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Practical 5: Objective:

Enzymes and Digestion a) To show the action of amylase on starch b) To study the effect of temperature on enzyme action c) To show the action of pepsin on egg white (protein) d) To show coagulation of milk e) To show emulsification of fats

Material and apparatus: Beaker Blue and red litmus paper Measuring cylinder Thermometer Test tube White tile Dropper Stopwatch 1% starch solution Dilute hydrochloric acid Dilute natrium hydroxide Benedict solution Iodine solution Egg Pepsin Rennin Milk Coconut oil Bile from guinea pig gall bladder Procedure: (a) 1. 2. 3. 4. 5. To show the action of amylase on starch. Rinse your mouth. Perform chewing movements to stimulate the flow of saliva and collect it in a test tube. Test the saliva with litmus paper to find out whether it is acidic or alkaline. Dilute with about an equal volume of distilled water. Divide the saliva preparation into 3 equal amounts in three test tubes and label them B, C and D. Fill each tube as follows: A 3 cm of distilled water (control experiment) B 3 cm of the saliva preparation C 3 cm of the saliva preparation and 3 cm dilute hydrochloric acid D 3 cm of the saliva preparation and 3 cm dilute natrium hydroxide To each of the test tubes and 5 cm of starch solution and stir thoroughly. After 30 minutes, test one half of the contents of each test tube with dilute iodine solution and boil the other half with an equal amount of Benedict solution. Tabulate your results as shown in the table.

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Results: Test tube A B C D Contents Starch + distilled water Starch + saliva Starch + saliva + dilute HCl Starch + saliva + dilute NAOH Iodine test Benedict test Inferences

From this experiment, what general conclusion can you draw regarding the nature of amylase activity? (b) 1. 2. 3. 4. 6. 7. 8. 9. 10. 11. To study the effect of temperature on enzyme action Prepare a solution of saliva as in experiment (a). Place 5 cm of this saliva solution in a test tube and 5 cm of 1 % starch solution in another. Allow both test tubes to stand at room temperature. Take a white tile and place a series of drops of dilute iodine solution on it. Mix the two solutions in the tubes and note the time of mixing. By means of a clean glass rod remove a drop of the properly stirred mixture and test with a drop of iodine on the white tile. A deep blue colour appears. Repeat this test at intervals of one minute, washing the glass rod with distilled water between each test until the mixture fails to give a blue colour with iodine. Record the total time taken between the mixing of the saliva and the starch solution and the end of the test. This is the time for all the starch to be converted to maltose by amylase at room temperature. Repeat the experiment at different temperatures examples at 5, 15, 25, 45, 55, 65 and 75. It is important that for each of these experiments the saliva and the starch solutions be warmed or cooled to the required temperature before they are allowed to mix. For temperatures higher than room temperature use a water bath, for temperatures lower than room temperature use ice cubes to bring the temperature down. For the above experiments, it may be necessary to work in groups, each group working at a particular temperature. Tabulate your results. Temperature Room temperature 5 15 25 45 55 65 75 Time in minutes (t) Activity (1/t)

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Since the shorter the time taken the more active the enzyme will be, the activity (rate of reaction) is denoted by 1/t ( the reciprocal of the time taken).

Questions: 1. 2. (c) 1. 2. 3. Plot a graph of 1/t against temperature. What is the optimum temperature? Describe how temperature affects enzyme catalysed biochemical reactions. To show the action of pepsin on egg white (protein). Prepare a suspension of egg white by beating up a little raw egg white with boiling water in a small beaker and allow the suspension to cool. To each of the three test tubes A, B and C, add a few cm of the cooled egg white suspension. Place the tubes in a water bath at about body temperature (about 37 C). Fill each tube as follows: A 5 cm of 0.2 % hydrochloric acid B 5 cm of artificial gastric juice ( prepared by dissolving 3.5 g of pepsin in 100 cm of 0.2 % hydrochloric acid C 5 cm of artificial gastric juice and 1 cm of dilute natrium hydroxide Plug the mouth of each tube with a piece of cotton wool after adding a little thymol to prevent decay. Leave the tubes to stand for about one hour and shake them periodically. What happens to the egg white suspension in tube B? Does it turn yellow, transparent and then gradually dissolve? Is there any change in the other tubes? Carry out the biuret test on the contents of each tube at the end of one hour. Tabulate your results. Contents Egg white + dilute HCl Egg white + artificial gastric juice Egg white + artificial gastric juice + dilute NaOH Observation Biuret test Inferences

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Test tube A B C

(d) 1. 2. 3. (e) 1. 2.

To show coagulation of milk Take a little rennin solution with about 5 cm of milk. Place the mixture in a warm water bath (about 37 C) After about one hour, compare it with a control in which rennin has not been added. Do you notice any difference? What is the value of such reaction to a mammal? To show emulsification of fats Shake a little coconut oil vigorously with some water in a test tube. What do you observe after the mixture has been left to stand for a while? Obtain a little bile from the gall bladder of a guinea pig and add to the mixture in (1). Shake vigorously, then leave the mixture to stand for some time. Do you notice any difference before adding and after adding the bile?