Enzyme Kinetics Experiments

Molecular Biology of Life Laboratory BIOL 123 ENZYME KINETICS: EXPERIMENTS Laboratory Supplies Construction of Standard Graph for PNP - All Groups PNP stock solution, 2 x 10-4 M Distilled water, 50 ml Small test tubes Pipets, 5 ml Pipets, 1 ml Small test tube rack Na2CO3, 1 M Determination pH Optimum-Group 1 pH 2, pH 4, pH 5, pH 6, pH7, pH 8 and pH 10 buffers NPP, 2 x 10-4 M Distilled water Na2CO3, 1 M Test tubes Pipets, 1 ml Pipets, 5 ml Undiluted enzyme solution Effect of Enzyme Concentration-Group 2 pH 5 buffer in a test tube NPP, 2 x 10-4 M Distilled water Na2CO3, 1 M Test tubes Pipets, 1 ml Undiluted enzyme solution Time Course (Kinetic Assay)-Group 3 pH 5 buffer in a test tube NPP, 2 x 10-4 M Undiluted enzyme solution Na2CO3, 1 M Distilled water Test tubes Pipets, 1 ml Pipets, 5 ml Pipets, 10 ml Beaker, 100 ml Effect of Substrate Concentration-Group 4 pH 5 buffer in a test tube NPP, 2 x 10-4 M Distilled water Test tubes Pipets, 1 ml, 5 ml Undiluted enzyme solution Na2CO3, 1 M 1 5 ml/group 1/group 7/group 2/group 8/group 1/group 6 ml/group each 2 ml/tt 8 ml 10 ml 6 ml 5 4 1 1 ml 15 ml 15 ml 15 ml 15 ml 13 8 5 ml 15 ml 15 ml 1 ml 10 ml 25 ml 9 9 9 3 1 10 ml 10 ml 15 ml 8 4 of each 1 ml 10 ml Molecular Biology of Life Laboratory BIOL 123 Procedures The class will be divided into 4 groups and each group will do a section of the experiment. However, every group should prepare a standard graph on its own and also, everyone should obtain all the data from the other experiments, interpret and plot the data, and discuss all of the work in the lab report, if one is assigned. To draw your graphs, divide each page of graph paper into 4 sections and use one section per experiment. In all of the following protocols, add the indicated components in the order listed, being especially careful to add the enzyme last to all reaction mixtures. Incubate all reaction mixtures at 37°C for 30 min unless otherwise indicated. Then add 1.0 ml Na2CO3 to each tube to stop the reaction and develop color in the product. The last tube in each protocol is the blank for that experiment. (Remember: the blank is used to zero the spectrophotometer. The λmax for PNP is 420 nm.) Construction of Standard Graph for PNP - All Groups 1. Obtain 7 small test tubes and label them 1 through 7. Add 2 ml of water to tubes 2 through 7. 2. The stock solution of PNP has a molarity of 2 x 10-4 M. Add 2 ml of this stock to tubes 1 and 2. Mix tube 2 thoroughly by a vortexer and transfer 2 ml of it to tube 3. Mix tube 3 well. Note that the concentrations of PNP in tubes 1, 2 and 3 are 2 x 10-4, 1 x 10-4 and 5.0 x 10-5 M, respectively. (See Fig. 8). Figure 8. A flow diagram of preparation of dilution tubes for use in standard graph construction. 3. Continue the dilution process for tubes 4 through 6 in the same manner as above. The idea is to halve the concentration of the PNP in each successive tube. Discard 2 ml from tube 6. Do not add any PNP to tube 7 since it is your "control" tube. 4. Add 1 ml of 1 M Na2CO3 to all tubes and mix well. Note that no incubation at 37°C is necessary because PNP is the product. 5. Zero the spectrophotometer using tube 7 as blank. Read both the absorbance (A420 nm) and transmittance (%T420 nm) of all tubes. This is the only time that you will record 2 Molecular Biology of Life Laboratory BIOL 123 %T; it is done to illustrate the mathematical relationship between transmittance and absorbance (A = O.D.). 