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Iodine Test on Enzymes

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Topics: Enzyme, Starch
62 Iodine test for starch Amount of starch remaining Enzyme activity level Dark blue-black All None (0) Blue Most Low (1) Light brown Some Moderate (2) Gold None High (3) Part 1: Effect of Enzyme Concentration 1. Label five test tubes 1-5. Place 4 mL of 1 % starch in each of the first four test tubes. Place 4 mL of amylase solution in the fifth tube. Place all of the tubes in the 37°C water bath for 5 minutes. Obtain 5 clean droppers and label them 1-5. (To avoid contamination of these solutions, you will use a separate dropper for each mixture.) 2. Tube 1 will be your control and it will not contain any enzyme. Remove the tubes from the water bath momentarily, and quickly add 3 drops of the warmed amylase solution to tube 2, add 6 drops amylase to tube 3, and add 10 drops amylase to tube 4. Mix the tubes quickly by shaking them gently, and immediately put them back in the 37°C water bath. Record the time at which you added enzyme. Go on immediately to the next step. 3. Transfer four drops of each reaction mixture (tubes 1-4) using a separate clean dropper for each to separate wells on a spot plate. (Keep the test tubes in the water bath.) Add one drop of the iodine reagent to each. Record your observations. Use the visual color reference table to assess the enzyme activity. After you have recorded your observations, rinse off the spot plate. 4. Repeat step 3 after the mixtures have had five minutes of reaction time. 5. Repeat step 3 after the mixtures have had ten minutes of reaction time. 6. In the test tubes where hydrolysis occurred, the presence of glucose can be confirmed with Benedict’s reagent. Add 3 mL of Benedict’s reagent to each test tube and put them in the boiling water bath for 3-4 minutes. The appearance of a green to orange or red precipitate indicates the presence of glucose. If the solution remains blue with no solid, then no glucose is present and no hydrolysis of the starch has taken place. 7. Make a graph of the enzyme activity vs. the amount of amylase solution using your results at 10 minutes.

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