Biologically Important Molecules
Part III – Enzymatic Digestion
The majority of the molecules in the food you ingest are too large to be absorbed. These macromolecules
must be chemically broken down into their smaller components through digestion. Digestion in animals
is facilitated through the use of enzymes, special proteins that allow chemical reactions to occur much
more quickly and readily than they might under normal circumstances.
To conduct enzymatic digestion of major nutrients and identify the products of those reactions.
Procedure 1: Starch Digestion by Salivary Amylase
Starch is considered a complex carbohydrate, or polysaccharide, because it is made of long chains of
glucose molecules bonded together. This makes starch an ideal molecule for plant energy storage and
explains the high caloric content of foods such as bread, rice, pasta, and potatoes. Starch can be detected
in a solution by adding iodine (I2KI), which turns it into a dark bluish black color.
Starch digestion begins right away through an enzyme present in saliva called salivary amylase. This
enzyme catalyzes a chemical reaction called hydrolysis that breaks down starch.
Two molecules are produced by the digestion of starch by amylase. The first is the monosaccharide
glucose. The second is the disaccharide maltose, which is made of two glucose molecules joined
together. Both of these molecules are reducing sugars, and will react with Benedict’s reagent to form a
green to reddish orange product.
Given the location of salivary amylase in the body, what do you think the optimal temperature and
pH of this enzyme would be?
Eight test tubes Benedict’s Reagent
Iodine (I2KI) Starch solution
Hot Plate Amylase solution
400mL beaker Distilled water
1. Set up a boiling hot water bath using distilled water, the beaker, and hot plate.
2. Number the test tubes 1-10.
Written by James Dauray http://www.aurumscience.com/biology.html Page 1
3. Individually add 5mL of the following materials to the test tubes.
Test tube 1: Distilled water + amylase
Test tube 2: Distilled water + amylase
Test tube 3: Starch
Test tube 4: Starch
Test tube 5: Starch + Amylase
Test tube 6: Starch + Amylase
Test tube 7: Glucose
Test tube 8: Glucose
Test tube 9: Glucose + Amylase
Test tube 10: Glucose + Amylase
4. Allow the reaction to occur for 10 minutes. While you are waiting, make a prediction of the
results of the iodine and Benedict’s test for each test tube:
Table 1: Predicted Results of Amylase Digestion
Water + Starch + Glucose +
Amylase Amylase Amylase
5. The Benedict’s test and iodine test must be conducted in separate test tubes.
6. Conduct the Benedict’s test by adding 5 drops of the reagent to every odd-numbered test tube and
heating in the hot water bath for 10 minutes.
7. Conduct the iodine test by adding 7-10 drops of iodine (I2KI) to every even-numbered test tube.
8. Record the results of your tests.
Table 1: Actual Results of Amylase Digestion
Water + Starch + Glucose +
Amylase Amylase Amylase
What test tubes had a hydrolysis reaction occur? How do you know?
Did any of the results differ from what you expected? Explain how.
What was the purpose of waiting 10 minutes before conducting the tests?
What other variables could have affected the reaction and the enzyme activity?
Procedure 2: Protein Digestion by Pepsin
Like starches, proteins are also made of repeating subunits. Instead of repeating monosaccharides, these
molecules are made of repeating amino acids. However, just like with starch, they are formed by
Protein digestion begins in the stomach with the addition of an enzyme called pepsin. This enzyme
catalyzes a chemical reaction called hydrolysis that breaks down protein. This is the same reaction that
breaks down starch, however, a different enzyme is required that is shaped specifically for protein
Enzymes are very specific molecules, and require environments to perform their role. Two of the biggest
factors that affect enzyme activity are temperature and pH.
Given the location of the pepsin enzyme, what pH and temperature might be ideal?
Recall that Biuret reagent reacts in the presence of polypeptides and forms a purple product. It also reacts
with smaller, simpler peptides to form a pink product. This reagent does not react with individual amino
Six test tubes Warm water bath
Albumin (protein) Biuret reagent
Pepsin Distilled water
0.2% Hydrochloric Acid (HCl)
1. Number the test tubes 1-6.
2. Individually add the following materials to the test tubes.
Test tube 1: 15mL distilled water only
Test tube 2: 5mL albumin + 5mL distilled water
Test tube 3: 5mL albumin + 5mL HCl + 5mL distilled water
Test tube 4: 5mL albumin + 5mL pepsin + 5mL distilled water
Test tube 5: 5mL albumin + 5mL HCl + 5mL pepsin
Test tube 6: 5mL distilled water + 5mL HCl + 5mL pepsin
Which one of these test tubes will be the control?
3. Shake each test tube gently to mix.
4. Allow the reaction to occur for 45 minutes in a warm water bath. While you are waiting, make a
prediction of the results of the biuret test for each test tube:
Table 3: Predicted Results of Biuret Test
Albumin + HCl +
Control Albumin Pepsin +
5. Add three drops of Biuret solution to each test tube. Mix.
6. Record the results of the Biuret test.
Table 4: Actual Results of Biuret Test
Albumin + Albumin +
Water Albumin HCl + Pepsin
HCl Pepsin + HCl
Which test tube(s) showed evidence that enzyme digestion had occurred?
Which test tube had the highest amount of hydrolysis of protein?
What conclusions can you draw about the necessary conditions for protein digestion?
Give another variable that could affect the rate of protein digestion and describe how you would