ne way to test the activity of amylase is to find the rate at which it breaks down a known quantity of starch. You may remember testing for the presence of starch with a solution of iodine (I2KI). Starch chains wrap around the tri-iodide ion in a coil. It is this shape, shown below, that produces the deep purple color.
iodine_in_starch
If we add amylase to the solution, it breaks down the starch until there are no chains long enough to wrap around the tri-iodide, and the color of the solution returns to the yellow tint of plain iodine ions.
1. Exercise 3.1: Enzyme Activity vs Substrate Concentration
a. purpose
In this exercise, the student will investigate the effect of substrate concentration on the rate of the enzymatic reaction. How do you think an increased substrate concentration versus a decreased substrate concentration will affect the rate (speed) of the reaction? Why?
b. procedure
1) Formulate a hypothesis about the effect of changing the substrate concentration on the speed of the enzymatic reaction. Record your hypothesis on Worksheet 2.1.
2) Using the "if-then" format, make a prediction for the proposed effect of substrate concentration versus the time required for substrate depletion. Record your prediction on Worksheet 2.1.
3) Before proceeding with the enzyme assay, perform the positive and negative controls as described below.
a) Take three test tubes from the shelf and place them on the counter.
b) Positive Control: To the first tube add 5ml of 2% starch solution and then 1ml of the iodine solution. Observe the color change and record it on the worksheet.
c) Negative Control: To the second tube add 5ml of water and then 1ml of the iodine solution. Observe the color and record it on the worksheet.
d) Negative Control: To the third tube add 5ml of the amylase solution followed by 1ml of iodine. Observe the resulting color and record it on the worksheet.
e) When finished place all test tubes in the recycle bin
4) In order to perform this experiment, you will need several substrate concentrations. In order to produce the different concentrations, perform a 2-fold serial dilution of the substrate stock as described below.
a) Place six (6) clean test tubes and an Erlenmeyer flask on the work bench.
b) Add 99ml of water to the flask followed by 1ml of the 2% starch solution. This will give you a stock solution of 0.02%.
c) To test tubes 2-5 add 2ml of water.
d) To the first test tube at 4ml of the .02% stock starch solution.
e) Transfer 2ml of the solution in tube 1 to tube 2 by dragging tube 1 over to tube 2.
f) Now transfer 2ml of the solution in tube 2 to tube 3. Continuously transfer 2ml from one test tube to the next until you have deposited 2ml of solution in test tube 6. Place tube 6 in the recycle bin. You now have five test tubes that contain starch solutions of various percents (0.02%, 0.01%, 0.005%, 0.0025%, 0.00125%).
5) Add 1ml of iodine to each of the test tubes (Tubes 1-5). Note the color change.
6) Add 0.5ml of amylase to test tube 1. Be sure to note the virtual labs clock at the bottom of the screen. Record the time of adding amylase on the worksheet
7) Observe the reactions. What happens to the color as time passes? Continue to time the reaction until the solution is yellow/brown. This signifies that the reaction has completed since no starch is present. Record your observations and time of completion on the worksheet.
8) Repeat steps 6 and 7 to test tubes 2-5.
9) When finished, all test tubes in the recycle bin but save your stock starch solution.
2. Exercise 3.2: Enzyme Activity vs Enzyme Concentration
a. purpose
In this exercise, the student will investigate the effect of enzyme concentration on the rate of the enzymatic reaction. How do you think an increased enzyme concentration versus a decreased enzyme concentration will affect the rate (speed) of the reaction? Why?
b. procedure
1) Formulate a hypothesis about the effect of changing the enzyme concentration on the speed of the enzymatic reaction. Record your hypothesis on Worksheet 2.1.
2) Using the "if-then" format, make a prediction for the proposed effect of enzyme concentration versus the time required for substrate depletion. Record your prediction on Worksheet 2.1.
3) Before proceeding with the enzyme assay, perform the positive and negative controls as described below.
a) Take three test tubes from the shelf and place them on the counter.
b) Positive Control: To the first tube add 5 ml of starch solution and then 1 ml of the iodine solution. Observe the color change and record it on the worksheet.
c) Negative Control: To the second tube add 5 ml of water and then 1 ml of the iodine solution. Observe the color and record it on the worksheet.
d) Negative Control: To the third tube add 5 ml of the amylase solution followed by 1 ml of iodine. Observe the resulting color and record it on the worksheet.
e) When finished place all test tubes in the recycle bin
4) In order to perform this experiment, you will need several enzyme concentrations. In order to produce the different concentrations, perform a 2-fold serial dilution of the substrate stock as described below.
a) Place six (6) clean test tubes on the work bench.
b) To test tubes 2-5 add 2 ml of water.
c) To the first test tube at 4 ml of the amylase solution.
d) Transfer 2 ml of the solution in tube 1 to tube 2 by dragging tube 1 over to tube 2.
e) Now transfer 2 ml of the solution in tube 2 to tube 3. Continuously transfer 2 ml from one test tube to the next until you have deposited 2 ml of solution in test tube 6. Place tube 6 in the recycle bin. You now have five test tubes that contain amylase solutions of various concentrations (100%, 50%, 25%, 12.5%, 6.25%).
5) Add 1 ml of iodine to each of the test tubes (Tubes 1-5). Note the color change.
6) Add 0.5 ml of starch solution to tube 1 and start the timer.
7) Observe the reactions. What happens to the color as time passes? Continue to time the reaction until the solution is yellow/brown. This signifies that the reaction has completed since no starch is present. Record your observations and time of completion on the worksheet.
8) Repeat steps 6 and 7 for test tubes 2-5.
9) When finished, place all test tubes in the recycle bin.
3. Exercise 3.3: Enzyme Activity vs Temperature
a. purpose
In this second exercise, the student will investigate the effect of reaction temperature on the rate of the enzymatic reaction. How do you think a decreased temperature versus an increased temperature will affect the rate (speed) of the reaction? Why?
b. procedure
1) Formulate a hypothesis about the effect of changing the reaction temperature on the speed of the enzymatic reaction. Record your hypothesis on the worksheet.
2) Using the "if-then" format, make a prediction for the proposed effect of reaction temperature versus the time required for substrate depletion. Record your prediction on the worksheet.
3) Before beginning, place the following items on your work bench:
a) One fresh Erlenmeyer flask
b) Four (5) test tubes
c) Four (4) constant temperature baths
i) Set the one bath to "Ice" and the others to 21.5˚C, 37˚C, 50˚C, and 80˚C by clicking on the arrow button located on the right side of each bath.
4) Create a 0.005% stock starch solution by adding 50ml of water to the fresh Erlenmeyer flask followed by 25ml from the 0.02% solution. Once you have made the 0.005% starch solution, you may place the 0,02% solution in the recycle bin.
5) Place one test tube in each of the temperature baths. To each tube add 1ml of Amylase followed by 1ml of iodine.
6) To the test tube incubated in ice, add 4ml of the 0.005% starch solution. Be sure to note the virtual lab clock at the bottom of the screen. Record the time of adding starch on the worksheet.
7) Observe the reactions. What happens to the color as time passes? Continue to time the reaction until the solution is yellow/brown. This signifies that the reaction has completed since no starch is present. Record your observations and time of completion on worksheet 2.1. If the solution has not turned yellow after 5 minutes, you can assume the enzyme has been denatured.
8) Repeat steps 6 and 7 to the remaining test tubes (make sure you go one at a time).
9) When finished, place all materials in the recycle bin.