AMYLASE LAB. STUDENT HANDOUT...pages 1-13 TEACHER REFERENCE PAGES...pages 14-21

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1 AMYLASE LAB STUDENT HANDOUT...pages 1-13 TEACHER REFERENCE PAGES...pages 14-21

2 EQUIPMENT SUPPLIES Factors Affecting the Activity of Salivary Amylase STUDENT HANDOUT Vortex mixers, water baths, UV-Vis spectrophotometers, pipet pump dispensers. Test tubes, test tube rack, µl pipettors with tips, µl pipettors with tips, styrofoam cups, ice, plastic cuvettes, buffer solutions (ph 4.0, 7.0, 10.0), amylopectin solution, amylase solution, maltose solution, NaK tartrate solution, DNS solution (color reagent), DI water, 50 ml beakers, grease pencils or markers, timer or watch with seconds readout. PROCEDURE Your teacher will tell you which parts (A, B, C, or D) of the procedure you should complete. Each part will require five different samples. After you have completed your part of the lab, obtain data for the other parts of the lab from your classmates and complete the student worksheet. CAUTION: Careful pipetting and mixing is the key to success of this laboratory. WARNING: DNS solution will cause burns and stain your skin. Wash it off your skin immediately. UV-Vis SPECTROPHOTOMETER When you are ready to use the UV-Vis Spectrophotometer, follow these instructions and the demonstrations of your teacher: 1. Place cuvette with first sample in UV-Vis. Remember to hold ribbed sides of the cuvette and position toward you. 2. Choose Scan (F1 key) (this will give you a table with sample number and absorbance ) 3. Repeat Steps 7 & 8 for each of your remaining samples to record data for each sample. 4. Choose Hardcopy (F9 Key) to print a hardcopy (optional.) 5. Press Delete (F3 Key) to clear data. Press "A" and enter to clear all. Biology Amylase Lab, Page 1 4/26/01

3 Part A. Measuring the amount of maltose present 1. Select five test tubes labeled A1 through A5. Be sure to label them with a permanent marker. 2. Place these test tubes in the test tube rack. 3. Using the pipet pump dispenser, add 1 ml of amylopectin solution, 1 ml of DNS reagent and 1 ml of tartrate solution to each tube. Caution: DNS reagent should be washed off your skin immediately. Check off each chemical in the table below as you add it. Test tube # Amylo-pectin DNS Reagent Tartrate Solution Maltose DI Water A1 1 ml 1 ml 1 ml 0 µl 1000 µl A2 1 ml 1 ml 1 ml 200 µl 800 µl A3 1 ml 1 ml 1 ml 400 µl 600 µl A4 1 ml 1 ml 1 ml 800 µl 200 µl A5 1 ml 1 ml 1 ml 1000 µl 0 µl 4. Using an adjustable pipettor, add amounts of maltose solution and distilled water to each tube as indicated by the table above. Check off each one as you add it. 5. Mix the contents of each tube using the vortex mixer. Check off this step in the table below as you complete it. Test Tube # Vortex Mixer ( ) Hot Water Bath ( ) Absorbance Measured A1 A2 A3 A4 A5 6. Place your tubes into a hot (95 C-use caution) water bath for five minutes. 7. Remove the tubes from the water bath, return to your lab station and allow them to cool to Biology Amylase Lab, Page 2 4/26/01

4 room temperature. Store tubes in the refrigerator until next lab period if necessary. 8. Take your samples to the spectrophotometer. Measure the absorbance at a wavelength of 540 nm of the contents of each tube. Record the absorbance in the above table. Your teacher will provide instructions on the use of the spectrophotometer. The absorbance is proportional to the amount of maltose present. 9. Prepare the graph of absorbance versus volume of maltose solution added as indicated in the worksheet. Biology Amylase Lab, Page 3 4/26/01

