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Download MrPaul : PP 2 Lab Action of digetive enzymes
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PP 2 Lab: Action of Digestive Enzymes Write down your results appropriately in a data table: Before you start make sure: 1. You have all your reagents with you 2. You have individual droppers for each solution (Do not mix droppers for different solutions) 3. Test pH using a litmus paper 1. Action of Amylase on starch Amylase is an enzyme which catalyzes the hydrolysis of the polysaccharide starch to the disaccharide maltose. Salivary amylase is produced by the salivary glands and pancreatic amylase is produced by the pancreas. If amylase is added to a solution of starch, the starch will be digested to form maltose. Substrate Amylase Products Starch - - - - -> Maltose Test I2KI Benedicts Set up the test-tubes in the following manner: Test each for starch (Iodine test) and each for sugar (Benedict’s test) Test-tube Content number 1. 1 mL of 1% starch solution 2. 1 mL of 1 % starch solution + 1 % active amylase + 2 mL of pH buffer 7 (check that the pH is 7 before starting the experiment) 3. 4. Leave at 37OC for 10 minutes and then test. 1 mL of 1 % maltose 1 mL of 1 % starch solution + 1 % active amylase Boil in water bath for 5 minutes then test. 5. 1 mL of 1 % starch solution + 1 % active amylase + 2 mL of pH 2 (check the pH is 2 before starting the experiment) Leave at 37oC for 10 minutes and then test. 2. Digestion by Lipase Lipase is a fat-digesting enzyme, catalyzing the hydrolysis of fat to fatty acids and glycerol. The main source of lipase is the pancreas. Substrate Lipase Lipid - - - -> Products Glycerol + Fatty Acid Test Phenol Red Acid In the following experiment you will use a solution of commercially available pancreatic lipase to study the hydrolysis of milk fat. To follow the reaction, you will make use of the fact that fats are neutral, while fatty acids are acidic. The release of fatty acids from fats by hydrolysis will increase the acidity (lower the pH) of the reaction mixture. This change can be observed by using the indicator dye, phenol red, which is useful for measuring pH values between 6.8 and 8.4. You will add NaOH (sodium hydroxide) to all tubes at the start making all tubes initially alkaline. The more enzyme activity, the more fatty acids, the more acid (lower pH) Set up the test-tubes in the following manner: Use pH indicator strips to check pH / (alternate use phenol red * must be placed at 37oC for 20 min if you do this). +++ Fuschia Alkaline None . . . . . . . ++++ Red Neutral Some ++ Yellow Acid Most Color Symbol Description pH Amount of Lipase Activity Test-tube number 1. Content 1 mL Whole milk + 1 mL of 2% lipase + 1 mL of 1 M NaOH 2. Place at 37oC wait 20 min - then test with pH strips or add 1ml of Phenol Red with the content before adding to water bath if not using pH strips 1 mL low fat milk + 1 mL of 2% lipase + 1 mL of 1 M NaOH 3. Place at 37oC wait 20 min - then test with pH strips or add 1ml of Phenol Red with the content before adding to water bath if not using pH strips 1mL cream + 1 mL of 2% lipase + 1 mL of 1 M NaOH Place at 37oC wait 20 min - then test with pH strips or add 1ml of Phenol Red with the content before adding to water bath if not using pH strips 3. Digestion by Protease (Pepsin) Pepsin is a protease that begins digestion of proteins, breaking them into peptides and amino acids. Pepsinogen, is secreted by gastric glands of the stomach into the stomach. There, in the acid environment of the stomach, pepsinogen is converted into pepsin. Although both pepsin and trypsin are proteases, they require quite different conditions of acidity and alkalinity for their action. Substrate Pepsin Protein - - - - -> Products Peptides Amino Acids Test Disappearance of egg white Set up the test-tubes in the following manner: Test visually for disappearing egg white Test-tube number 1. Content 1 mL of egg white + 2mL pH buffer 7 + 2 mL of 2 % Pepsin (check that the pH is 7 before starting the experiment) 2. Place at 37OC - wait at least 10 minutes before checking 1 mL of egg white + 2 mL of pH buffer 2 + 2 mL of 2 % Pepsin (check that the pH is 2 before starting the experiment) 3. Place at 37OC - wait at least 10 minutes before checking 1 mL of egg white + 2 mL of pH buffer 2 + 2 mL of 2 % Pepsin (check that the pH is 2 before starting the experiment) Place in boiling water bath for 5 minutes before checking.
