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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°C
Incubation Time: 60 minutes
Incubation Temperature: 37°C
Incubation Time: 30 minutes
Incubation Temperature: 10°C
Incubation Time: 60 minutes
1. Which pH provided the
highest pepsin activity? How
does this correlate to the
location of pepsin in the
body?



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
2. Would pepsin be active in
the mouth? Explain.


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?



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)
What effect did boiling have
on pepsin?

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
Pancreatic Lipase Digestion of Fats and the Action of Bile
CHART 5
Tube no.
1
2
3
4
5
Lipase
Lipase
Lipase
D.I. water
Veg. oil D.I. water
Lipase
Veg. oil
Veg. oil
D.I.
Bile salts
Veg.oil
Additives Bile salts
Bile salts
water
pH 9.0
Bile salts
pH 7.0
pH 7.0
pH 7.0
buffer
pH 2.0 buffer
buffer
buffer
buffer
Incubation 37degreesC 37degreesC
condition
60 min
60 min
pH
37degreesC
60 min
37degreesC
60 min
37degreesC
60 min
6
Lipase
Veg. oil
Bile salts
pH 9.0 buffer
37degreesC
60 min
Pancreatic Lipase Digestion of Fats and the Action of Bile
CHART 5
Tube no.
1
2
3
4
5
Lipase
Lipase
Lipase
D.I. water
Veg. oil D.I. water
Lipase
Veg. oil
Veg. oil
D.I.
Bile salts
Veg.oil
Additives Bile salts
Bile salts
water
pH 9.0
Bile salts
pH 7.0
pH 7.0
pH 7.0
buffer
pH 2.0 buffer
buffer
buffer
buffer
Incubation 37degreesC 37degreesC
condition
60 min
60 min
pH
6.21
6.72
6
Lipase
Veg. oil
Bile salts
pH 9.0 buffer
37degreesC
60 min
37degreesC
60 min
37degreesC
60 min
37degreesC
60 min
9
7
2
8.97
1. Explain the difference in
activity between test tubes 1
and 2.



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.
2. Can we determine if fat
hydrolysis has occurred in
tube 6? Explain.



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?

pH 7.0 resulted in maximum
lipase activity.
4. Can we determine if fat
hydrolysis has occured in
tube 5? Explain.



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?

Mouth - pH 7
• Yes, lipase can be active in the
mouth because the pH is near
the optimum pH

Stomach – pH 2-3
6. What is the substrate, and
what subunit is formed in this
experiment?.
Vegetable
oil (fat)
Bile salts
Lipase
• No, lipase would be inactive in
the stomach as it is well below
the optimum pH
Monoglycerides
and fatty acids
The enzyme lipase is located in the small intestine, where the pH is neutral.
16. Describe the activity of
lipase with & without the bile
salts.

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.
18. What pH resulted in the
maximum pancreatic lipase
activity?.

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.
 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/lifescience/metabolomics/enzymeexplorer/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