1
Amino Acids, Peptides, and Proteins
Introduction
Amino Acids
Amino acids are the building blocks of proteins. In class you learned the structures of the 20
common amino acids that make up proteins. All amino acids have the general structure shown
below.
H O
H 3N + C C O R
Peptides
Peptides are short chains of amino acids, each one connected to the next by an amide linkage
called a peptide bond.
Below is the chemical reaction by which two amino acids become connected by an amide
linkage (the circled O and two H‟s are eliminated as a water molecule during the reaction):
H O
H H O
+
-
H3N C C O
+
+
H O
-
H N C C O
R
H R
+
H3N C C
R
H O
N C C O-
+ H2O
H R
peptide bond
A tripeptide (composed of three amino acids):
H O
H O
H O
H3N+ C C
R
N C C
N C C O-
H R
H R
two peptide bonds
The artificial sweetener aspartame (brand name Nutrasweet) is an example of a modified
peptide. The structure of aspartame is shown below.
H O
+
H3N C C
CH2
O
C
O-
H O
N C C O CH3
H CH2
2
Proteins
Proteins are the work horses of living cells. They act as microscopic cellular machines that
function in much the same manner as human-built machines. Protein molecules are long chains
of amino acids connected to each other in the same manner as in peptides. Very small proteins
may be composed of 50 to 100 amino acids, while large proteins may contain thousands of
amino acids. In a normal functional protein, the long chain of amino acids is “folded” into a 3dimensional shape (imagine the long amino acid chain as a piece of string that has been
crumpled up into a 3-D ball/blob). Every protein has a unique 3-dimensional shape that is suited
to its biological function in a living organism. If the 3-dimensional shape “unfolds” for some
reason, the protein will no longer be able to carry out its biological function and will usually be
destroyed by the cell. (Unfolding is like the crumpled up blob of string being unwound again.)
The unfolding of a protein structure is called denaturation.
Chemical Tests
In this experiment you will perform three chemical tests to distinguish between free amino acids,
peptides, and proteins in the lab.
Biuret Test
The Biuret reagent contains copper ions which give it a blue color. The copper ions will interact
with a compound that contains two or more peptide bonds, resulting in the formation of a
violet/purple-colored product. When a compound does not have at least two peptide bonds, it
will not react with the Biuret reagent, and no purple color will appear (solution will remain a
shade of blue due to the copper ions).
Positive Biuret test: violet/purple product forms
Negative Biuret test: no violet/purple product formed
Ninhydrin Test
The ninhydrin reagent will react specifically with a primary (1o) amino functional group on a
compound, resulting in the formation of a violet/purple-colored product. When a compound does
not have a primary amino group, it will not react with the ninhydrin reagent, and no purple color
will appear (solution will remain colorless).
Positive ninhydrin test: violet/purple product forms
Negative ninhydrin test: no violet/purple product formed
Denaturation Test
Strong acid will often denature (unfold) proteins. Proteins that are properly folded into their
normal 3-dimensional shape tend to be soluble in aqueous solution. However, proteins that
have been denatured tend to clump together and come out of solution as a precipitate because
they are no longer soluble when they are denatured and clumped together. In the denaturation
test, strong acid is used to test a solution for the presence of protein. When strong acid is
added, the formation of a white precipitate (composed of denatured protein molecules) is
considered a positive denaturation test and indicates the presence of protein in the solution. If
no precipitate forms, the denaturation test is negative, and indicates that no protein is present in
the solution.
3
Amino Acids, Peptides, and Proteins
Prelab
Name_______________
1. Draw the structures of alanine and glycine. (Refer to the figure showing the 20 amino acids
which is included in your In-Class Biochemistry Notebook.)
2. Show the structure of the peptide that would result from a chemical reaction between glycine
and alanine.
3. A. Indicate whether each of the following is an amino acid, a peptide, or a protein.
B. Indicate whether each will give a positive or negative Biuret test.
A. aa, pept, prot?
B. Biuret test? (+ or - )
aspartic acid (a.k.a. aspartate) ____________
___________
phenylalanine
____________
___________
Nutrasweet (aspartame)
____________
___________
4. Examine the structure of aspartame (Nutrasweet) in the introduction, paying careful attention
to the side chains. Which two amino acids would be released if the peptide bond was
„hydrolyzed‟ (broken by a reaction with H2O)?
