SBI4U Factors Influencing Enzyme Activity Introduction: Enzymes act as specific catalysts for chemical reactions in living cells, by lowering the activation energy required for a reaction to occur. The enzyme to be used in this lab is called catalase. This is an enzyme used to decompose hydrogen peroxide (H2O2), a toxic waste product of metabolism. catalase 2H2O2 (aq) 2H2O (l) + O2 (g) In this experiment, the effect of various factors upon the rate of catalase activity will be studied. Question: How do changes in temperature, pH and enzyme concentration affect the rate of enzyme activity? Hypotheses: Materials: - 100 mL graduated cylinder - 2 x 10 mL graduated cylinder - 5 test tubes - 100 mL beaker - thermometer - 1.0 M HCl and 1.0 M NaOH 500 mL beaker (for water bath) test tube tongs or hot gloves 3% H2O2 solution prepared diluted liver puree - hot plate crushed ice test tube rack distilled water Please label (using tape) the base of one 10 mL graduated cylinder “H2O2” and the base of the other 10 mL graduated cylinder “Liver”. This will prevent you from mixing up the solutions! Safety: Hydrogen peroxide is corrosive and may cause burns to the respiratory tract, skin and eyes. Do not inhale vapors. Always wear safety goggles. NaOH and HCl are corrosive so avoid direct contact with these chemicals. Procedure: Teacher Preparation: Your teacher will prepare a stock solution for the class to share as follows: 150 g of beef liver pureed with 1 L of distilled water. Each group will TAKE 2.0 mL of the liver puree and put it in a clean 100 mL beaker (labeled “Liver Puree Solution”) then ADD 78 mL distilled water to this beaker. This is the liver puree solution you will be using for your trials! PART A: Effect of Enzyme Concentration 1. Using a 10 mL graduated cylinder, add 8.0 mL of H2O2 to the 100 mL graduated cylinder. 2. Using a 10 mL graduated cylinder, measure 1.0 mL of the diluted liver puree. Add the liver puree to the H2O2 in the 100 mL graduated cylinder. 3. Record the maximum height (in mL) of the foam produced. Be sure to subtract the volume of solution at the bottom of the graduated cylinder. 4. Rinse the 100 mL graduated cylinder in the waste bin. Repeat steps 1, 2, and 3 using 2 mL of diluted liver puree, then 4 mL, then 8 mL and 10 mL. PART B: Effect of pH 1. Prepare the following mixtures in three separate test tubes: Test Tube A B C Mixture 5 mL diluted liver puree and 2 drops of 1.0 M HCl (pH = 1) 5 mL diluted liver puree and 2 drops H2O (pH = 7) 5 mL diluted liver puree and 2 drops of 1.0 M NaOH (pH = 14) 2. Add 8 mL of H2O2 to the 100 mL graduated cylinder. Add the contents of test tube A to the 100 mL graduated cylinder. Observe and record the maximum height of foam produced. 3. Rinse the 100 mL graduated cylinder and empty the contents in the waste bin. Repeat steps 2 and 3 with test tubes B and C. PART C: Effect of Temperature Note – you MUST record the ACTUAL temperature that you read on the thermometer just when you are mixing the H2O2 and liver puree. If it says 22C, write it down as 22C and not 20C. The temperatures below are suggested values only! Do not waste time waiting! 1. Add 8 mL of H2O2 to the 100 mL graduated cylinder. Add 5 mL of the liver puree to a test tube. Immerse the test tube in an ice bath until the liver puree reaches a temperature of approximately 10C. 2. Pour the contents of the test tube into the 100 mL graduated cylinder. Observe and record the maximum height of foam produced. 3. Repeat the above procedure with the liver puree at room temperature (approximately 20C). 4. Fill up a 500 mL beaker with tap water. Place this beaker on a hot plate and turn the hot plate on to start a rolling water bath. This will be your water bath. Have three test tubes ready and put them aside. Add 5 mL of the liver puree to a test tube. Repeat this for the other two test tubes. Place the three test tubes in the rolling water bath to warm them up. 5. Add 8 mL of H2O2 to a 100 mL graduated cylinder. Put aside. 6. When the temperature has reached approximately 40C, take ONE of the test tubes out of the beaker using hot gloves or test tube tongs. Pout the contents (carefully!) of this test tube into the 100 mL graduated you had set aside. Observe and record the maximum height of foam produced. 