Enzyme Activity Lab (Biochemistry) PDF

46 CHEM 132.2: Biochemistry Laboratory Exercise No. 7: Enzyme Activity Introduction Enzymes are biological catalysts. They participate in...

46 CHEM 132.2: Biochemistry Laboratory Exercise No. 7: Enzyme Activity Introduction Enzymes are biological catalysts. They participate in essentially every biological reaction necessary for the maintenance of a living system. In this experiment, you will use a readily available enzyme, salivary amylase, which begins the hydrolysis of carbohydrates (amylose) in the saliva of the mouth. An enzyme acts upon the reactants or substrates of a reaction to give an enzyme-substrate intermediate. New compounds called products result. The enzymes are used over and over during a reaction. Enzyme + Substrate Enzyme-substrate Enzyme + Product E + S ⇆ ES → E + P Enzymes catalyze reactions in a cell at body temperature and mild conditions. The rates of enzyme-catalyzed reactions are much faster than they would be without the enzymes. Enzymes speed up a reaction by lowering the energy of activation required to make the reaction occur. To follow the action of an enzyme, it is necessary to test for the appearance of a product, or the disappearance of a reactant over a measured period of time. AMYLASE Amylase is an enzyme that is found in the saliva. In the reactions of amylase with starch, you will test for the disappearance of starch by reacting samples of the reaction mixtures with iodine. Initially, a starch solution gives a blue- black color with iodine. The amylase catalyzes the hydrolysis of the α-1, 4 glycosidic bonds forming smaller polysaccharides, dextrins, maltose, and eventually glucose. Starch Smaller polysaccharides Glucose Dextrins Maltose After the starch is hydrolyzed, the blue-black color produced with iodine no longer occurs, and only the red or gold color of the iodine solution is seen. The faster the amylase hydrolyzes the starch, the more quickly the blue-black color disappears. If the blue-black color does not fade, you may conclude that the enzyme is no longer active and that no hydrolysis of starch has occurred. Page 46 of 102 Vision: A globally competitive university for science, technology, and environmental conservation. Mission: Development of a highly competitive human resource, cutting-edge scientific knowledge TP-IMD-04 and innovative technologies for sustainable communities and environment. V0 07-15-2020 No. For instructional purposes only 1st Semester SY 2020-2021 47 FACTORS AFFECTING ENZYME ACTIVITY Enzyme activity depends upon several factors including enzyme concentration, substrate concentration, pH, temperature and heavy metals. Your own saliva containing the amylase enzyme will be used for this experiment although the levels of amylase vary considerably from one person to another. Each experiment must be timed. As you proceed with each experiment, you will check enzyme activity by reacting a few drops of the reaction mixture with iodine. The time at which the blue-black color of starch disappears will be noted in each experiment. The time required for the disappearance of starch will be correlated to the relative enzyme activity. When enzyme activity is high, the time for the starch to disappear will be very short. When the enzyme is operating poorly or not at all, the activity will be low and more time will be required for the starch to disappear. In some cases, the enzyme will be completely inactivated and the blue-black color of starch and iodine will persist throughout the entire experiment. Graphs will be prepared showing the effects of concentration, pH, temperature and heavy metals upon the relative enzyme activity. Learning Outcomes 1. To associate the presence of enzymes with the catalysis of chemical reactions in living cells. 2. To determine the effect of enzyme concentration, substrate concentration, temperature, pH and heavy-metal salts upon the activity of salivary amylase. Materials  Test tubes Temperature baths 0.1 M AgNO3  Test tube rack Warm water bath (50°C) 0.1 M NaCl  Thermometer Boiling water bath (100°C) Ethanol  Beakers Ice-water bath (0°C) Crushed ice  Eyedropper 1%starch (buffered pH 7.0) Buffers (pH 3, 5,  Spot plate, or wax Iodine reagent 7, 9, 11) paper Page 47 of 102 Vision: A globally competitive university for science, technology, and environmental conservation. Mission: Development of a highly competitive human resource, cutting-edge scientific knowledge TP-IMD-04 and innovative technologies for sustainable communities and environment. V0 07-15-2020 No.  48 CHEM 132.2: Biochemistry Laboratory Procedure A. ENZYME CONCENTRATION A-1 Collect 2-3 mL of saliva in a small, clean beaker. Add an equal volume of distilled water to the saliva and mix. Chewing gum or rubber bands may help your secretion of saliva. Using 5 test tubes, place 5 mL portions of 1% starch solution each. Set up a water bath and heat to about 37°C. If it gets too warm, add tap water to achieve a water bath temperature close to 37°C. Place the test tubes in the 37°C water bath for 5 min. Add the following amounts of saliva to the test tubes as quickly as you can, mix thoroughly, and replace the test tubes in the 37°C water bath. Record the time that you place the test tubes in the 37°C water bath. Watch the temperature of the water bath adding more warm water as needed. Test tube Drops of Saliva Solution 1 1 2 5 3 10 4 15 5 30 Starch-Iodine Test Prepare a spot plate or sheet of wax paper for testing in the samples. Place 1 drop of iodine reagent in each depression of the spot plate or on the wax paper. Add a drop of starch solution to the first drop of iodine. The reaction should give a deep-blue-black color which indicates the presence of starch. A-2 Five minutes after addition of enzyme, remove a drop of each mixture (use clean droppers each time) and add to a drop of iodine in the spot plate or on the wax paper. A blue-black color indicates that starch is present after 5 