6. Complete the following table and draw graphs of (a) mM PNP (x-axis) vs. A (y-axis), (b) %T (y-axis) vs. mM PNP (x-axis) and (c) % T (x-axis) vs. A (y-axis). Try the third graph on semi-log paper. The A vs. mM PNP is your standard graph. Tube number 1 2 3 4 5 6 7 ml PNP added ml water ml 1M Na2CO3 2.0 0.0 1.0 2.0 2.0 1.0 0.0 2.0 1.0 0.0 2.0 1.0 0.0 2.0 1.0 0.0 2.0 1.0 0.0 2.0 1.0 mM PNP present ____ ____ ____ ____ ____ ____ ____ A420 nm ____ ____ ____ ____ ____ ____ ____ %T420 ____ ____ ____ ____ ____ ____ ____ Note: For simplicity, calculate the mM PNP present without regard to the amount of Na2CO3 added. Determination of pH Optimum-Group 1 1. Prepare a 1/2 dilution of the enzyme; i.e., mix 0.5 ml of the enzyme and 0.5 ml of water. 2. Prepare 7 small test tubes and add the proper amounts of buffer, NPP and water as shown in the following table. Add the enzyme solution last. Mix well. Note: First line of table in bold numbers shows pHs of buffers and not their amounts. 3. Incubate the tubes 30 min at 37°C and then add 1.0 ml of 1 M Na2CO3 to all tubes and mix well. 4. Measure the absorbance value of each tube using tube 7 as your blank. Plot mM PNP produced/30 min (y-axis) vs. pH (x-axis). What is the pH optimum of your enzyme? Tube number 1 2 3 4 5 6 7 1.0 ml of buffer of pH ml NPP stock ml water ml 1/2 enzyme 2.0 1.0 1.9 0.1 4.0 1.0 1.9 0.1 5.0 1.0 1.9 0.1 6.0 1.0 1.9 0.1 8.0 1.0 1.9 0.1 10.0 1.0 1.9 0.1 7.0 1.0 2.0 0.0 Incubate at 37°C for 30 min ml 1M Na2CO3 1.0 1.0 1.0 1.0 1.0 1.0 1.0 A420 nm ____ ____ ____ ____ ____ ____ ____ mM PNP produced ____ ____ ____ ____ ____ ____ ____ 3 Molecular Biology of Life Laboratory BIOL 123 Effect of Enzyme Concentration-Group 2 1. Prepare 1.0 ml each of 1/2, 1/3, and 1/5 dilutions of the enzyme. 2. Label 10 small test tubes and add buffer, NPP and water as shown in the following table. Add the proper amount of the enzyme solutions and incubate the tubes at 37°C for 30 min. Tube number 1 2 3 4 5 6 7 8 9 10 ml pH 5 buffer ml NPP stock ml water ml 1/5 enzyme ml 1/3 enzyme ml 1/2 enzyme ml undil. enzyme 1.0 1.0 1.9 0.1 0.0 0.0 0.0 1.0 1.0 1.9 0.0 0.1 0.0 0.0 1.0 1.0 1.9 0.0 0.0 0.1 0.0 1.0 1.0 1.9 0.0 0.0 0.0 0.1 1.0 1.0 1.8 0.0 0.0 0.0 0.2 1.0 1.0 1.6 0.0 0.0 0.0 0.4 1.0 1.0 1.4 0.0 0.0 0.0 0.6 1.0 1.0 1.2 0.0 0.0 0.0 0.8 1.0 1.0 1.0 0.0 0.0 0.0 1.0 1.0 1.0 2.0 0.0 0.0 0.0 0.0 Incubate at 37°C for 30 min ml 1M Na2CO3 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 A420 nm ____ ____ ____ ____ ____ ____ ____ ____ ____ ____ mM PNP ____ ____ ____ ____ ____ ____ ____ ____ ____ ____ 3. After incubation, add 1.0 ml of 1 M Na2CO3 to all tubes. 4. Record the absorbance values of each tube. Use tube 10 as the blank. Plot mM of PNP produced/30 min (y-axis) vs. ml of undiluted enzyme (x-axis). Calculate how much undiluted enzyme is represented by the dilution used in the first 3 experimental tubes. Time Course (Kinetic) Assay-Group 3 1. Prepare 9 test tubes and place 1.0 ml of 1 M Na2CO3 in all and 0.4 ml water in the first 8 tubes. Also add 1.0 ml pH 5 buffer, 1.0 ml of 2 x 10-4 M NPP, 2.0 ml of distilled water and 1.0 ml Na2CO3 in the last tube and use it as a blank to zero the spectrophotometer with it. Tube number ml pH 5 buffer ml NPP stock ml water ml 1M Na2CO3 2. 