Activity 3

GI Laboratory Simulation: DigestionGroup 1:Aguila, Alog, Alejandro, Almajar, Angeles, Arao, Balictar, Buemio, De los Arcos, Escobillo, Manuel, Pea, Rabanal, Rivera,Ronquillo, Siazon,Sutingco,Tagalog, Tamayo, C. Uy, Yeo, YuActivity 3Protein Digestion by PepsinPepsinA protein-digesting enzyme Produced by chief cells of the stomach glandsHydrolyzes proteins to small fragmentsAmino acids and some polypeptides

Gastric Secretion: Background2 types of glands in the stomach mucosa:Oxyntic (acid-forming) glands aka gastric glandsInside surfaces of the body and fundusSecrete Hydrochloric acid, pepsinogen, intrinsic factor, mucus, gastrinComposed of 3 types of cellsMucous neck cells: secrete mucousPeptic chief cells: secrete pepsinogenParietal (oxyntic) cells: secrete HCl and intrinsic factorPyloric glandsPepsinogen and PepsinPepsinogen: inactive form of PepsinBecomes activated into pepsin when it comes into contact with Hydrochloric acidPepsinAn active proteolytic enzyme in a highly acidic mediumOptimum pH 1.8 3.5Above pH of 5: almost no proteolytic activity and may become completely inactivated

Protein Digestion in StomachRequires stomach juices to be acidic for it to be activeHCl secreted by parietal (oxyntic) cells in glandsWhen mixed with stomach contents, pH becomes 2.0-3.0 (highly favorable for pepsin activity)Protein Digestion by PepsinPepsin can digest collagenImportant for digestion of meat and other meat proteins Only initiates protein digestion20% of total protein digestionConverts protein to:ProteasesPeptonesFew polypeptidesBreaks down protein by hydrolysis at peptide linkages between amino acidsLab Simulation Acitivity 3Protein Digestion by PepsinProcedureBAPNA: synthetic proteinTransparent and colorless in solutionWill turn yellow if active pepsin (or any protein digesting enzyme) is presentPepsin will digest BAPNAPepsin IncubationBoil Test tube 1Pepsin AssaySpectrophotometer: to measure optical density

Lab Results and AnalysisRun 1 Incubation Temperature: 37CIncubation Time: 60 minutes

Run 1

Run 2Incubation Temperature: 37CIncubation Time: 30 minutes

Run 2

Run 3Incubation Temperature: 10CIncubation Time: 60 minutes

Run 3

Analysis1. Which pH provided the highest pepsin activity? How does this correlate to the location of pepsin in the body?2. Would pepsin be active in the mouth? Explain.pH 2.0Pepsin obtains optimal activity in a highly acidic medium (pH 1.8-3.5). When pH exceeds 5.0, very little or no proteolytic activity will take place.This pH correlates with the pH in the stomach which has an acidic pH of 2.0-3.0 when HClmixes with stomach contents

No.The normal pH of the mouth stays close to neutral (7.0). This pH however, can change temporarily when certain foods are digested. The pH of the mouth does not reach the ideal acidity in order for pepsin activity to take place (pH 1.8 3.5).

Analysis3. How did the results of tube 1 compare with those of tube 2?

Test tube 1 displayed an optical density of 0.00 while test tube 2 displayed an optical density of 0.40. The solution in test tube 1 remained clear whereas the solution in test tube 2 turned yellow, evidence that BAPNA in test tube 2 was digested.

4. Tubes 1 and 2 contained the same substances. Explain why their optical density measurements were different.

The boiling of the solution in test tube 1 gave rise to the difference in optical density measurements between tubes 1 and 2. The boiling of the solution in test tube 1 caused the denaturing of pepsin, rendering this inactive and unable to digest BAPNA.Analysis5. Did the pepsin or deionized water contain any contaminating digested BAPNA? Which tubes confirm this?

No. The pepsin or deionized water did not contain contaminating digested BAPNA. Test tubes 3 and 4 confirm this with 0.00 optical density.

6. What do you think would happen if you reduced the incubation time to 30 minutes? How did this affect optical density results?

Reducing the incubation time to 30 minutes reduced the optical density due to the reduced amount of time for digestion of BAPNA.Test tube 5 turned yellow despite the neutral pH because pepsin can still be active at neutral pH and becomes irreversible inactive only when pH levels reach 8.0.19Analysis7. What do you think would happen if you decreased the temperature to 10C? What effect would this have on pepsin activity? Why?What effect did boiling have on pepsin?Decreasing the temperature to 10C would decrease pepsin activity and would decrease the amount of digested BAPNA.Decreasing the temperature would, in effect, decrease optical density.Pepsin works best at body temperature (~37C)Boiling led to the denaturation of pepsin rendering this inactive.AnalysisWhat is the substrate in the experiment?