5 Part B. Effect of different amounts of amylase 1. Select 5 test tubes labeled B1 through B5 and place them in a test tube rack. 2. Using an adjustable pipettor, add the amount of deionized water indicated in the table below to each tube. Check off each chemical as you add it. 3. Using an adjustable pipettor, add the amount of amylase indicated in the table to each tube. CAUTION: Be careful to fully add the small volumes of B2. Test tube # DI Water Amylase Amylo-pectin B µl 0 µl 1 ml B2 950 µl 50 µl 1 ml B3 900 µl 100 µl 1 ml B4 700 µl 300 µl 1 ml B5 0 µl 1000 µl 1 ml CAUTION: The next step is important. Please read and follow the directions carefully. 4. Using a pipet pump dispenser, add 1 ml of amylopectin to each tube. Add the amylopectin solution as quickly as possible to all tubes and note the time when the solution is added to your last tube. 5. After exactly 10 minutes, use the pipet pump dispensers to add 1 ml of DNS reagent and 1 ml tartrate solution to each tube. Caution: DNS reagent should be washed off your skin immediately. Test Tube # DNS Solution Tartrate Solution Vortex Mixer ( ) Hot Water Bath ( ) Absorbance Measured B1 1 ml 1 ml B2 1 ml 1 ml B3 1 ml 1 ml B4 1 ml 1 ml B5 1 ml 1 ml 6. Mix the contents of each tube using the vortex mixer and place them into a hot (use caution) water bath for five min. Check off each step as you complete it in the table above. Biology Amylase Lab, Page 4 4/26/01

6 7. Remove the tubes from the water bath and allow them to cool to room temperature. Store the tubes in the refrigerator until the next lab period. 8. Take your samples to the spectrophotometer. Measure the absorbance at a wavelength of 540 nm of the contents of each tube. Record the absorbance in the above table. Your teacher will provide instructions on the use of the spectrophotometer. The absorbance is proportional to the amount of maltose produced by the enzyme in ten minutes. 9. Prepare a graph of absorbance versus volume of amylase solution added as indicated in the worksheet. Biology Amylase Lab, Page 5 4/26/01

7 Part C. Effect of different amounts of substrate 1. Select 5 test tubes labeled C1 through C5 and place them in a test tube rack. Be sure to label test tubes with a permanent marker. 2. Using an adjustable pipettor, add the amount of deionized water indicated in the table below to each tube. Check off each chemical as you add it. Test tube # DI Water Amylo-pectin Amylase C µl 0 µl 0.2 ml C2 900 µl 100 µl 0.2 ml C3 800 µl 200 µl 0.2 ml C4 400 µl 600 µl 0.2 ml C5 0 µl 1000 µl 0.2 ml 3. Using the adjustable pipettor, add the amount of amylopectin solution (substrate) indicated in the table to each tube. CAUTION: Be careful to fully add the small volumes of C2. 4. Using a pipet pump dispenser, add 0.2 ml of amylase solution to each tube as quickly as possible. Note the time when the amylase is added to the last tube. 5. After exactly 10 minutes, use the pipet dispensers to add 1 ml of DNS reagent and 1 ml tartrate solution to each tube. Caution: DNS reagent should be washed off your skin at once. 6. Mix the contents of each tube using the laboratory mixer and place each tube into a hot (use caution) water bath for five minutes. Check off each step in the table below. Test Tube # DNS Solution Tartrate Solution Vortex Mixer ( ) Hot Water Bath ( ) Absorbance Measured C1 1 ml 1 ml C2 1 ml 1 ml C3 1 ml 1 ml C4 1 ml 1 ml C5 1 ml 1 ml Biology Amylase Lab, Page 6 4/26/01

8 7. Remove the tubes from the water bath and allow them to cool to room temperature. Store the tubes in the refrigerator until your next lab period. 8. Take your samples to the spectrophotometer. Measure the absorbance at a wavelength of 540 nm of the contents of each tube. Record the absorbance in the above table. Your teacher will provide instructions on the use of the spectrophotometer. The absorbance is proportional to the amount of maltose produced by the enzyme in ten minutes. 9. Prepare a graph of absorbance versus volume of amylopectin solution added as indicated in the worksheet. Biology Amylase Lab, Page 7 4/26/01