1 0.4 1.0 2 0.4 1.0 3 0.4 1.0 4 0.4 1.0 5 0.4 1.0 6 0.4 1.0 7 0.4 1.0 8 0.4 1.0 9 1.0 1.0 2.0 1.0 Prepare a 100 ml beaker containing 10 ml of pH 5 buffer, 10 ml of 2.0 x 10-4 M NPP and 20 ml of distilled water. 4 Molecular Biology of Life Laboratory BIOL 123 3. Prepare a 1/2 dilution of the enzyme in a clean test tube. 4. At time 0 min, pipet 1.0 ml of diluted enzyme into the beaker, swirl immediately to mix the contents. Immediately pipet 3.6 ml of the reaction mixture into tube 1 and mix well. Return the beaker to 37°C water bath. Record A420 value for tube 1 using tube 9 as blank. 5. Continue taking 3.6 ml samples at 5 min intervals for 35 min and repeat the procedure as in step 4. Incubation (min) 6. A420 nm 0' ____ 5' ____ 10' ____ 15' ____ 20' ____ 25' ____ 30' ____ 35' ____ none ____ mM PNP ____ ____ ____ ____ ____ ____ ____ ____ ____ Plot mM of PNP produced (y-axis) vs. time of incubation (x-axis) on graph paper. Determine the slope of the linear portion of the graph (in fact, the whole assay may be linear). Effect of Substrate Concentration-Group 4 1. Dilute 1.0 ml of the undiluted enzyme with 1.0 ml of water to get a two-fold dilution of the enzyme. 2. Prepare 8 test tubes and add the proper amounts of buffer, NPP, water and enzyme as outlined in the following table. 3. Incubate tubes at 37°C for 30 min. 4. After incubation, add 1.0 ml of 1 M Na2CO3 to all tubes. Record the absorbance value for each tube using tube 8 as blank. Plot mM PNP produced/30 min vs. mM of substrate. 5. Take the reciprocals of all the substrate concentrations and their corresponding O.D.s and construct a Lineweaver-Burk plot on graph paper. Determine the Km and Vmax of the enzyme-substrate complex from this graph. Tube number 1 2 3 4 5 6 7 8 ml pH 5 buffer ml NPP stock ml water ml 1/2 enzyme 1.0 0.1 2.8 0.1 1.0 0.2 2.7 0.1 1.0 0.5 2.4 0.1 1.0 1.0 1.9 0.1 1.0 1.5 1.4 0.1 1.0 2.0 0.9 0.1 1.0 2.5 0.4 0.1 1.0 0.0 2.9 0.1 Incubate at 37°C for 30 min ml 1M Na2CO3 A420 nm 1.0 ____ 1.0 ____ 1.0 ____ 1.0 ____ 1.0 ____ 1.0 ____ 1.0 ____ 1.0 ____ mM PNP ____ ____ ____ ____ ____ ____ ____ ____ 5 Molecular Biology of Life Laboratory BIOL 123 6 Molecular Biology of Life Laboratory BIOL 123 Results of Enzyme Kinetics Lab Exercise Name ______________________________________ Date _____________ Section _____ Name of partners ___________________________________________________________ Due in one week: 1. Fill in the blanks in the following tables: (a) Construction of standard graph for PNP Tube number 1 2 3 4 5 6 7 ml PNP added ml water ml 1M Na2CO3 2.0 0.0 1.0 2.0 2.0 1.0 0.0 2.0 1.0 0.0 2.0 1.0 0.0 2.0 1.0 0.0 2.0 1.0 0.0 2.0 1.0 mM PNP present ____ ____ ____ ____ ____ ____ ____ A420 nm ____ ____ ____ ____ ____ ____ ____ %T420 ____ ____ ____ ____ ____ ____ ____ (b) Determination of pH optimum Tube number 1 2 3 4 5 6 7 1.0 ml of buffer of pH ml NPP stock ml water ml 1/2 enzyme 2.0 1.0 1.9 0.1 4.0 1.0 1.9 0.1 5.0 1.0 1.9 0.1 6.0 1.0 1.9 0.1 8.0 1.0 1.9 0.1 10.0 1.0 1.9 0.1 7.0 1.0 2.0 0.0 Incubate at 37°C for 30 min ml 1M Na2CO3 1.0 1.0 1.0 1.0 1.0 1.0 1.0 A420 nm ____ ____ ____ ____ ____ ____ ____ mM PNP produced ____ ____ ____ ____ ____ ____ ____ (c) Effect of enzyme concentration Tube number 1 2 3 4 5 6 7 8 9 10 ml pH 5 