Exercise 4: Biologically Important Molecules
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OBJECTIVES:
 Learn the structure and importance of the four types of biological molecules.
 Understand hydrolysis and dehydration synthesis.
 Become familiar with different tests used to identify macromolecules.
 Recognize the difference between a negative and a positive control, and the purpose of
each type in an experimental design.
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INTRODUCTION:
Biological organisms use four classes of macromolecules: carbohydrates, proteins,
lipids and nucleic acids. These organic compounds (i.e., carbon-based molecules) are important
for proper cellular functioning and each plays a different role within the cell. Carbohydrates
provide the primary source of energy or “fuel” for cells and are used to support cell walls of
bacteria, fungi and plants. Proteins function as “building blocks” or structural elements within
the cell and aid in the transport of molecules across membranes. Proteins also regulate cellular
activities (enzymes and hormones) and are important components of the immune system
(antibodies). Lipids are used for “storage” of excess fuel and are an integral part of cell
membranes. Nucleic acids comprise our genes (DNA, RNA), regulate cell function, and are
involved in energy transfer. In addition, both DNA and RNA participate in cellular replication.
Many biological molecules are polymers comprised of small subunits (monomers) held
together by covalent bonds. Carbohydrates, for example, are composed of varying combinations
of monosaccharides (e.g. glucose) that can be joined to form disaccharides (e.g. sucrose) and
polysaccharides (e.g. starch, glycogen). Similarly, proteins are made from unique combinations
of amino acids. Monomers are linked together by dehydration synthesis (condensation)
reactions in which energy is used to remove a water molecule, resulting in the covalent bonding
of two subunits (Fig. 1a). Conversely, the bond between monomers can be broken by the
addition of a water molecule and the release of energy - hydrolysis (Fig. 1b).
Although all macromolecules are characterized by the presence of a carbon backbone, the
four classes vary in their elemental structure and chemical properties. The disparate functional
groups impart different solubilities and polarities to each type of macromolecule. For instance,
lipids, which are made of fatty acids and have very little oxygen, are nonpolar and insoluble in
water. Proteins, on the other hand, are polymers of amino acids covalently linked by peptide
bonds. Because amino acids are polar, non-polar, charged or aromatic, the properties of the
resulting proteins vary in accordance with the type of amino acids that comprise them.
In today’s lab, you will perform several biochemical tests to detect the presence of
organic molecules in known substances. Throughout this process, you will learn about the use of
controls as standards for comparison and their role in identifying unknown solutions. Controls
are an essential component of every experiment because they help eliminate alternate
explanations of experimental results. In general, a control is any variable kept constant during the
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experiment, and it is compared to the experimental sample(s) being tested. There are two types
of controls: negative and positive. A negative control helps minimize false positives by
providing a known negative result for a given experimental treatment. Thus, a negative control
provides an example of what the results should look like if the experimental manipulation had no
effect on the variable of interest. A positive control helps minimize false negatives by
demonstrating what a positive result should look like if the experimental manipulation produces
a change. For example, when testing for the presence of salt in a substance, a salt solution would
serve as the appropriate positive control and distilled water as the negative control.
Figure 1. Dehydration synthesis and hydrolysis reactions
Task 1: CARBOHYDRATES
Carbohydrates are molecules made of Carbon (C), Hydrogen (H) and Oxygen (O) in a ratio of
1:2:1. Monosaccharides (Fig. 2A) are made of single sugar molecules while disaccharides (Fig.
2B) and polysaccharides (Fig. 2C) are composed of two or more sugar molecules, respectively.
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A
B
C
Figure 3. Carbohydrate Molecules
Monosaccharides contain either aldehyde (-CHO) or ketone (-C=O) side groups that
reduce oxidizing compounds. A molecule is oxidized if it loses an electron or hydrogen atom and
is reduced when it gains an electron or hydrogen atom (Fig. 4). Collectively, the two processes
are referred to as a redox reaction because when one molecule is oxidized, another is reduced.
Figure 4. Redox reactions
I. Examine Reducing Sugars
Benedict’s reagent can be used to identify the presence of reducing sugars and is a good
indicator for the presence of some carbohydrates. At basic/alkaline pHs (8-14) the copper ions
(Cu2+) in Benedict’s reagent are reduced by the monosaccharide functional groups (i.e. -CHO or
–C=O) to form cuprous oxide. In the Benedict’s test for reducing sugars, the Benedict’s reagent
is reduced while the reducing sugar is oxidized. This redox reaction results in a tractable color
change going from a light blue solution to a green/reddish orange one. The intensity of the
color change is indicative of the amount of reducing sugar present (Fig. 5).