4
Amino Acids, Peptides, and Proteins
Procedure
Isolation of the Protein Casein from Milk
1. Put 50 mL of skim milk into a 250 mL beaker. While stirring gently, add a solution of 10%
acetic acid dropwise to the milk. As the mixture becomes acidic (at approximately pH 5.5), the
milk protein casein becomes insoluble and begins to precipitate. Add acetic acid until you see
no further precipitation forming.
2. Prepare a simple filtration apparatus as follows. Place a square of cheese cloth (about 3
layers thick) over the top of a large beaker—the cheesecloth square should be just large enough
to cover the top of the beaker with a little overlap at the edges. Secure the cheesecloth using a
rubber band.
3. Pour your precipitated casein mixture through the cheesecloth. Wash the casein with 25 mL
of ethanol. Scrape the casein off the cheesecloth and press it between some paper towels to
dry it.
4. Take about half of your casein and put it in a 50 mL beaker. Add 10 mL of 0.1 M NaOH to
the casein and stir to dissolve. This is the solution of dissolved casein you will use in the
following steps.
Biuret Test
1. Use the dilute soap solution in the bottles near the sinks (bottles with blue lids) to thoroughly
clean 5 test tubes. Rinse them well with distilled water. It is very important that the test tubes be
clean in order to prevent anomalous results in the chemical tests you will carry out. The test
tubes do not have to be completely dry, but shake them to remove as much water as possible.
2. NOTE: glycylglycine is a dipeptide composed of two glycine‟s linked together.
Place the following chemicals into four test tubes as indicated:
Tube 1:
1 mL distilled water (water will serve as a control)
Tube 2:
1 mL 1% alanine solution
Tube 3:
1mL 1% glycylglycine solution
Tube 4:
5 drops of your casein solution (from step 4 above) + 1 mL distilled water
NOTE: It may take more than 5 drops of the casein. Start with 5 drops and
show your results to the instructor to see if this is enough.
Tube 5:
1 mL of the unknown
Then add 1 mL of the biuret solution to each of the five test tubes. Allow the contents to stand
for 10 minutes. While waiting, fill a 250 mL beaker about 1/3 full with distilled water and place it
on a hot plate, but don‟t turn on the hot plate yet (you‟ll need it for the ninhydrin test below). If
you wish, you may also do step 1 of the ninhydrin test below while you are waiting.
5
3. At the end of 10 minutes, observe and record the color of the test tube contents on the report
sheet. Answer the questions regarding interpretation of the test results. The contents of the test
tubes can then be discarded down the drain.
Ninhydrin Test
1. Your casein solution is acidic (pH below 7), but it needs to be neutral (pH ≈ 7) to conduct a
ninhydrin test. Neutralize your casein solution as follows. Put 3 drops of the casein solution
from step 4 above into a clean test tube and add 1 mL of distilled water.
Test the pH of the solution by dipping a glass stirring rod into the solution and then touching the
end of the rod onto a piece of pH paper.
If the pH is too high (above 7-8), add a drop of 0.3 M HCl to the test tube, mix the
contents of the tube, and test the pH again.
If the pH is too low (below 7-8), add a drop of 0.3 M NaOH to the test tube, mix the
contents of the tube, and test the pH again.
Use more drops of HCl or NaOH as needed to achieve a pH of 7 to 8.
2. Turn the hot plate on high to start heating the water—you will need boiling water for the
ninhydrin test.
3. Wash the five test tubes from the Biuret test thoroughly so they can be re-used now. They
need not be completely dry. Place the following chemicals into the five clean test tubes:
Tube 1:
1 mL distilled water (water will serve as a control)
Tube 2:
1 mL 1% alanine solution
Tube 3:
1mL 1% glycylglycine solution
Tube 4:
1 mL of neutralized casein solution from step 1 directly above
Tube 5:
1 mL of the unknown
Then add 1 mL of 0.2% ninhydrin solution to each test tube. Label the tubes clearly using a
grease pencil. Don‟t use tape to label the tubes because the tape will fall off in the boiling water
bath in the next step.