7. Rinse the 100 mL graduated cylinder and empty the contents in the waste bin. 8. Repeats steps 5, 6 and 7 for the other two test tubes (approximately 60C and 80C). Observations: Record your lab data in the spaces provided. Part A – Effect of Enzyme Concentration Vol. of H2O2 (mL) Notes: Vol. of Liver Puree (mL) Total Vol. (mL) (H2O2 + Puree) Max. Height of Foam (mL) Change in Vol. (mL) Visual Observations Part B – Effect of pH Vol. of H2O2 (mL) Vol. of Liver Puree (mL) Total Vol. (mL) (H2O2 + Puree) Max. Height of Foam (mL) Change in Vol. (mL) pH Visual Observations Temp (°C) Visual Observations Notes: Part C – Effect of Temperature Vol. of H2O2 Vol. of Liver (mL) Puree (mL) Notes: Total Vol. (mL) (H2O2 + Puree) Max. Height of Foam (mL) Change in Vol. (mL) Results: You will create THREE graphs (one for each factor). The graphs should be formatted according to the SBI4U lab write-up guidelines. It is recommended you use Microsoft Excel to create the graphs. Discussion: *** This is a FORMAL lab report. Follow the SBI4U Formal Lab Report Guidelines thoroughly *** For the Discussion section of your formal lab report, you should have ALL components of the Discussion section as requested in the lab report guidelines (re-state purpose of lab, state observations/results, explain the results using course connections, account for errors, refer to hypotheses and provide a general conclusion, etc). Within the body of your Discussion section, include well-composed (and supported) answers (in paragraphs) to the following questions for EACH factor that you tested. Compare the data your group collected to your hypothesis for the factor. State whether your hypothesis was correct. If not, why do you think this is? (consider sources of error in the design or made during conducting the lab) How does the factor affect the enzyme activity? Explain scientifically using course content. What trends did you observe in your graphical results for the factor? How would you explain the trends? Make specific reference to the data collected (and your graph) to help you explain. What is the optimum value for this factor? (Do this ONLY for temperature and pH) If you were to repeat this experiment, what would you change to improve it, and why? What errors did you encounter? Why? What proposed solutions do you have in mind? Evaluation Scheme: Code A1 B2 Level Mark Expectation Demonstrate scientific investigation skills (related to both inquiry and research) in the four areas of skills (initiating and planning, performing and recording, analyzing and interpreting, and communicating). Investigate the chemical structures, functions, and chemical properties of biological molecules involved in some common cellular processes and biochemical reactions. I R 1- 1 1+ 2- 2 2+ 3- 3 3+ 4- 4 4+ 4++ A1. Demonstrate scientific investigation skills (related to both inquiry and research) in the four areas of skills (initiating and planning, performing and recording, analysing and interpreting, and communicating). Make informed educated hypotheses to focus inquiries or research. Select appropriate instruments and materials, and identify appropriate methods, techniques, and procedures. Apply knowledge and understanding of safe laboratory practices and procedures. Using appropriate materials and equipment safely, accurately, and effectively, to collect observations and data. Compile accurate data from laboratory and other sources, and organize and Limited Approaching Sufficient Insightful record the data using tables. Synthesize, analyze, interpret and evaluate qualitative and/or quantitative data to determine whether the evidence supports or refutes the initial hypotheses; identify sources of error; and suggest improvements to the inquiry to reduce the likelihood of error. Draw conclusions based on inquiry results and justify their conclusions with reference to scientific knowledge. Communicate ideas, results and conclusions in writing using appropriate language and a variety of formats. I R 1- 1 1+ 2- 2 2+ 3- 3 3+ 4- 4 4+ B2. Investigate the chemical structures, functions, and chemical properties of biological molecules involved in some common cellular processes and biochemical reactions Use appropriate terminology related to biochemistry. Plan and conduct an investigation related to a cellular process (factors that Limited Approaching Sufficient Insightful affect enzyme activity) using appropriate laboratory equipment and techniques, and report the results in an appropriate format. 4++