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A = negative (no reducing sugars present)
B = positive (small amount of reducing
sugars present)
C = positive (larger amount of reducing
sugars present)
D = positive (abundance of reducing sugars
present)
Figure 5. Expected test results for Benedict’s test for reducing sugars.
Procedure:
1. Obtain seven test tubes and label them 1-7.
2. Add the materials listed in Table 1 to each of your tubes.
3. Half fill a 250mL beaker with water. Place it on the hot plate at your station and allow it
to come to a gentle boil.
4. In the meantime, predict the color changes you expect to occur in each tube and record
them in Table 1 in the “Benedict’s Test Results Expected (color)” column. Also mark
which tube you think is the positive control and which is the negative control.
5. Add 2mL of Benedict’s reagent to each tube.
6. Place all 7 tubes in the gently boiling water bath for 3 minutes. Observe the tubes for any
change in color during this time.
7. After 3 minutes, remove the tubes and allow them to cool to room temperature. Record
the color of each tube in Table 1 in the “Benedict’s Test Results Observed
(color)”column.
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Table 1:
Benedict’s Test Results
Tube #
Solution
1
10 drops onion juice
2
10 drops potato juice
3
10 drops sucrose
4
10 drops glucose
5
10 drops distilled water
6
10 drops reducing sugar
7
10 drops starch
Expected
(color)
Observed
(color)
Iodine Test Results
Expected
(color)
Observed
(color)
Questions:
a. How do mono-, di- and polysaccharides differ?
b. Explain, in your own words, what are negative and positive controls? Why are they
used in experiments?
o Which of the solutions in Table 1 were your positive and negative controls?
Explain.
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c. How does the Benedict’s test work? What chemical reactions does it test for, and
what does this have to do with carbohydrates?
d. Will a Benedict’s test be able to detect ALL sugars? Explain.
II. Examine Starch
Starch is a polysaccharide often used by organisms for storage of metabolic energy.
Unlike the simpler mono- and disaccharides, starch is a structurally complex polymer (Fig. 6).
Iodine (iodine-potassium iodide, I2KI) reacts with the three-dimensional (3D) structure of this
molecule, resulting in a color change (going from yellow to blue-black, Fig. 7). In this exercise,
we will test the substances previously examined for the presence of reducing sugars for starch.
Figure 6. Starch Molecule
A = negative (no starch present)
B = positive (starch present)
Figure 7. Expected test results for Iodine test for starch.
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Procedure:
1. Obtain seven test tubes and label them 1-7.
2. Add the materials listed in Table 1 to each of your tubes.
3. Predict the color changes you expect to occur in each tube and record them in Table 1 in
the “Iodine Test Results Expected (color)” column. Also mark which tube you think is
the positive control and which is the negative control.
4. Add 7-10 drops of iodine to each tube.
5. Record the color of each tube in Table 1 in the “Iodine Test Results Observed (color)”
column.
Questions:
a. What type of carbohydrate are you testing for when you use the Iodine test? Is this type
of carbohydrate a mono-, di- or polysaccharide?
b. Based on both your answer to part a, as well as your test results, what is the predominant
carbohydrate in onion juice? What about potato juice?
c. Which of the solutions in Table 1 were your positive and negative controls for starch?
Explain.
Task 2: PROTEINS
Proteins are composed of amino acids covalently linked by peptide bonds (Fig. 9). All
amino acids contain an amino group (-NH2), a carboxyl group (-COOH), and a variable side
chain (R-group, Fig. 8) by which they are categorized. Peptide bonds (C-N) form when the
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Fig. 3.20
amino group of one amino acid reacts with the carboxyl group of another (Fig. 9). The Biuret
reagent, regularly colored blue, is used to identify proteins. When the copper ions (Cu2+) in the
reagent interact with peptide bonds, a violet color is produced (Fig. 10). In order for the
interaction between Cu2+ and the peptide bonds to result in a color change, a minimum of 4-6
peptide bonds is required. In general, the longer the protein chain, the greater the intensity of the
reaction.
