efficient enzyme activity: 2 enzyme activity. (The disc should be flat on top of the solution at the end.) Materials  100 mL graduated cylinders  2.1 cm filter paper discs (or uniform-cut discs made by identical paper punches)  50 mL beakers (or small disposable paper cups)  Rulers (if paper cups are used)  Blender  Cheesecloth  Distilled water  Forceps  Hot plate  Hydrogen peroxide  Ice  Potatoes  Thermometer 5 PROCEDURE Extraction of Potato Catalase (Your instructor may do this in advance) 1. Peel a fresh potato tuber and cut the tissue into small cubes. 2. Weigh out 50 grams of tissue. 3. Place the tissue, 50 mL of cold distilled water, and a small amount of crushed ice in a pre-chilled blender. 4. Homogenize for 30 seconds at high speed. FROM THIS POINT ON, THE ENZYME PREPARATION MUST BE CARRIED OUT IN AN ICE BATH. 5. Filter the potato extract, using cheesecloth. 6. Pour the filtrate into a 100 mL graduated cylinder and add cold distilled water to bring up the final volume to 100 mL. NOTE This extract will arbitrarily be labeled 100 units of enzymes per mL, or 100 units/mL, and will be used in the tests. Independent Variables & Measurements & Recordings You will be manipulating the independent variables. So as to be able to plot more than two data points (i.e. a line) any experiment needs a minimum of three measurements, which usually works out as: standard conditions, conditions above normal, conditions below normal. In these tests, you will work with five variations of any one condition. You and your lab partner(s) will be responsible for conducting an experiment measuring rate changes for one of the conditions, which your instructor will assign. However, you need to observe how the other conditions are being manipulated and what can be learned about enzyme function. All recordings are to be kept in the Data Table at the end of these instructions. Those results will be submitted along with your graphs and answers to questions. 6 EVERY STUDENT IS RESPONSIBLE FOR RECORDING AND GRAPHING THE CLASS AVERAGE RESULTS OF ALL THE EXPERIMENTS. 7 and mix equal volumes of 1.5% and 0.0% to create your 0.75% solution of H2O2. You need equal amounts to do the assay. Use this table as a guide. Beaker Hydrogen Peroxide Dilution mL of 3% H2O2 mL of water 0.0% 0.0% 0.0 40.0 0.75% 0.75% 10.0 30.0 1.5% 1.5% 20.0 20.0 2.25% 2.25% 30.0 10.0 3.0% 3.0% 40.0 0.0 IMPORTANT: It is very important that your substrate solutions are accurately prepared. 10 III. EFFECT OF TEMPERATURE 1. Use 40 mL of a 3.0% hydrogen peroxide solution as the substrate. 2. Use 100% (100 units/mL) enzyme concentration. 3. Run the reactions in five water baths at five different temperatures. Follow the same procedure of using standard conditions (i.e. room temperature) and then two temperatures above and two below. The following temperatures are suggested:  40C  100C  220C (room temperature)  400C  650C + NOTE: At the time of each assay record the exact temperature of the water bath. 4. Use the same assay procedure as outlined on page 5 (I, steps 3 – 8). 5. Record your results in Table 4. Plot your data on a graph. IV. EFFECT OF ENZYME INHIBITION Hydroxylamine attaches to the iron atom of the catalase molecule, thereby interfering with the formation of the enzyme-substrate complex. 1. Add 5 drops of 10% hydroxylamine to 1 mL of enzyme extract, and let it stand for 1 minute. 2. Prepare a control. 3. Using the same assay procedure as outlined on page 5 (I, steps 3 – 8), measure the activity of each of the solutions. Use 40 mL of 3.0% H2O2 for the substrate. 4. Record the results in Table 4. 11 DISCUSSION and/or QUESTIONS for REPORT 1. Basic questions of the experiment: a. What is the enzyme of this reaction? __________________________ b. What is the substrate of this reaction? __________________________ c. What is the product of this reaction? __________________________ d. What is the gas produced and how could you demonstrate that? ________________________________________________________ 2. How does enzyme activity vary with enzyme concentration? 3. How is the rate of enzyme activity affected by increasing the concentration of the substrate? 4. What do you think would happen if you increased the substrate concentration to 40.0% hydrogen peroxide? 5. How does changing the substrate concentration compare to changing the enzyme concentration in this experiment? 6. Explain the results when the hydroxylamine is added to the enzyme. 7. From the temperature data, what can you conclude about how temperature affects enzyme activity? How would you explain the results? 8. What is meant by optimum temperature? According to the class data, what is the optimum data for the potato catalase and why? 9. What would you predict if you had first mixed the catalase (100%) in serial solutions of varying pHs of 3, 5, 7, 9, 11? What would you predict would be optimum pH? 10. Design a controlled experiment to test the effect of varying pH, temperature, or enzyme concentration. 12 Submit these tables and your graphs along with any questions the instructor asks you to answer. 15 COLLEGE BOARD – ENZYME CATALYSIS LABORATORY ACTIVITY Overview In this laboratory activity, students will observe the conversion of hydrogen peroxide to water and oxygen gas by the enzyme catalase. They will then measure the amount of oxygen generated and calculate the rate of enzyme-catalyzed reaction. Objectives Before doing this laboratory activity students should understand:  the general functions and activities of enzymes  the relationship between the structure and function of enzymes  the concept of initial reaction rates of enzymes  how the concept of free energy relates to enzyme activity  that changes in temperature, pH, enzyme concentration, and substrate concentration can affect the initial reaction rates of enzyme-catalyzed reactions After performing this laboratory activity students should be able to:  measure the effects of changes of temperature, pH, enzyme concentration, substrate concentration on reaction rates of an enzyme-catalyzed reaction in a controlled experiment  explain how environmental factors affect the rate of enzyme- catalyzed reactions 16