Enzymes_Lab-protocol_F2020

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University of Nevada, Las Vegas *

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Chemistry

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Dec 6, 2023

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Laboratory Exercise: Enzymes Laboratory Objectives 1. Understand and explore how enzymes work. 2. More practice using the spectrophotometer. 3. Explore how enzymatic function is influenced by environmental conditions. Enzymes – An Introduction ***Remember to answer all questions in bold.*** Enzymes are proteins that catalyze biochemical reactions in all living organisms. For example, the reactions necessary for photosynthesis and respiration are catalyzed by many enzymes. Enzymes allow living organisms to carry out rapid and complex chemical activities at relatively low temperatures; however, enzymes cannot initiate a reaction that would not have occurred in their absence. Enzymes are highly specific; they tend to accelerate only one or a group of related reactions. Therefore, there are many different enzymes in a cell, but because they are unchanged by chemical reactions, only a small amount of any given enzyme may be present. Enzymes speed up reactions by bringing reacting molecules (substrates) together to increase the chances that a reaction will occur. Each enzyme has a specific active site, a portion of the molecule where the substrates adhere. Many factors may affect enzyme activity such as temperature, pH, concentration of enzyme, concentration of substrate, concentration of products, and the presence of inhibitors or activators. In this exercise we will use the enzyme peroxidase from turnips. Peroxidases are common in plant and animal cells, and are often found in a special organelle, the peroxisome. The generic reaction catalyzed by peroxidase can be symbolized as follows: RH 2 + H 2 O 2 -> R + 2 H 2 O What is the name for H 2 O 2 ? Using oxygen in metabolism generates small amounts of H 2 O 2 , which is very toxic. Peroxidase (among other enzymes) speeds up the conversion of H 2 O 2 to water so that the cell is not damaged. Today we will use a substance called guaiacol as our hydrogen donor (or reducing agent) because it changes color after it loses hydrogens, so we will easily be able to tell if the reaction has taken place. 4 guaiacol + 2 H 2 O 2 -> tetraguaiacol + 8 H 2 O (brown) What are redox reactions? 1

Experiment A Question: How can we measure the rate of the reaction mediated by turnip peroxidase? Hypothesis: The activity rate of turnip peroxidase can be determined using a spectrophotometer. Prediction: As the turnip peroxidase catalyzes the conversion of hydrogen peroxide to water, more tetraguaiacol will be produced and absorbance will increase through time. Procedure: You will use pipettes to measure out solutions. Use a clean glass pipette or new pipette tip for each solution. Use a pipette pump with glass pipettes. 1. At your table you should have two labelled test tubes containing 5 mL each of the following solutions 1% H 2 O 2 and 1X turnip extract. Use these as your stock solutions . Do not contaminate them with each other or other solutions. 2. Label one clean test tube “Guaiacol” and measure out 5 mL guiacol from the jar at your table. This will be your stock guaiacol. Do not contaminate with other solutions. 3. Label three clean test tubes as “1”, “2”, and “3”, and pipette in the amounts of solutions given in Setup Table A . 4. Obtain 2 clean cuvettes and read the rest of the directions before you do anything else! Do not mix any reagents until you are clear with the procedure, since this is a timed experiment. Setup Table A: Tube Guaiacol Turnip Ext. 0.1%H 2 O 2 Distilled water 1 100 µl 1.0 ml - 8.9 ml 2 100 µl 200 µl 4.7 ml 3 - 1.0 ml - 4.0 ml 5. Transfer some of the solution in Tube 1 to a cuvette and use it as a blank to zero your spectrophotometer. This is called a reagent blank. Q1. Why do we use a reagent blank instead of distilled water? 6. Mix the contents of tubes 2 and 3 quickly because you only have 20 seconds to mix the tubes before the reaction begins. Do not try to mix tubes 2 and 3 in the cuvette, as you’ll overflow the cuvette. 2

7. Pour some of the contents of the mixed tubes 2 & 3 into the clean cuvette, wipe the sides, and take your first reading. 8. You will then record the absorbance every 20 seconds for 2 minutes. Ask your TA for assistance with reading the graph. 9. You only need two cuvettes: one for the blank and one for the reaction. Begin when you are ready. Time (sec) A 500 0 0 20 40 60 80 100 120 Q2. Excel Data: Plot a graph of the dependent variable - absorbance ( y -axis) versus the independent variable - time ( x -axis). This curve will represent your baseline of enzyme activity. Q3. Do your data support or reject your hypothesis? Q4. Is there a control in this experiment? If so, what? Q5. Can you be absolutely sure that you are testing the effects of peroxidase on the reaction, and if so, how? Experiment B Question: Is the reaction catalyzed by a substance in the extract? Hypothesis: The reaction will not proceed in the absence of extract. Prediction: 3

Procedure: Groups 1–3 will set up reactions as given in Setup Table B , and exchange data for this with groups 4–6, who will do the reactions in Setup Table C . Setup Table B Tube Guaiacol Turnip Extract 0.1% H 2 O 2 Distilled water (ml) Rxn 1: No extract (Group 1) 1 100 µl - - 9.9 2 100 µl - - 4.9 3 - - 200 µl 4.8 Rxn 2: 0.5X extract (Groups 2) 1 100 µl 500 µl - 9.4 2 100 µl - 200 µl 4.7 3 - 500 µl - 4.5 Rxn 3: 2X extract (Group 3) 1 100 µl 2.0 ml - 7.9 2 100 µl - 200 µl 4.7 3 - 2.0 ml - 3.0 Use Tube #1 in each reaction as a blank. Mix the contents of tubes 2 and 3 to start the reaction, then quickly (but carefully) pour the mixture into the cuvette and take the reading. ( Follow the procedure given in Experiment A. ) Experiment B Results : Data Table B Time (sec) Rxn 1: No Extract Rxn 2: 0.5 X Extract Rxn 3: 2X Extract 0 0 0 0 20 40 60 80 100 120 1. Excel Data: Plot a graph of absorbance ( y -axis) versus time ( x -axis) for your baseline data collected in Experiment A, no extract, 0.5X extract concentration, and 2X extract concentration . Include all the data on one graph. 4

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