9 Part D. Determine the effect of ph and temperature. 1. Select 5 test tubes labeled D1 through D5 place them in a test tube rack. 2. Using the pipet pump dispenser, add 1 ml of amylopectin to each tube. Check off each chemical in the table below as you add it. 3. Using an adjustable pipettor, add the amount of the appropriate buffer as indicated in the table to each tube. Be sure to switch tips when you change buffers. Check off each one as you add it. 4. Obtain a styrofoam cup of ice and place tube D5 in the ice. 5. Using the pipet pump dispenser quickly add 0.2 ml of amylase solution to each tube as quickly as possible. Immediately place tube D4 in the hot water bath and leave D5 in the ice bath. Note the time when the amylase is added to tube D5. Test tube # Amylo-pectin 1000 µl Buffer Amylase D1 1 ml ph ml D2 1 ml ph ml D3 1 ml ph ml D4 1 ml ph ml D5 1 ml ph ml 6. After exactly 10 minutes, use the pipet pump dispensers to add 1 ml of DNS reagent and 1 ml tartrate solution to each tube. Caution: DNS reagent should be washed off your skin immediately. 7. Mix the contents of each tube using the laboratory mixer and place each tube into a hot (use caution) water bath for five minutes. Check off each step in the table below. Test Tube # DNS Solution Tartrate Solution Vortex Mixer ( ) Temperature D1 1 ml 1 ml Room temp. D2 1 ml 1 ml Room temp. D3 1 ml 1 ml Room temp. D4 1 ml 1 ml 95 C Waterbath D5 1 ml 1 ml 0 C Ice Hot Water Bath ( ) Absorbance Measured Biology Amylase Lab, Page 8 4/26/01

10 8. Remove the tubes from the water bath and allow them to cool to room temperature. Store the tubes in the refrigerator until the next lab period. 9. Take your samples to the spectrophotometer. Measure the absorbance at a wavelength of 540 nm of the contents of each tube. Record the absorbance in the above table. Your teacher will provide instructions on the use of the spectrophotometer. The absorbance is proportional to the amount of maltose produced by the enzyme in ten minutes. 10. Prepare a graph of absorbance versus ph for samples D1, D2, and D3 as indicated in the student worksheet. Biology Amylase Lab, Page 9 4/26/01

11 TEACHER REFERENCE PAGES - AMYLASE ACTIVITY LAB STUDENT WORKSHEET Name Define the following terms: 1. enzyme: 2. amylase: 3. enzyme activity: List 4 different characteristic variables that can cause a change in, but not totally destroy enzyme activity. Indicate whether that change would be an increase (+) or a decrease (-) in enzyme action Prepare graphs of your experimental results on graph paper: GRAPH #A Standard curve: absorbance versus [maltose] Prepare a graph of absorbance (Y axis) versus volume of maltose solution added (X axis). Draw a smooth curve through the points. This graph is called a standard curve. Sample Graph Biology Amylase Lab, Page 10 4/26/01

12 TEACHER REFERENCE PAGES - AMYLASE ACTIVITY LAB For the following graphs, use the standard curve (Graph A) prepared by the groups which do Part A to convert from absorbance to amount of maltose. See the example above. In this example, an absorbance of 0.6 converts to 700 µl of maltose. Do not use this graph for your data; it is an example only. Use the graph prepared by your classmates who did part A. GRAPH #B Rate of reaction (maltose produced in 10 min) versus amylase [enzyme]. Prepare a graph of amount of maltose (Y axis) versus volume of amylase solution added (X axis). Draw a smooth curve through the points. GRAPH #C: Rate of reaction (maltose produced in 10 min) versus amylopectin [substrate]. Prepare a graph of amount of maltose (Y axis)versus volume of amylopectin solution added (X axis). Draw a smooth curve through the points. GRAPH #D-1: Rate of reaction (maltose produced in 10 min) versus ph Prepare a graph of amount of maltose (Y axis) versus ph (X axis) for samples D1, D2, and D3. Draw a smooth curve between the points. GRAPH #D-2: Rate of reaction (maltose produced in 10 min) versus temperature. Prepare a graph of amount of maltose (Y axis) versus temperature (X axis) for samples D1, D4, and D5. Draw a smooth curve between the points. Biology Amylase Lab, Page 11 4/26/01

13 TEACHER REFERENCE PAGES - AMYLASE ACTIVITY LAB DATA ANALYSIS Carefully examine your experimental data plotted on the graphs and then answer the following questions as concisely as possible. 1. The effect of increasing enzyme concentration on rate of amylase reaction was: 2. The control tubes (tubes #A1, B1, C1) without maltose, enzyme, or substrate showed that: 3. The effect of changing ph on the rate of the amylase reaction was: Biology Amylase Lab, Page 12 4/26/01

14 TEACHER REFERENCE PAGES - AMYLASE ACTIVITY LAB 4. Changing substrate concentration (amylopectin) had the following effect(s) on the rate of reaction catalyzed by amylase: 5. Did low and high temperatures have the same reversible effects on regulating the rate of reaction catalyzed by amylase? Explain why changing temperature can produce a change in rate of product formation. Biology Amylase Lab, Page 13 4/26/01