Materials Included In Kit

Additional Materials Required

Prelab Preparation

1. Prepare 2% protein (albumin and gelatin) and 1% amino acid (arginine, cysteine and tyrosine) solutions by adding 100 mL of distilled water directly to each sample bottle. The correct amount of each sample has been provided to prepare the concentrations needed.
2. The protein casein is insoluble in water, but soluble in dilute base. To prepare 2% casein solution: add 98 mL of distilled water followed by 2 mL of 3 M NaOH solution directly to the casein sample bottle.
3. Cap all of the sample bottles and gently shake to dissolve. More extreme shaking or agitation of the protein solutions may cause the proteins to denature and precipitate out of solution. The amino acid and protein solutions can be prepared up to one week before use.
4. The sodium nitroferricyanide solution should be freshly prepared the day of lab. Add 75 mL of distilled water to the sample bottle. Cap tightly and shake gently to dissolve. The minimum amount of material has been provided to avoid unnecessary exposure and disposal problems.

Safety Precautions

Biuret solution contains copper sulfate, which is moderately toxic by ingestion, and sodium hydroxide, which is severely corrosive to eye and body tissue. α-Naphthol solution contains ethyl alcohol and is a flammable liquid. Avoid contact with flames or other sources of ignition. Nitric acid, sodium hydroxide and sodium hypochlorite solutions are corrosive liquids and can cause skin burns. Do not allow sodium hypochlorite (bleach) to come in contact with acids—toxic chlorine gas may be generated. Sodium nitroferricyanide solution is highly toxic by ingestion and inhalation. Do not allow this solution to come in contact with acids and do not heat the solution. Ferricyanides react with concentrated acids to generate a toxic gas. Dispense and use sodium nitroferricyanide in the hood or in a well-ventilated lab only. Avoid exposure of all chemicals to eyes and skin. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Please review current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. All protein and amino acid samples can be flushed down the drain with excess water according to Flinn Suggested Disposal Method #26b. The waste solutions collected in the waste disposal beakers can be flushed down the drain with 20-fold excess water according to Flinn Suggested Disposal Method #26b. Excess sodium nitroferricyanide solution can be disposed of according to Flinn Suggested Disposal Method #14. Excess biuret test solution, sodium hydroxide, and sodium hypochlorite can be disposed of according to Flinn Suggested Disposal Method #10.

Teacher Tips

• Enough materials are provided in this kit for 30 students working in pairs or for 15 groups of students. The experimental work for this lab can reasonably be completed in one 50-minute lab period.
• The classification tests in this study can be performed in any order. To avoid congestion at the materials bench, consider staggering the starting points for different student groups. Set up separate stations for the four different tests in different locations and have the students rotate among the stations to complete the lab activity. Another suggestion—dispense beforehand smaller amounts of all of the solutions needed for each lab table or bench to use separately to prevent possible contamination of protein and amino acid samples.
• The name of the biuret test is frequently confusing to students and teachers alike. The test solution consists of copper sulfate in basic solution. The name of the test actually derives from the name of the reagent chemical, called biuret, that gives a characteristic positive control test with the test solution. Biuret is a derivative of urea that contains two amide (–CONH₂) groups in its structure and thus forms a purple coordination complex with copper ions. The biuret test solution itself does not contain any biuret!
• The xanthoproteic test was the subject of a famous public “Christmas” lecture by Michael Faraday, who demonstrated the reaction of silk and feathers with concentrated nitric acid. Consider performing this demonstration in class before the lab to stimulate student interest in the protein and amino acid classification tests.
• The xanthoproteic test is normally performed with boiling, concentrated nitric acid. We have significantly improved the safety of this test by reducing the concentration of nitric acid to 3 M. The results are very similar, except the color change to yellow is not always accompanied by a precipitate.
• Sodium nitroferricyanide (sodium nitroprusside) is highly toxic by ingestion and inhalation. Do not allow sodium nitroferricyanide solution to come in contact with strong acids. Do not heat the solution. Sodium nitroprusside is a pharmaceutical drug used in the treatment of high blood pressure and congestive heart disease. The nitroprusside test for cysteine is also a clinical test used in medical technology laboratories to test for excessive amounts of cysteine in urine, which can be a symptom of disease.
• In order to make the most efficient use of lab time, the ninhydrin reaction, a popular protein and amino acid test, has not been included in this kit. Ninhydrin is used to identify both amino acids and proteins and gives a positive, purple color test result with ALL of the samples included in this study. The ninhydrin test thus does not provide any additional information concerning the structure and properties of different amino acids and proteins. The test is easy to perform: to 2 mL of protein or amino acid solution in a test tube, add 1 mL of 0.5% ninhydrin solution. Heat the samples to 80–90 °C—a blue-purple color develops within 3–5 minutes. The ninhydrin test is most widely used to identify “spots” in the separation of amino acids or proteins by chromatography. Ninhydrin solution can be prepared in ethyl alcohol, isopropyl alcohol, or butyl alcohol.