4. Once the water is boiling moderately, place the tubes in the boiling water bath for 5
minutes—be sure that the water level is not so high that water boils over into the test tubes.
At the end of 5 minutes, turn off the hot plate. Observe and record the color of the tube contents
on the report sheet. Answer the questions regarding interpretation of the test results. The
contents of the test tubes can then be discarded down the drain.
6
Denaturation with Acid
1. Wash the five test tubes from the ninhydrin test thoroughly so they can be re-used now. Be
sure to use a test tube brush and soap in order to remove any purple residue from the ninhydrin
test. The tubes need not be completely dry. Place the following chemicals into the five clean
test tubes:
Tube 1:
1 mL distilled water (water will serve as a control)
Tube 2:
1 mL 1% alanine solution
Tube 3:
1mL 1% glycylglycine solution
Tube 4:
10 drops of your solution of dissolved casein in the 50 mL beaker from step
4 under “Isolation of the Protein Casein from Milk”
Tube 5:
1 mL of the unknown
Then add 1 mL of 20% trichloroacetic acid to each tube. Mix the contents of the tubes by flicking
them with your finger. NOTE: if you see purple color in any of the solutions, the test tube was
not thoroughly cleaned after the ninhydrin test.
2. Observe and record your observations on the report sheet. (There may be nothing to
observe—no changes—in some of the tubes.) Answer the questions regarding interpretation of
the test results. The contents of the test tubes can then be discarded down the drain.
3. All other solutions can be discarded down the drain. The solid casein can be discarded in the
trash.
7
Amino Acids, Peptides, and Proteins
Biuret Test
Report Sheet
Name ___________
Color observed
water (control)
1% alanine
1% glycylglycine
casein
unknown
Interpretation of Biuret Test Results
It will be helpful to review the structures of alanine and glycylglycine (see Postlab #1, #2) in
order to interpret the results of the test.
What do the results of the Biuret test tell you about alanine?
What do the results of the Biuret test tell you about glycylglycine?
What do the results of the Biuret test tell you about casein?
Ninhydrin Test
water (control)
1% alanine
1% glycylglycine
casein
unknown
Color observed
8
Amino Acids, Peptides, and Proteins
Report Sheet
Name ___________
Interpretation of Ninhydrin Test Results
It will be helpful to review the structures of alanine and glycylglycine (see Postlab #1, #2) in
order to interpret the results of the test.
What do the results of the ninhydrin test tell you about alanine?
What do the results of the ninhydrin test tell you about glycylglycine?
What do the results of the ninhydrin test tell you about casein?
Denaturation Test
Observations
water (control)
1% alanine
1% glycylglycine
casein
unknown
Interpretation of Denaturation Test Results
What do the results of the denaturation test tell you about alanine?
What do the results of the denaturation test tell you about glycylglycine?
What do the results of the denaturation test tell you about casein?
9
Amino Acids, Peptides, and Proteins
Postlab
Name_______________
1. Draw the structure of glycylglycine (first refer to the “NOTE” on step 2 on page 4). Use an
arrow and a brief description to indicate the structural feature that caused the glycylglycine
solution to produce the Biuret test result that it did.
2. Draw the structure of alanine. Circle the structural feature that caused the alanine solution to
produce the ninhydrin test result that it did.
3. Look at the structure of aspartame (Nutrasweet) in the introduction. Predict the results
(+ or -) of the following tests on aspartame:
Biuret test:___
Ninhydrin test:___
Denaturation test:___
4. Oxytocin is a hormone; it is a 9-amino acid peptide. Predict the results (+ or -) of the
following tests on oxytocin:
Biuret test:___
Ninhydrin test:___
Denaturation test:___
5. Hemoglobin is a protein composed of over 100 amino acids linked together by peptide bonds.
Predict the results (+ or -) of the following tests on hemoglobin:
Biuret test:___
Ninhydrin test:___
Denaturation test:___
6. Look back at the test results you obtained for the uknown. Based on the test results, check
the identity of your unknown.
An amino acid ___
A pentapeptide (composed of 5 amino acids) ___
A protein (composed of several hundred amino acids) ___
7. What was the purpose of having water as a control for each test?
8. You have a test tube containing either a dipeptide or a large protein. What tests could you
use to determine which is in the test tube?