buffer ml NPP stock ml water ml 1/5 enzyme ml 1/3 enzyme ml 1/2 enzyme ml undil. enzyme 1.0 1.0 1.9 0.1 0.0 0.0 0.0 1.0 1.0 1.9 0.0 0.1 0.0 0.0 1.0 1.0 1.9 0.0 0.0 0.1 0.0 1.0 1.0 1.9 0.0 0.0 0.0 0.1 1.0 1.0 1.8 0.0 0.0 0.0 0.2 1.0 1.0 1.6 0.0 0.0 0.0 0.4 1.0 1.0 1.4 0.0 0.0 0.0 0.6 1.0 1.0 1.2 0.0 0.0 0.0 0.8 1.0 1.0 1.0 0.0 0.0 0.0 1.0 1.0 1.0 2.0 0.0 0.0 0.0 0.0 7 Molecular Biology of Life Laboratory BIOL 123 Continuation of table from previous page Incubate at 37°C for 30 min ml 1M Na2CO3 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 A420 nm ____ ____ ____ ____ ____ ____ ____ ____ ____ ____ mM PNP ____ ____ ____ ____ ____ ____ ____ ____ ____ ____ (d) Time course (kinetic) assay Tube number 1 2 3 4 5 6 7 8 9 ml pH 5 buffer ml NPP stock ml water ml 1M Na2CO3 0.4 1.0 0.4 1.0 0.4 1.0 0.4 1.0 0.4 1.0 0.4 1.0 0.4 1.0 0.4 1.0 1.0 1.0 2.0 1.0 Incubation (min) 0' 5' 10' 15' 20' 25' 30' 35' none A420 nm ____ ____ ____ ____ ____ ____ ____ ____ ____ mM PNP ____ ____ ____ ____ ____ ____ ____ ____ ____ (e) Effect of substrate concentration: Tube number 1 2 3 4 5 6 7 8 ml pH 5 buffer ml NPP stock ml water ml 1/2 enzyme 1.0 0.1 2.8 0.1 1.0 0.2 2.7 0.1 1.0 0.5 2.4 0.1 1.0 1.0 1.9 0.1 1.0 1.5 1.4 0.1 1.0 2.0 0.9 0.1 1.0 2.5 0.4 0.1 1.0 0.0 2.9 0.1 Incubate at 37°C for 30 min ml 1M Na2CO3 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 A420 nm ____ ____ ____ ____ ____ ____ ____ ____ mM PNP ____ ____ ____ ____ ____ ____ ____ ____ Use extra sheets to answer the following questions and attach them to these pages. 2. Why must enzyme be added last to all reaction mixtures? 3. How can one determine that the λmax of PNP is 420 nm? 4. You have determined the pH optimum of your enzyme. Discuss the effect(s) that raising the pH to 10 might have upon the structure and activity of the enzyme. 8 Molecular Biology of Life Laboratory BIOL 123 Due in two weeks: You are required to write a paper on Enzyme Kinetics lab exercises and submit a hard copy as well as an electronically copy of it within two weeks from the time you perform the experiments. Make sure you understand the Lab Report Format as given below. For the hard copy that you submit to your TA, include all your graphs (your standard graph, your experiment and those of other groups in the class and the Lineweaver-Burk graph) and a discussion of each graph in detail. The electronic copy submitted through the BlackBoard (see instructions on next page) is an exact duplicate of your report but it should not include any graphs. If you have any questions about the format of either report, ask your TA. ********** Lab Report Format (For This Topic As Well As Others Throughout the Semester) Any lab report that is called “a paper” should be typed in double-spaced format and should contain 5 parts. These parts should be clearly labeled: Introduction: Provide some background information on the subject of the paper and then present the a null hypothesis for each experiment clearly. Materials and methods: For the purpose of these experiments, assume your reader is familiar with general lab procedures so you don't have to describe them in detail. It suffices to include flow diagrams with some short descriptions directly within them. Results: Show the results in figures (graphs or histograms) on a separate page and refer to it in the text: "As