In this lab, you will determine, through observation, which protease is secreted into the stomach, and which is secreted into the small intestine.

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1 Lab 2: Protein and Fat Digestion LABORATORY OBJECTIVES To investigate the effect of ph and digestive enzymes on the digestion of proteins To investigate the action of lipase on the breakdown of fats INTRODUCTION: Proteins: During digestion, food is broken down in stages by a series of chemical reactions. These chemical reactions are catalyzed by digestive enzymes. A protease is an enzyme that breaks down proteins. Two proteases pepsin and trypsin (trypsin is one of the enzymes found in pancreatin), are secreted at different stages and at different sites during digestion. Each digestive enzyme works best at an optimum ph. The stomach has a ph of about 2, while the small intestine has a ph of between 7 and 8. In this lab, you will determine, through observation, which protease is secreted into the stomach, and which is secreted into the small intestine. Fats: Lipase, an enzyme produced in the pancreas, acts specifically on fats to split (hydrolyze) them into fatty acids and glycerol. Lipase (lipase is one of the enzymes found in pancreatin) hydrolyses the fat in milk to fatty acids which react with sodium carbonate to lower the ph of the mixture. This ph change is observed by using phenolphthalein. Phenolphthalein is used only as a chemical indicator of ph. Phenolphthalein turns red in a neutral solution and colorless in an acidic solution. From: Burns, Karen. Year?. Protein Digestion/The action of lipase (pancreatin) on fats. Vancouver Island University, Biology 067 Laboratory experiment.

2 ACTIVITY : Protein Digestion MATERIALS 6 x 25 ml test tubes Test tube stand Test tube marking pencil Boiled egg white in small cubes Pepsin solution (=5g pepsin in 00ml H2O) Pancreatin solution Dilute NaOH (ph 8) Dilute HCl (ph 2) METHODS. Label 6 test tubes from to 6 2. Label all test tubes so they are identifiable as yours (initials?) 3. Add small pieces of egg white to six clean test tubes 4. Add 3 ml amounts of the solutions listed below: Test tube : egg white + pepsin Test tube 2: egg white + pepsin + HCl Test tube 3: egg white + pepsin + NaOH Test tube 4: egg white + pancreatin Test tube 5: egg white + pancreatin + HCl Test tube 6: egg white + pancreatin + NaOH 5. Incubate the tubes for about 24 hours at 37 o C 6. Complete the table below (fill in last column [final observations] after incubation period). This table should be redone and put into your data/observations section of your lab report. 2

3 DATA AND OBSERVATIONS Test Tube # Contents Acid, Base, or neutral Observation before digestion Observation after (degree of digestion) DISCUSSION QUESTIONS. Why was egg white selected as the test material? 2. Under what ph conditions does pepsin act most efficiently? 3. What conditions are most effective for the action of pancreatin? 4. Compare and comment on your results and the actual ph conditions in the stomach and small intestine. 5. What constants were used in this experiment (What is a constant?)? 3

4 ACTIVITY 2: Action of Lipase on Fats MATERIALS 2 x 25 ml test tubes Test tube stand Test tube marking pencil Fresh whipping cream Pancreatin solid Phenolphthalein solution Detergent mol sodium carbonate solution (0.6 g Na2CO3/00 ml soltn) Hotplate Thermometer 600 ml beaker METHODS. Turn on hot plate and start warming a 600 ml beaker of water. 2. Label 2 test tubes as A and B 3. To each of both test tubes, add 0 ml fresh cream and two pinches of detergent and shake. 4. Add a few drops of phenolphthalein indicator If this does not produce a deep red color, add a few drops of sodium carbonate (instructor will add this) until the red color is distinct. 5. To test tube A, add a small amount of pancreatin (solid) from the tip of the spatula. 6. Warm both tubes in a 37 o C water bath (in 600 ml beaker). 7. Note the time that you place the tubes in the bath and 8. Note the time that it takes for the indicator to become colorless. 4

5 DATA AND OBSERVATIONS: Test Tube A Test Tube B 0 ml cream 0 ml cream Detergent (pinch) Detergent (pinch) Phenolphthalein (few drops) Phenolphthalein (few drops) + Pancreatin Color before warm = Color after warm = Color before warm = Color after warm = Time for color change from red to colorless in tube A = minutes Time for color change from red to colorless in tube B = minutes DISCUSSION QUESTIONS. What was the purpose of using the detergent? 2. What was the ph of the initial samples (After step 4 in procedure) 3. What was the purpose of adding sodium carbonate? 4. What does the color change indicate concerning the actions of pancreatin on the fats present in the cream? 5. Is there any significance to the 37 o C temperature of the water bath and the time required for the color change to take place? 6. What are the final products of fat digestion? (describe with reference to observations made in this lab) 5