Group 1:Aguila, Alog, Alejandro, Almajar, Angeles, Araño, Balictar,

Buemio, De los Arcos, Escobillo, Manuel, Peña, Rabanal, Rivera,Ronquillo, Siazon,Sutingco,Tagalog, Tamayo, C. Uy,

Yeo, Yu

Protein Digestion by Pepsin

A protein-digesting enzyme •Produced by chief cells of the stomach

glands Hydrolyzes proteins to small

fragments•Amino acids and some polypeptides

2 types of glands in the stomach mucosa:• Oxyntic (acid-forming) glands

aka gastric glands Inside surfaces of the body and fundus Secrete Hydrochloric acid, pepsinogen, intrinsic

factor, mucus, gastrin Composed of 3 types of cells

Mucous neck cells: secrete mucous Peptic chief cells: secrete pepsinogen Parietal (oxyntic) cells: secrete HCl and intrinsic factor

• Pyloric glands

Pepsinogen: inactive form of Pepsin• Becomes activated into pepsin when it

comes into contact with Hydrochloric acid Pepsin

• An active proteolytic enzyme in a highly acidic medium Optimum pH 1.8 – 3.5 Above pH of 5: almost no proteolytic activity and

may become completely inactivated

Requires stomach juices to be acidic for it to be active

HCl secreted by parietal (oxyntic) cells in glands• When mixed with stomach contents, pH

becomes 2.0-3.0 (highly favorable for pepsin activity)

Pepsin can digest collagen• Important for digestion of meat and other

meat proteins Only initiates protein digestion

• 20% of total protein digestion• Converts protein to:

Proteases Peptones Few polypeptides

• Breaks down protein by hydrolysis at peptide linkages between amino acids

Protein Digestion by Pepsin

BAPNA: synthetic protein• Transparent and colorless in solution• Will turn yellow if active pepsin (or any

protein digesting enzyme) is present Pepsin will digest BAPNA

Pepsin Incubation• Boil Test tube 1

Pepsin Assay• Spectrophotometer: to measure optical

density

Incubation Temperature: 37°CIncubation Time: 60 minutes

Incubation Temperature: 37°CIncubation Time: 30 minutes

Incubation Temperature: 10°CIncubation Time: 60 minutes

1. 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 2.0 Pepsin 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).

3. 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.

5. 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.

7. What do you think would happen if you decreased the temperature to 10°C? What effect would this have on pepsin activity? Why?

What effect did boiling have on pepsin?

Decreasing the temperature to 10°C 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 (~37°C)

Boiling led to the denaturation of pepsin rendering this inactive.

What is the substrate in the experiment?

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

What was the significance of using 37°C for incubation?

• 37°C was used for incubation because the experiment tried to simulate the activity of pepsin in the body. 37°C is representative of normal body temperature.

Pancreatic Lipase Digestion of Fats and the Action of Bile

Necessary 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.

Pancreatic Lipase Digestion of Fats and the Action of Bile

CHART 5 Pancreatic Lipase Digestion of Fats and the Action of Bile

Tube no. 1 2 3 4 5 6

Additives

LipaseVeg. oil

Bile saltspH 7.0 buffer

LipaseVeg. oil

D.I. waterpH 7.0buffer

LipaseD.I. waterBile saltspH 9.0 buffer

D.I. waterVeg. oil

Bile saltspH 7.0 buffer

LipaseVeg.oil

Bile saltspH 2.0 buffer

LipaseVeg. oil

Bile saltspH 9.0 buffer

Incubation condition

37degreesC60 min

37degreesC60 min

37degreesC60 min

37degreesC60 min

37degreesC60 min

37degreesC60 min

pH

CHART 5 Pancreatic Lipase Digestion of Fats and the Action of Bile

Tube no. 1 2 3 4 5 6

Additives

LipaseVeg. oil

Bile saltspH 7.0 buffer

LipaseVeg. oil

D.I. waterpH 7.0buffer

LipaseD.I. waterBile saltspH 9.0 buffer

D.I. waterVeg. oil

Bile saltspH 7.0 buffer

LipaseVeg.oil

Bile saltspH 2.0 buffer

LipaseVeg. oil

Bile saltspH 9.0 buffer

Incubation condition

37degreesC60 min

37degreesC60 min

37degreesC60 min

37degreesC60 min

37degreesC60 min

37degreesC60 min

pH 6.21 6.72 9 7 2 8.97

1. 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

3. 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

5. 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 7• Yes, lipase can be active in

the mouth because the pH is near the optimum pH

Stomach – pH 2-3• No, lipase would be inactive

in the stomach as it is well below the optimum pH

Vegetable oil (fat)

Bile salts

Lipase

Monoglycerides and fatty acids

16. 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 enzyme’s 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.

Guyton, 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.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/analytical-enzymes/pepsin.html>.

http://www.worthington-biochem.com/introbiochem/effectspH.html

Ranaldi, 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 630–638