Biology 160 Enzyme Lab Name: _______________________________________ Introduction: Living systems function under very mild conditions (pH 7, atmospheric pressure = 1, temperature = 20°C, give or take). Chemical reactions generally occur slowly under these conditions, so cells have evolved enzymes to accelerate their chemical reactions. In enzyme-catalyzed reactions, the chemical substance that the enzyme acts upon is the substrate, and the substance into which it is transformed is the product. Because enzymes can denature when exposed to variations in temperature, pH, and salt concentration, enzyme-catalyzed reactions are usually studied by placing the substrate in a buffered (to keep pH constant) aqueous solution. The buffer together with the substrate is referred to as the reaction mixture. Some enzymes are so vigorous (the enzyme under study in this exercise, catalase, is one of them) that their activity can be explored in pure, unbuffered water. Enzymes are, almost without exception, protein molecules, and they are very specific; that is, enzymes are specialized to speed up a particular chemical reaction involving the same reactants and products every time. The relationship between an enzyme and a substrate has often been compared to the relationship between a lock and a key; only a particular type of key can activate a particular lock. By analogy, only a particular substrate can be converted to product by a particular enzyme. If you placed any one of several thousand substrates in solution with catalase, it would only interact with hydrogen peroxide (H2O2). It can be difficult to get a feel for how much enzymes accelerate chemical reactions. Often the acceleration is 7 to 9 orders of magnitude (107 to 109). For a reaction that is speeded up by 109 through the activity of an enzyme, the same quantity of reactant that would be transformed to product in one minute by the enzyme would take 1903 years without that enzyme catalyst! You can understand why catalysis is such an important biological phenomenon (how would you like to stretch a 2-hour biology lab into one lasting 228,000 years?). There are thousands of enzymes and substrates that they convert to products. The one we will investigate today is catalase, an enzyme that converts hydrogen peroxide to water and oxygen gas through the following reaction: 2H2O2 → 2H2O + O2 The fact that one of the products of this reaction (oxygen) forms a gas, it is convenient to observe the conversion from reactants to products. A small piece of filter paper can be soaked in a solution of catalase and immersed in a solution of hydrogen peroxide (H2O2). It should immediately sink to the bottom of the vessel. As the catalase converts the hydrogen peroxide to water and oxygen, some of the oxygen gas accumulates on the disk, changing the buoyancy of the disk and floating it to the surface. The elapsed time from immersion to flotation is thus proportional to the rate of the reaction (the time required to produce sufficient product (O2) to float the disk). This allows us to explore the effect of a number of variables on the rate of a reaction. Today we will investigate the effects of substrate concentration and temperature. Materials: Work in groups of 2-4 people. 1. 2. 3. 4. 5. 6. Tape for labeling 5 test tubes, all the same size Stock 3% H2O2 Catalase solution Filter paper Water baths at room temperature (approximately 20°), 40°, 60°, 80°, and 100° C. These will be beakers on hot plates with temperature monitored by a thermometer. Be sure to check temperatures frequently and adjust the hot plate settings accordingly. Before beginning, make some hypotheses and predictions about the scientific questions we are asking: Question 1: How does substrate concentration affect the speed at which hydrogen peroxide converts into water and oxygen? Hypothesis:___________________________________________________________________________ _____________________________________________________________________________________ Write your scientific prediction here (remember to use the “if, then” form):________________________ _____________________________________________________________________________________ _____________________________________________________________________________________ _____________________________________________________________________________________ What are the independent and dependent variables we are testing in this experiment? _____________________________________________________________________________________ Methods: Part 1: The effect of substrate concentration on the rate of reaction 1. Place 20 ml stock 3% H2O2 in one of the beakers or test tubes and label with the tape. Make sure your label identifies your group and the concentration of H2O2. 2. Dilute stock 3% H2O2 to make 1.5%, 0.75%, 0.38%, and 0.19% H2O2. You should figure out how to do this by serial dilution of your stock 3% H2O2. All beakers or test tubes should contain 20 ml. 3. Make 15 small filter paper disks with a hole punch. 4. With forceps, grab a disk and immerse it in the catalase solution for about 3 seconds. Immediately drop it into one of the solutions of H2O2 and record the time to the nearest second. If you look closely you will see bubbles of O2 forming along the disk. Record the time again the moment that the paper disk rises to the surface of the H2O2 solution and record the elapsed time from immersion to flotation. Remove the paper disk and repeat this process 2 more times with the same beaker or test tube. 5. Repeat step 4 for each of the concentrations of H2O2. You should have timed 3 filter paper reactions for each of your beakers or test tubes. 6. Graph your results on a separate sheet of paper. Plot the independent variable on the X axis and the dependent variable on the Y axis. Use the averages of your replicates for the graph. 3% H2O2 Elapsed time from immersion to flotation of filter paper 1.5% H2O2 0.75% H2O2 0.38% H2O2 0.19% H2O2 Average: Average: Average: Average: Average: Use the bottom row to record the average elapsed time for your 5 replicates in each H2O2 concentration. Make hypotheses and predictions about part 2 before beginning the procedure. Question 2: How does temperature affect the speed at which hydrogen peroxide converts into water and oxygen? Hypothesis:___________________________________________________________________________ _____________________________________________________________________________________ Write your scientific prediction here (remember to use the “if, then” form):________________________ _____________________________________________________________________________________ _____________________________________________________________________________________ _____________________________________________________________________________________ What are the independent and dependent variables we are testing in this experiment? _____________________________________________________________________________________ Part 2: The effect of temperature on the rate of reaction 1. Prepare 5 test tubes with 20 ml each of 1% H2O2 solution, and label them with tape to identify your group and the temperature each one will be kept at. Keep one of your test tubes immersed in water at room temperature, and place the others in water baths at 40°, 60°, 80°, and 100° C, respectively. Leave the test tubes in the water baths for at least 5 minutes. Do not remove a test tube from a water bath until you are ready to use it immediately. 2. Make 15 more filter paper disks. 3. Measure the reaction rate as you did in part 1, by dipping each filter paper into catalase for 3 seconds, then recording the time it takes for a disk of filter paper to rise to the top of each test tube after being dropped to the bottom. As in Part 1, repeat this procedure with 3 filter paper disks per test tube. 4. Record your data in the table below. 5. Graph your results, with the independent variable on the x-axis and the dependent variable on the y-axis. Use the averages from each test tube on your graph. Room Temperature Elapsed time from immersion to flotation of filter paper 40° C 60° C 80° C 100° C Average: Average: Average: Average: Average: Use the bottom row to record the averages of your 3 replicates for each temperature. Questions: 1. What is the name of the enzyme and what is the name of the substrate in this reaction? 2. After the reaction has finished and your paper floats to the top, is the concentration of enzymes in your test tube decreased at all? What about the concentration of the substrate molecules? 3. You have explored the effect of substrate concentration and temperature on enzymatic activity. What OTHER factors might affect enzyme activity to speed or slow a reaction? List at least 2. 4. If you were to do these two experiments again, how could you improve your data? What are some possible sources of experimental error, and how could that error be reduced? 5. Describe the patterns you see on each of your two graphs. Do they match your predictions?