shown in Figure 1, the measurements indicate that...". Do not repeat the actual results in a table and also in a graph; you will lose points if you do so. The results need to be represented clearly only once. Keep the results section brief. Discussion: A brief essay of how your results reflect on the axioms and predictions. If your results contradict what other researchers have shown, i.e., what is in the textbook or lab manual, try to rationalize why your results are different. Was there something different with your assumptions, your equipment or your technique that would produce different results? 9 Molecular Biology of Life Laboratory BIOL 123 The following are some more explanations by the Head TA, Jessica Ardis, for writing your report: 1. Aim for your introduction to be about one double-spaced page. You should have a little background information about enzymes and how they work. You do not have to explicitly state: “My first hypothesis is…” but you should introduce the questions that you sought to answer by doing the four experiments. You must cite references within the body of your introduction. 2. If you want to cite the lab manual, use Eby Bassiri for author, and University of Pennsylvania for publisher and cite the website of the Lab Manual in its entirety. 3. You must write about all four experiments even though your group did only one. 4. You do not need to show calculations for concentrations in the report. 5. Trend lines should only be shown on the standard graph and the Lineweaver-Burk plot. When you perform a linear regression, display the equation on the graph. 6. If your results do not make sense, speculate as to why they turned out the way they did in the discussion section. It is more important to be able to intelligently discuss results than it is for everything to have turned out perfectly. 10 Molecular Biology of Life Laboratory BIOL 123 Now log in to BlackBoard and click on the “Assignments” button on the left panel. Next click on the appropriate report to get a page that allows you to read the instructions, follow them and attach your 123sssWW-TYY-XxxxxxZ.doc report under “2. Assignment Materials”. Finally click the submit button to send your report. If you need any help with the submission process, your TA will be happy to help you. Please do not leave submissions to the last minute so you have time to alleviate any problems if they arise. Reports not submitted by the due time will lose points no matter what the excuse is. Grading Criteria Your TA uses quite a number of criteria to grade your reports. These include quality of reasoning and clarity of exposition, correct interpretation of data and conclusions, ability to employ the hypothetico-deductive approach and quality of presentation (even neatness, spelling, grammar, etc). The lab report is an individual effort. You will necessarily share results (data) in the lab, but any similarity in writing and reasoning between lab reports will be investigated. We have the ability to use software that can unequivocally show if a student’s paper or parts of it have been copied from another source and is not totally the effort of the student. Be prepared to produce documentation of your work (lab notebook, rough draft, worksheets, etc.). Please note that any plagiarism will be dealt with severely. 11