Figure 9. Protein composed of two amino acids and
linked by a peptide bond (red).
Figure 8. Amino Acid Structure
A = negative (no protein containing more
than 4-6 peptide bonds present)
B = positive (small amount of protein
containing more than 4-6 peptide bonds
present)
A
B
C
C = positive (large amount of protein
containing more than 4-6 peptide bonds
present)
Figure 10. Expected test results for Biuret test for protein.
Procedure:
1. Obtain 5 test tubes and label them 1-5.
2. Add the substances listed in Table 2 to each test tube.
3. Predict the color changes you expect to occur in each tube and record them in Table 2 in
the “Expected Results (color)” column. Also mark which tube you think is the positive
control and which is the negative control.
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4. Add 2mL of 2.5% sodium hydroxide followed by 3 drops of Biuret reagent.
5. Record the color of each tube in Table 1 in the “Observed Results (color)” column.
Table 2:
Tube #
1
Solution
1
2mL egg albumen1
2
2mL honey
3
2mL amino acid solution
4
2mL distilled water
5
2mL protein solution
Expected Results
(color)
Observed Results
(color)
Albumen = clear liquid inside of an egg (“egg white”)
Questions:
a. What monomer comprises a polypeptide? What molecular structures indicate a molecule
is a protein?
b. Using the Biuret test, what would a positive and a negative result indicate?
c. Hypothetically, you test two different substances for the presence of protein. One test
provides a strong positive result and the other a weak, but still positive result. What does
this indicate about the two samples?
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Task 3: LIPIDS
Lipids, which include triglycerides (fats), steroids, waxes, and oils, vary in function.
Similar to carbohydrates, fatty acids bond to glycerol with the input of energy and the formation
of water. While triglycerides and oils serve as energy-storage molecules, phospholipids
aggregate to form cellular membranes which are important sources of cholesterol, a necessary
component of steroid hormones. All lipids share one characteristic; they are insoluble in water
(i.e. hydrophobic) because they have a high proportion of non-polar carbon-hydrogen bonds and
can only dissolve in non-polar solvents such as ether, ethanol and acetone. This property can be
used to test unknown solutions for the presence of lipids. One indicator commonly used is Sudan
IV, a fat-soluble dye that binds to lipids when added to a solution (Fig. 11).
A.
B.
A = positive (lipids present)
B = negative (no lipids present)
Figure 11. Expected test results for Sudan IV test for lipids
I. Examine Lipid Solubility
Procedure:
1. Obtain two test tubes and label them 1 and 2.
2. In this exercise, you will assess the solubility of lipids in polar and non-polar solvents.
Predict what you expect to occur in each tube and record your predictions in Table 3 in
the “Expected Results” column.
3. Add 1mL of vegetable oil to each tube followed by the solutions listed in Table 3.
4. Record your observations in Table 3 in the “Observed Results” column.
Table 3:
Tube #
1
2
Solution
5mL water
Expected Results
5mL acetone
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Observed Results
Questions:
a. Can a lipid dissolve in water? Why or why not?
b. How did this test incorporate what you know about a lipid’s structure and its solubility
properties?
II. Sudan IV Test for Lipids
Procedure:
1. Obtain a filter paper and label it as shown in figure 12. You must use pencil; pen may
affect the results.
1
2
6
5
3
4
Figure 12. Filter paper for Sudan IV test
2. Blot a small amount of test substance onto each numbered circle. Match each substance
to a number according to Table 4. Allow the drops to dry completely, use a hair dryer if
available.
3. Predict the color changes you expect to occur for each substance and record them in
Table 2 in the “Expected Result (color)” column. Also indicate which substance you
think is the positive control and which is the negative control.
4. Soak the filter paper in a petri dish containing 0.2% Sundan IV for 5 minutes, rinse and
dry.
5. Record the color of each spot in Table 4 in the “Observed Results (color)” column.
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Table 4:
Tube #
Solution
1
Known lipid
2
Distilled water
3
Honey
4
Skim milk
5
Cream
6
Salad oil
Expected Result
(color)
Observed Result
(color)
Questions:
a. What observation(s) indicates a positive test for lipids?
b. Lipids contain twice as many calories per gram compared to carbohydrates. Based on
your test results, which has more calories, the salad oil or the honey?
c. Compare the observed results of cream and skim milk. What result might you expect if
2% milk was tested?
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