IDENTIFICATION OF PROTEINS AND AMINO ACIDS
Purpose:
The purpose of this lab experiment is to identify and classify both proteins and amino
acids using chemical tests.
Discussion:
Certain qualitative chemical tests can be used in biochemistry to reveal properties of
proteins and amino acids. Provide general background knowledge of proteins and amino acids,
and how the two are related.
Conduct research on the following tests to determine how they will be used to identify
and classify proteins and amino acids. Be sure to identify what structural aspect allows the tests
to be effective.
1. Biuret test
2. Xanthoproteic test
3. Sakaguchi test
4. Nitroprusside test
Objectives:
In this lab, you will…
1. use chemical tests to identify and classify three proteins.
2. use chemical tests to identify and classify three amino acids.
Materials:
Beakers, 400 mL and 600 mL
Hot plate
Pipets
Test tubes, small, 7
Test tube holder
Test tube rack
Chemicals:
Protein solutions
Albumin, 2%
Casein, 2%
Gelatin, 2%
Amino acid solutions
Arginine, 1%
Cysteine, 1%
Tyrosine, 1%
Chemicals (cont.):
Test solutions
Biuret reagent
-naphthol, 0.1% in EtOH
HNO3, 3 M
NaOH, 3 M
NaClO, 5 %
Na2Fe(CN)5NO∙2H2O, 2%
Water, distilled
Safety:
 Biuret reagent is moderately toxic by ingestion and corrosive to eye and body tissue.
 -naphthol is slightly toxic by ingestion, inhalation and skin absorption and is a body
tissue irritant. It also is in solution with ethanol (EtOH), which is flammable.
 Nitric acid, sodium hydroxide and sodium hypochlorite solutions are corrosive liquids
and can cause skin burns.
 Sodium nitroferricyanide is highly toxic by ingestion and inhalation. Do not let it come
in contact with acids, nor should it be heated. Leave the reagent bottle in the fume hood.
 Wear goggles!!!
Procedure:
 Prepare a boiling water bath for use in the Xanthoproteic Test. Fill a 400 mL beaker halffull with water and add a boiling stone. Heat the water bath to boiling on a hot plate.
Biuret Test
1. Label the test tubes 1-7.
2. Add 1 mL of each solution as follows:
Test tube 1: water
Test tube 2: albumin
Test tube 3: casein
Test tube 4: gelatin
Test tube 5: arginine
Test tube 6: cysteine
Test tube 7: tyrosine
3. Add 1 mL of biuret reagent to each test tube.
4. Observe the color and appearance of each solution and record the results in the data table.
5. Pour 25 mL of 5% NaClO solution into a 600 mL beaker for waste disposal. Set the
beaker aside for steps 6, 11, 15 and 19.
6. Rinse the contents of the test tubes with a large amount of water into the waste disposal
beaker. Wash the contents of the test tubes and rinse well with distilled water. Relabel
them 1-7, if necessary, for the next test.
Xanthoproteic Test
7. Repeat step 2 to prepare a set of protein and amino acid samples to be tested.
8. To each test tube add 1 mL of 3 M HNO3.
9. Place the test tubes in the boiling water bath for 3-5 minutes.
10. Use a test tube holder to remove the test tubes from the boiling water bath. Allow the
solutions to cool and then record your observations of their color and appearance in the
data table.
11. Rinse the contents of the test tubes with a large amount of water into the waste disposal
beaker. Wash the contents of the test tubes and rinse well with distilled water. Relabel
them 1-7, if necessary, for the next test.
Sakaguchi Test
12. Repeat step 2 to prepare a set of protein and amino acid samples to be tested.
13. To each test tube add 3 drops of 3 M NaOH, followed by 5 drops of 1% -naphthol
solution.
14. Add 10 drops of NaClO to each test tube and record the color and appearance of each
solution in the data table.
15. Rinse the contents of the test tubes with a large amount of water into the waste disposal
beaker. Wash the contents of the test tubes and rinse well with distilled water. Relabel
them 1-7, if necessary, for the next test.
Nitroprusside Test
16. Repeat step 2 to prepare a set of protein and amino acid samples to be tested.
17. To each test tube add 20 drops of 3 M NaOH, followed by 10 drops of sodium
nitroferricyanide solution.
18. Record the color and appearance of each solution in the data table.
19. Rinse the contents of the test tubes with a large amount of water into the waste disposal
beaker. Wash the contents of the test tubes and rinse well with distilled water.
Cleanup:
 Rinse the contents of the waste beaker down the drain with approximately 20-fold
volume of running water.
 Return all equipment to its proper location.
Analysis:
 Use the research you conducted to identify and classify the proteins.
 Use the research you conducted to identify and classify the amino acids.