minutes. If the color with iodine is red or gold, the starch has been completely hydrolyzed. Record the time for disappearance of the blue-black color in any of the samples. Clean the spot plate or wax paper. Repeat the testing with fresh drops of iodine in the spot plate or on the wax paper every 5 minutes thereafter. Continue testing for 30 minutes. Record the time when the blue-black color no longer appears for each sample. Page 48 of 102 Vision: A globally competitive university for science, technology, and environmental conservation. Mission: Development of a highly competitive human resource, cutting-edge scientific knowledge TP-IMD-04 and innovative technologies for sustainable communities and environment. V0 07-15-2020 No. For instructional purposes only 1st Semester SY 2020-2021 49 A-3 Calculate the amount of time in minutes for the hydrolysis of starch in each sample. A-4 Plot the time (minutes) for the starch to hydrolyze (disappearance of blue-black color) against the amount (drops) of saliva solution. B. EFFECT OF pH B-1 Place 5 mL of buffer solutions (pH 3, 5, 7, 9, 11) in separate test tubes. Label. Add 5 drops of saliva solution to each test tube. Place the test tubes all together in a 37°C water bath for five minutes. Prepare a spot plate or wax paper with drops (1 each) of iodine for testing the reaction progress. To start the reaction, add 5 mL OF 1% starch solution to each test tube. Record the time you added the starch solution. Five minutes after the starch was added, remove a drop of the reaction mixture from each test tube and perform the starch-iodine test. Repeat the test for the starch every 5 minutes thereafter. Continue testing as long as starch is present up to 30 minutes. In some test tubes, the enzyme will be inactive, and the test will be positive for starch the entire time. B-2 Calculate the time required for the disappearance of starch (blue- black color) at each pH. B-3 Plot the time for starch to disappear (no longer gives a blue-black color) against the pH of the samples. C. EFFECT OF TEMPERATURE C-1 Work with your lab neighbors to prepare temperature baths using larger beakers as follows: 0°C ice water mixture 20-25°C tap water 37°C warmed tap water 70°C warmed water bath 100°C boiling hot water bath Place 5 mL of 1% starch solution in each of five test tubes. Place one test tube in each of the five water baths and leave for 5 min. Then add 10 drops of saliva solution to each test tube, and record the time. Leave the test tubes in the water baths another 5 minutes. Five minutes after the first addition of enzyme, remove a drop from each test tube and test for starch. Test for starch every 5 minutes Page 49 of 102 Vision: A globally competitive university for science, technology, and environmental conservation. Mission: Development of a highly competitive human resource, cutting-edge scientific knowledge TP-IMD-04 and innovative technologies for sustainable communities and environment. V0 07-15-2020 No.  50 CHEM 132.2: Biochemistry Laboratory thereafter. Record the time at which the blue-black color disappears for each sample. Continue testing for 30 minutes. C-2 Calculate the time (minutes) required for the disappearance of starch in each sample. C-3 Plot the relative enzyme activity (hydrolysis time of starch) against the temperature. D. INHIBITION OF ENZYME ACTIVITY D-1 Prepare 4 test tubes with the following: Test Tube 1 2 mL of 0.1 M AgNO3 2 2 mL of 0.1 M NaCl 3 2 mL of ethanol 4 2 mL water Add 10 drops of the saliva solution to each. Place the test tubes in a 37°C water bath and leave for 5 min. Prepare a spot plate or wax paper with drops of iodine reagent. To start the reaction, add 4 mL of starch solution to each test tube and mix thoroughly. D-2 Five minutes after the addition of enzyme, remove a drop of solution from each test tube and test for starch. Repeat the test every 5 minutes thereafter. Continue testing for 20 minutes. D-3 Calculate the time required for the blue color to disappear. Guide questions and instructions on how to prepare and submit the laboratory 1. (A) How does the activity of the enzyme change as its concentration is increased? What are some reasons for these changes in activity? 2. (B) What is the effect of pH upon the relative enzyme activity? What are some reasons for this effect? 3. (C) How is the enzyme affected by low temperatures? By high temperatures? What are some reasons for these temperature effects? According to your experiment, what is the optimum temperature for salivary amylate? 4. (D) Which of the compounds added to the reaction tubes are inhibitors? Why do these compounds act as inhibitors of enzyme activity? Why is an alcohol swab used prior to giving an injection to a patient? Page 50 of 102 Vision: A globally competitive university for science, technology, and environmental conservation. Mission: Development of a highly competitive human resource, cutting-edge scientific knowledge TP-IMD-04 and innovative technologies for sustainable communities and environment. V0 07-15-2020 No. For instructional purposes only 1st Semester SY 2020-2021 51 This guide questions will serve as your quiz and to be submitted along with your lab report. The laboratory report sheets provided will be submitted by the student after every exercise through email or courier. See abridged for the schedule of submission. Additional Resources Use References style here References Robyt, J.F. and B.J. White. 1987. Biochemical Techniques: Theory and Practice. Brooks/Cole Pub. Co.: Monterey, California; pp. 407. Timberlake, K. 1988. Laboratory Manual for Chemistry. Harper Collins Publishers Inc.: New York, pp. 277-372. Page 51 of 102 Vision: A globally competitive university for science, technology, and environmental conservation. Mission: Development of a highly competitive human resource, cutting-edge scientific knowledge TP-IMD-04 and innovative technologies for sustainable communities and environment. V0 07-15-2020 No.