The substrate used in the experiment was BAPNA BAPNA: N-benzoyl-DL-arginine-p-nitroaniline

What was the significance of using 37C for incubation?

37C was used for incubation because the experiment tried to simulate the activity of pepsin in the body. 37C is representative of normal body temperature. Activity 4Pancreatic Lipase Digestion of Fats and the Action of BileLipaseNecessary for the absorption and digestion of nutrients in the intestines.

Responsible for breaking down lipids (fats), in particular triglycerides. Once broken down into smaller components, triglycerides are more easily absorbed in the intestines.

Produced in the pancreas but is also produced in the mouth and stomach. Most people produce sufficient amounts of pancreatic lipase. Lipid Digestion by Lipase

Lab Simulation Acitivity 4Pancreatic Lipase Digestion of Fats and the Action of BileProcedureCHART 5Pancreatic Lipase Digestion of Fats and the Action of BileTube no. 123456AdditivesLipaseVeg. oilBile saltspH 7.0 bufferLipaseVeg. oilD.I. waterpH 7.0bufferLipaseD.I. waterBile saltspH 9.0 buffer

D.I. waterVeg. oilBile saltspH 7.0 bufferLipaseVeg.oilBile saltspH 2.0 bufferLipaseVeg. oilBile saltspH 9.0 bufferIncubation condition37degreesC60 min37degreesC60 min37degreesC60 min37degreesC60 min37degreesC60 min37degreesC60 minpHLab Results and AnalysisResultsCHART 5Pancreatic Lipase Digestion of Fats and the Action of BileTube no. 123456AdditivesLipaseVeg. oilBile saltspH 7.0 bufferLipaseVeg. oilD.I. waterpH 7.0bufferLipaseD.I. waterBile saltspH 9.0 buffer

D.I. waterVeg. oilBile saltspH 7.0 bufferLipaseVeg.oilBile saltspH 2.0 bufferLipaseVeg. oilBile saltspH 9.0 bufferIncubation condition37degreesC60 min37degreesC60 min37degreesC60 min37degreesC60 min37degreesC60 min37degreesC60 minpH6.216.729728.97Analysis1. Explain the difference in activity between test tubes 1 and 2.2. Can we determine if fat hydrolysis has occurred in tube 6? Explain.The presence of bile is needed to emulsify fat into droplets

As fat is digested, it produces organic acid end products that lower pH levels.

Bile salts in test tube 1 more effectively broke down fat and produced organic acid end products, and thus lower the solution's pH more than test tube 2.

Some fat hydrolysis has still occurred

Bile salts and lipase are still present, fat was still hydrolyzed into its component fatty acids.

Minimal change in pH is caused by organic acids produced in the reaction

Analysis3. Which pH resulted in maximum lipase activity?4. Can we determine if fat hydrolysis has occured in tube 5? Explain.pH 7.0 resulted in maximum lipase activity.

This pH correlates with the optimum pH for pancreatic lipase [8.0]

No.

However, studies have shown that below pH 3.0, human pancreatic lipase has irreversibly lost enzyme activity and its ability to bind to lipid emulsions is abolished

Most likely, no reactions occurred

Analysis5. In theory, would lipase be active in the mouth? In the stomach?6. What is the substrate, and what subunit is formed in this experiment?.Mouth - pH 7Yes, lipase can be active in the mouth because the pH is near the optimum pH

Stomach pH 2-3No, lipase would be inactive in the stomach as it is well below the optimum pH

Vegetable oil (fat)Bile saltsLipaseMonoglycerides and fatty acidsAnalysis16. Describe the activity of lipase with & without the bile salts. 18. What pH resulted in the maximum pancreatic lipase activity?.The activity of lipase is enhanced with the addition of bile salts. This can be seen in the greater decrease in pH of tube 1 over that of tube 2.

17. Is the activity of bile a chemical or a physical process?Bile activity is a physical process. The fat particles are simply being broken down into smaller fat particles, to aid the subsequent activity of lipase.

pH 7.0

19. How does this optimal pH correlate to the enzymes location in the body?.The enzyme lipase is located in the small intestine, where the pH is neutral.The enzyme lipase is located in the small intestine, where the pH is neutral.

ReferencesGuyton, Arthur, and John Hall. Textbook of Medical Physiology. 11th ed. Philadelphia, Pennsylvania: Elsevier Saunders, 2006. Print.

"Pepsin." Enzyme Explorer. 2009. Sigma-Aldrich Co., Web. 25 Nov 2009. . http://www.worthington-biochem.com/introbiochem/effectspH.htmlRanaldi, S et al. 2008. Lid Opening and Unfolding in Human Pancreatic Lipase at Low pH Revealed by Site-Directed Spin Labeling EPR and FTIR Spectroscopy. Biochem 48 (3), pp 630638