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IDENTIFYING PROTEINS AND LIPIDS
PROBLEM:
To determine whether one set of substances contains proteins and whether a different set of substances contains
lipids.
BACKGROUND INFORMATION:
PROTEINS
Proteins are polymers (chains) of amino acids held together by peptide bonds which are a special type of covalent
bond. Each amino acid has a carboxyl group and an amino group.
H
\
H
/
N
amino
group
H H
\ /
H N
O
| |
//
H–C–C–C
| |
\
H H OH
alanine
O
C
⁄⁄
\
OH
carboxyl
group
During formation of peptide bonds the amine group of one amino acid reacts with the carboxyl group of the other.
Two hydrogens and an oxygen are lost as a water molecule. This is known as a dehydration synthesis or condensation reaction.
The biuret reagent has two active components: sodium hydroxide (NaOH) and copper sulfate (CuSO4). The NaOH,
a strong base, will denature the proteins, causing them to “uncoil” and lose their native conformation. This exposes
the peptide bonds to the (CuSO4). The copper ions of the (CuSO4) react with the bound amino group of the peptide
bond causing a color change from blue to violet. Thus this test checks for the presence of peptide bonds. The presence of peptide bonds results in a positive test. Since the intensity of violet is related to the concentration of protein,
the greater the quantity of protein, the more intense the shade of violet. See Table 1 below.
H H H
| | |
H–C–C–C–H
| | |
H | H
|
H–C–H
H
|
O
\
|
//
N –– C –– C
/
|
\
H
H
OH
leucine
H H H
| | |
H–C–C–C–H
| | |
H
H | H
|
|
H – C –H
H–C–H
H
|
O
|
O
\
|
||
|
//
N ––C –– C –– N –– C –– C
/
|
|
|
\
H
H
H H
OH
dipeptide
Identifying Proteins and Lipids – Page 1
Table 1: Relationship of color and quantity of protein in the Biuret test
Color of Solution
No Color Change (Pale Blue)
Pale Violet
Medium Violet
Deep Violet
Biuret Test
+++, ++, +/+
++
+++
Concentration of Protein
No protein
Low Concentration of Protein
Medium Concentration of Protein
High Concentration of Protein
HYPOTHESIS:
Discuss with your lab partner which substances will test positive for protein and which substances will have a positive lipid test. Record your predictions in the data table. Remember that your hypothesis does not have to be correct. The purpose for doing the experiment is to test the hypothesis.
MATERIALS AND EQUIPMENT:
Test tube rack with 5 test tubes
Filter paper
CuSO4
Sudan III stain
10% NaOH
Test solutions
PROCEDURE:
A. BIURET TEST
1. Obtain a test tube rack with 5 test tubes. Label the test tubes.
2. Place 15 drops of each sample to be tested in the corresponding labeled test tube.
3. To each test tube add 15 drops of 10% NaOH and 5 drops of 1% copper sulfate (CuSO4).
4. Tap the side of each test tube to mix all solutions.
5. Note the color and its intensity, and record in Table 2.
6. If you are having trouble distinguishing between pale blue and pale violet, hold the tube in question
against a white background with your control. If the tube in question looks the same as the control it must
be pale blue.
Identifying Proteins and Lipids – Page 2
LIPIDS
Lipids, like carbohydrates, are complex molecules consisting of carbon, hydrogen, and oxygen. Whereas carbohydrates
have a 1:2:1 ratio of carbon to hydrogen to oxygen, lipids have much less oxygen and proportionately more carbon
and hydrogen. The carbon-to-hydrogen bonds are nonpolar covalent, making the molecule hydrophobic and accounting for its insolubility in water, a polar solvent. Like lipids, Sudan III is nonpolar and thus has an affinity for lipids
resulting in their staining red. This is referred to as “hydrophobic
interaction” and provides the basis for the Sudan III test.
B. SUDAN III TEST
1. Obtain 1 piece of filter paper. Label the center with your period number – group number. Draw small circles
with a pencil and label as follows:
1
= Ethyl alcohol
2 = Flour
3
= Margarine
4
= Butter
5
= Salad oil
6
= Skim milk
7
= Whole Milk
8
= Soy milk
2. Place a drop of each solution to be tested in the circle on the filter paper. All solutions are extracted in
ethyl alcohol. What does this tell us about what our control should be?
3. Allow all drops to dry thoroughly. When dry, add a second drop of each solution. This will concentrate
the solution & make your results clearer.
4. Place filter paper disk in the Sudan III stain & leave for about 20 min. It will not hurt to leave it longer.
5. Remove filter paper with forceps and rinse under water. Record the results in Table 2.
6. Rate the color and intensity according to the following scale:
(–) No difference in color compared with the background (the filter paper itself will absorb some of the
stain)
(+) Pale orange color
(++) Strong orange color
Identifying Proteins and Lipids – Page 3