Enzyme Lab -Digestion
Introduction
As we have learned, enzymes are specialized proteins that catalyze biochemical reactions within and
outside the cell. We have learned that enzymes are specific to an individual bond-type and activity is
regulated in a variety of ways (i.e. co-enzymes, co-factors, temperature, pH, etc.). In this lab we will
discover how specific enzymes act on macromolecules and how changing the conditions of the
environment affect enzyme action. Because these enzymes are human enzymes they require an
operating temperature of 370C. We will accomplish this with hot water baths.
The material an enzyme acts upon is a substrate. The enzyme pepsin facilitates the breakdown of
proteins so the enzyme is pepsin and the substrate is protein (actually the peptide bond). We can test
for enzyme activity by testing for the substrate concentration or testing for changes in the environment.
Lipase acts on fats by breaking them into glycerol and fatty acids. Since fatty acids are acids it is a logical
conclusion that as pepsin acts on fats the pH of the environment would decrease (i.e. be lower than 7).
Indicators like phenolphthalein is pink/deep pink colour in a slightly basic environment (pH=8) which is
the conditions within the small intestine. If the pH drops, phenolphthalein becomes clear. Therefore,
when we want to test for pepsin activity we must begin with a slightly basic environment. We can
accomplish this by adding a “base” like sodium hydroxide to solution. Lipase stored in the bile salts of
your gall bladder
Amylase acts upon complex carbohydrates (starches) and breaks them into simpler saccharides. When a
solution containing iodine or potassium iodide is added to starch it has a deep blue colour. If no
starches are present the solution will remain a brown colour. If we allow amylase to work on a starch
then when we add the iodine solution (like Lugol’s solution even) there should be a negative result (No
starch present) .
Pepsin breaks down proteins into smaller peptides and amino acids. Pepsin is released in the stomach
which has an acidic environment (low Ph). Therefore, pepsin would be activated/regulated based upon
site specific conditions. Biuret reagent is an indicator that detects the presence of protein. Proteins
cause Biuret to display a purple/violet colour. When peptides and amino acids are present the indicator
takes on more of a pink/lavender colour. We can create an acidic environment by using a dilute solution
of hydrochloric acid (the same acid produced in the stomach).
Purpose:
To observe the breakdown of macromolecules by enzyme catalyzed reactions and observe how
changing temperature affects enzyme activity.
Materials:



Test tubes
Test tube rack
Starch Solution
Enzyme Lab -Digestion













Graduated Cylinders
Lipase
Albumin Solution
Pepsin Solution
Amylase Solution
Rubber stoppers
0.1 M sodium hydroxide
0.1 M Hydrochloric acid solution
Olive oil
Hot plate
Plastic Droppers
Biuret Solution
Potassium Iodide Solution
Procedure:
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
Create a data table for your rough observations (see Results for the template)
Obtain 4 test tubes and matching stoppers.
Label them 1 through 4 with initials and the letter A.
Add 5 drops of sodium hydroxide solution to test tubes 1 and 2
Add 5 drops of phenolphthalein to test tubes 1 and 2.
Use a dropper to add 15 drops of olive oil to test tubes 1 and 2.
Add 5 drops of lipase to test tube 2. Cap test tubes 1 and 2 with stoppers and shake to
thoroughly mix the Lipase solution.
Record your observations in your data table.
Place the test tubes in the water bath at 370C . Note the time. Let them incubate for 60
minutes before making your final observations.
Repeat steps 3 to 7 for test tube 3. Add the test tube to the ice bath for 60 minutes
Repeat steps 3 to 7 for test tube 4. Add the test tube to the hot water bath at 1000C for 60
minutes before making final observations.
Obtain 4 more test tubes.
Label them 1 through 4 with initials and the letter B
Use a graduated cylinder to add 4ml of albumin solution to all test tubes.
Add 25 drops of 0.1M HCl to all test tubes.
Use a graduated cylinder to add 1 ml of pepsin solution to test tube 2-4.
Tap the bottom of the test tubes gently to mix the solution. NO SHAKING
Make initial observations in the data table.
Add test tubes 1 and 2 to the 370C water bath and note the time. Incubate for 60 minutes. .
Add test tube 3 to the ice bath and test tube 4 to the 1000C water bath. Leave for 60 minutes .
After 60 minutes has passed add 20 drops of Biuret reagent to each of the test tubes “B” and tap
the bottom of the test tube to mix.
Observe the test tubes and make your final observations in the data table.
Enzyme Lab -Digestion
23.
24.
25.
26.
27.
28.
29.
Obtain 4 more test tubes.
Label them 1 through 4 with initials and the letter C
Using a graduated cylinder add 4ml of starch solution to all test tubes.
Add 5 drops of amylase solution to test tubes 2-4. Tap the bottoms gently to mix.
Make initial observations for all test tubes.
Place test tube 1 and 2 in the 370C water bath for 30 minutes.
Place test tube 3 in the ice bath for 30 minutes and test tube 4 in the 1000C water bath for 30
minutes.
30. After 30 minutes add3-4 drops of potassium iodide solution to all test tubes and tap the
bottoms of the test tubes to mix thoroughly. Make final observations for all test tubes.
31. All solutions can be poured down the sink. Clean all lab equipment and return.
Results
Table: Enzyme Activity
Lipid Digestion
Test Tube
1* (370C)
2(370C)
3 (Ice Bath)
4 (1000C)
Summary Comments
Initial
Color
Final
Colour
Protein Digestion
Initial
Color
Final
Colour
Carbohydrate digestion
Initial
Color
Final
Colour
*Test Tube one is the control for the experiment.
Conclusion
This lab requires a self-directed conclusion. See your lab write-up instructions to help formulate this
section. Be mindful of the purpose of this lab, your hypothesis and your actual results when composing
this conclusion.