Answers to Questions Activity 1: Assessing Starch Digestion by Salivary Amylase 10. Tubes Tubes 2, 6, and 7 showed the effect effect of pH on amylase activity activity.. The results of this this experiment indicate that that the activity maximum maximum of amylase is at pH 7.0, whereas whereas pH 2.0 and pH 9.0 demonstrated very little activity. In this experiment, pH 7.0 showed the highest level of amylase amylase activity. activity. Tube 3 showed that amylase did not contain maltose contamination. Tubes 3, 4, and 5 showed that water had no starch or maltose contamination. Tube 3 directly showed that water did not have maltose or starch contamination. Tube 4 was a starch control (with the same water) that showed no maltose, and tube 5 was a maltose control (also with water) that showed no starch. If control tubes 3, 4, or 5 were not done, then what is perceived as digestion might really be starch or maltose contamination. Saliva would not be active in the stomach because the stomach pH is too low. Boiling inactivates, or denatures, enzymes.
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PhysioEx™ Exercise 8
Activity 2: Assessing Cellulose Digestion Tubes #4, 5, and 6 showed that starch or cellulose was still present. Tubes #1, 2, 3, and 7 showed positive tests for the Benedict’s reagent, indicating the presence of reducing sugar. Freezing had no effect. Freezing does not restrict enzyme activity, unlike boiling. Amylase had no effect on the cellulose in tube #4. Cellulose is digestible by bacteria. Peptidase had no effect on animal starch. Peptidase does not work on carbohydrate substrates so has no effect on digestion of these molecules.
Activity 3: Assessing Protein Digestion by Pepsin 8. pH 2.0 allowed the highest pepsin activity. Pepsin would not be active in the mouth. The pH optimum of pepsin is pH 2.0 and the pH in the mouth is relatively neutral. Boiling tube 1 inactivated pepsin. Pepsin digested BAPNA in tube 2. Because there was no activity in tube 1, its optical density was 0.0. In contrast, there was relatively high activity in tube 2. Tubes 3 and 4 proved that neither pepsin nor deionized water contain any contaminating digested BAPNA since the optical density in both of these tubes was 0.0. If the incubation time were decreased to 30 minutes, slightly less BAPNA would be digested, and the optical density reading would be slightly lower. Because digestive enzymes work best at body temperature, incubating at a cooler temperature would decrease the amount of BAPNA digested.
Activity 4: Assessing Fat Digestion by Pancreatic Lipase and the Action of Bile 7. Tube 1 investigated the action of bile on enzyme activity, and tube 2 examined lipase activity without bile. Bile enhances fat digestion by lipase. Yes, you can determine if activity occurred in tube 6 because the pH would drop below pH 9.0. A small amount of fat digestion occurred because pH decreased from 9.0 to 8.97. The optimim pH for lipase activity was pH 7.0. Using a pH method to assay for activity at pH 2.0 does not work because the buffer is already quite acidic. There could have been activity at pH 2.0 that was not detectable by this method. In theory, lipase would be active in the mouth because its pH optimum is relatively neutral. However, it would not be active in the stomach because of the acidic pH condition. The substrate is vegetable oil (fat). The subunit formed is fatty acid (and monoglycerides).
PhysioEx™ Exercise 8
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R E V I E W
S H E E T EXERCISE
NAME ____________________________________ LAB TIME/DATE _______________________
8
Chemical and Physical Processes of Digestion
Carbohydrate Digestion The following questions refer to Activity 1: Assessing Starch Digestion by Sali vary Amylase. 1.
At what pH did you see the highest activity of salivary amylase? 7
Why?
Salivary amylase is active in the mouth, which has a pH of 7. 2.
How do you know that the amylase did not have any contaminating maltose? Tube 3 showed that amylase had no contaminating maltose.
3.
What effect did boiling have on enzyme activity? Why? Boiling caused the protein salivary amylase to be denatured, thus inactivating the enzyme.
4.
Describe the substrate and the subunit product of amylase.
The substrate is starch; the product is maltose.
The following questions refer to Activity 2: Assessing Cellulose Digestion. 5.
Does amylase use cellulose as a substrate? Explain. No; amylase is an enzyme that does not digest cellulose, only starch.
6.
Did freezing have an effect on the activi ty of amylase? Explain. No; the enzyme activity was the same at freezing as it was at 37°.
7.
Do you think that the bacterial suspension contained the enzyme cellulase (an enzyme that digests cellulose)? Why or why not? Yes; cellulose had been digested to maltose in tube 7.
8.
What is the substrate of peptidase? Explain, based upon your results. In this simulation, BAPNA was the substrate of peptidase because the optical density (indicating enzyme activity) increased in the solutions containing both BAPNA and peptidase.
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Protein Digestion by Pepsin The following questions refer to Activity 3: Assessing Protein Digestion by Pepsin. 9.
At which pH did you see the highest activity of pepsin? 2
How does this correlate to the location of pepsin in the
body? Pepsin is found in the stomach where the pH is 2.
10.
What effect did boiling have on pepsin? Pepsin did not digest BAPNA in tube 1 so it can be concluded that boiling denatures pepsin.
11.
Was there any digested BAPNA contaminating the pepsin or deionized (DI) water? No How can you tell? Tubes 3 and 4 had an optical density reading of 0.0.
12.
What is the substrate in this experiment? BAPNA What is the usual substrate for pepsin, and what subunits are formed with pepsin activity? Substrate—proteins Subunits formed—proteoses, peptones, peptides (all small protein fragments), and free amino acids
13.
What was the effect of decreasing the incubation ti me on the optical density results? Decreasing incubation time resulted in a slightly lower optical density.
14.
What effect would decreased incubation temperature have on pepsin activity? Why? Decreased incubation temperature reduced pepsin activity because the optimal temperature for pepsin activity is body temperature.
15.
What was the significance of using 37°C for the incubation? 37°C is body temperature.
Fat Digestion by Pancreatic Lipase and the Action of Bile The following questions refer to Activity 4: Assessing Fat Digestion by Pancreatic Lipase and the Action of Bile. 16.
Describe the activity of lipase with and without the addition of bile salts. Refer to Chart 4 for pH values. The activity of lipase is greater with the addition of bile salts.
17.
Is the activity of bile a chemical or a physical process? Explain. Bile activity is a physical process; it breaks fat particles into smaller fat particles.
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Review Sheet 8
18.
What pH resulted in the maximum pancreatic lipase activity? 7.0 How does this optimal pH correlate to the enzyme’s location in the body? The pH of the small intestines is neutral.
19.
Explain whether or not we can determine fat hydrolysis in tube 5. Why or why not? We cannot determine if fat hydrolysis occurred in tube 5, which had a buffer of 2.0, and the pH of the solution after incubation was still 2.0. There could have been activity at pH 2.0 that was not detectable by this assay method.
20.
What is the substrate in this experiment? vegetable oil (fat) What subunits does lipase form? fatty acids and monoglyceride
Physical Process: Mechanisms of Food Propulsion and Mixing The following questions refer to Activity 5: Studying Mechanisms of Food Propulsion and Mixing: Deglutition (Swallowing). 21.
Explain the significance of the movement of the tongue during swallowing. The tongue initiates and controls the buccal phase of swallowing by pushing contents into the pharynx.
22.
Describe three events that occur during the pharyngeal-esophageal phase of deglutition. Mouth, nasopharynx, and larynx are blocked; upper esophageal sphincter relaxes to open esophagus; food moves through esophagus by pressure gradients created by peristalsis.
23.
What was the time interval that you recorded between the first and second sound? 1–2 seconds
Review Sheet 8
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