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Chenzhe Cao Jyoti Suryadevara 4/25/13 E1 Digestive Enzymes Post-lab Questions 1.1 Tube B2 serves as the control because it is the one that contains cooked starch solution and distilled water. Because the saliva-water mixture was not added to the tube, no salivary amylase was provided in order to break up the starch into maltose. A positive test only occurs when maltose is present. Because no maltose is present, the result for this test tube will be a negative test, and our experimental tubes can be compared to this control to determine whether or not starch was broken down and to what extent. 1.2 Results. Boiling -Denatures enzymes. HCl - wrong pH and enzyme cannot work efficiently 1.3 Results. Depending on how much the starch has been hydrolyzed, there could be a positive test for both the Benedicts and Lugols test. If all the starch is completely hydrolyzed into maltose, Benedicts will be positive and Lugols will be negative. If none of the starch is hydrolyzed Lugols will be positive and Benedicts will be negative. If there is some unhydrolyzed starch and some hydrolyzed starch the Benedicts test can vary in color, from green, yellow, orange, to red; however still displacing a positive test. The Lugols test can also vary in color; the intensity of the dark, blackish color, depending on how much unhydrolyzed starch is present. 1.4 An ideal environment for salivary amylase is one that has a pH around 6-7. This is because once the pH drops below 4, it will inhibit the activity of the enzyme. 2.1 Tube B1 serves as the control. It only contains egg whites and distilled water. Because no pepsin solution was added, the proteins in the egg whites will not be hydrolyzed. The Biuret test will give a positive result. For all the other test tubes, however, pepsin is added so some of the proteins will begin to be hydrolyzed, decreasing the number of peptide bonds, thus giving a negative Biuret test. One is able to compare the color intensity of the other test tubes to tube B1 to determine if proteins are present and approximately the amount of protein present compared to when there is when protein is not hydrolyzed. If the color in the other test tubes is the same as B1 then there are proteins present. If there is no color or blue, then the proteins were broken down.

2.2 The Biuret test contains copper (II) sulfate which reacts with the peptide bonds in unhydrolyzed proteins producing a color. Results. Antacid neutralizes pH decreasing activity of pepsin. HCl is optimal because it creates optimal pH environment for pepsin to work in. 2.3 Antacids neutralize pH, bringing the pH to about 6 or 7. Pepsin works optimally in an acidic environment of about pH 2-3, so when the pH rises above pH of 5 pepsin becomes inactive. When pepsin becomes inactive, less proteins in the egg whites are broken down. the copper (II) sulfate in the Biuret test reacts with more peptide bonds in the unhydrolyzed proteins producing a darker pink, purple color, compared to when no antacid is present. The color is very similar to the Tube B1, where no pepsin was added. 2.4 Depending on what types of proteins were present in the egg whites, large polypeptides or small polypeptides, If trypsin was used instead of pepsin, the Biuret test would yield a positive or negative result. Trypsin digests peptides and smaller polypeptides, products from pepsin digestion, into dipeptides and amino acids. If there were smaller polypeptides present in the egg whites trypsin would be able to break them down, resulting in a negative Biuret test. If there were large polypeptides, trypsin would not be able to digest them and the Biuret test would be positive showing the presence of peptide bonds. 3.1 Tube B2 serves as the control. This tube only contains heavy cream, distilled water, and bile salts. No pancreatin solution was added, thus no lipases were added to the test tube. If no lipase was added, the fats are not digested and the litmus test yields a violet-blue color (neutral pH). In the other test tubes pancreatin solution is added which in turn breaks down the fats, depending on the environment of the test tube (temperature and bile salts). The other test tubes can be compared to tube B2 to determine the if the fats were broken down and if they were, to what extent. If the other tubes match the color of tube B2, then no fats were broken down. If the colors are closer to red then fat has been broken down. 3.2 Results. Bile salts help emulsify fats helping to increase the ability of lipase to break down the fats. 3.3 Pancreatic lipase facilitates fat digestion in the small intestine and works at an optimal pH of about 8. If HCl is added to the ideal environment for lipase activity, lipase activity will decrease. HCl will decrease the pH of the environment, making it more acidic. In a more acidic environment then the lipases optimal environment, lipase will become inactive and not work efficiently. If a Litmus test was done, the results would be a blue color. Lipase would not be able to break down fats. There would be no free fatty acids from the broken down fat to decrease the pH. Therefore the pH would be neutral. 3.4 As you increase the temperature the activity will also increase, so the reaction will go faster. This also occurs for digestive enzymes. The rate of enzymatic activity increases up to a certain temperature. The rate of an enzyme reaction doubles for each 10 oC rise in temperature, until the optimum temperature is reached. At the optimum temperature, the enzyme will work at its maximum rate. Above the optimum temperature the enzyme activity decreases. This is due to the loss of shape by the enzyme molecules, called denaturation.

Table 1 Topic 1: Carbohydrate digestion Benedicts Tube Content # +/- Color 1 2 3 4 5 Rank is 1-5: 1 = most maltose, 5 = most starch

+/-

Lugols Color

Rank

Table 2 Topic 2: Protein digestion Tube Content +/Color Rank # 1 2 3 4 5 Rank is 1-5: 1 = most smaller polypeptides, 5 = most unhydrolyzed protein Table 3 Topic 3: Triglyceride digestion Color, minutes after incubation Tube Content # 0 15 30 45 1 2 3 4 5 Rank is 1-5: 1 = most fatty acid, 5 = most triglycerides

60

Rank