Answers to Prelab Questions

1. See figure.
2. See figure.
   {13377_PreLabAnswers_Figure_4}
3. Aspartame will not give a positive biuret test. The biuret reaction requires at least two peptide linkages in the reacting molecule.

Sample Data

Answers to Questions

1. Which samples gave positive results and which gave negative results in the biuret test? Were there any differences in the color and intensity of the solutions that gave positive test results? How general is the biuret test for detecting proteins of different types?

   All of the proteins gave positive test results (lavender or purple solutions) with the biuret test. All of the amino acids examined in this study (arginine, cysteine and tyrosine) gave negative results in the biuret test. Cysteine gave an anomalous, negative result. Most negative tests were the same pale blue as the distilled water blank. Cysteine gave a yellow solution, possibly due to some other reaction with copper ion. The different proteins did give qualitatively different results; the purple color was most intense for albumin and least intense for gelatin. The biuret test is a reliable general method for identifying proteins, since all of the proteins tested gave positive results. In order to be used for quantitative analysis of the amount of protein in a sample, however, the biuret test should first be “standardized” using known amounts of the protein to be analyzed. The intensity of the purple color that develops can then be compared against standard solutions to determine how much protein is present.

2. Which amino acids are identified by means of the xanthoproteic test? Which protein samples gave positive xanthoproteic test results? Comment on the composition of the protein samples based on the results of this test.

   The xanthoproteic test identifies the aromatic amino acid tyrosine. The proteins albumin and casein also gave positive results with this test. The fact that gelatin gave negative test results suggests that gelatin does not contain noticeable amounts of the amino acid tyrosine.

3. Which amino acids are identified by means of the Sakaguchi test? Which protein samples gave positive Sakaguchi test results? Comment on the composition of the protein samples based on the results of this test.

   The Sakaguchi test identifies the basic amino acid arginine. All of the proteins tested (albumin, casein, and gelatin) also gave positive results with this test. This suggests that all of these proteins contain the amino acid arginine in amounts great enough to give a positive Sakaguchi test.

4. Which amino acids are identified by means of the nitroprusside test? Which protein samples gave positive nitroprusside test results? Comment on the composition of the protein samples based on the results of this test.

   The nitroprusside test identifies the sulfur-containing amino acid cysteine. The proteins albumin and casein also gave positive results with this test, as evidenced by the color change from yellow to brown. The fact that gelatin gave negative test results suggests that gelatin does not contain noticeable amounts of the amino acid cysteine.

Introduction

What are the characteristics of protein structure? What are the roles of amino acids in the structure and properties of proteins? Let’s explore the structure and properties of proteins and amino acids and learn how these biological molecules can be identified in the lab.

Concepts

• Protein
• Amino acid
• Peptide linkage
• Biuret test
• Xanthoproteic test

Background

Proteins represent the most diverse class of biological compounds within cells. It is estimated that a single bacteria cell contains more than 3,000 different types of proteins. The word protein is derived from the Greek word “proteios,” meaning first or primary. Proteins are of primary importance in terms of both their occurrence within cells and their function in cell activities. The functions of proteins are at the very center of life itself—proteins catalyze all of our metabolic reactions, carry oxygen to our body tissues, protect the body from infection and maintain cell and tissue structure.

Proteins are composed of amino acid molecules joined together in a chain-like fashion via peptide linkages. Amino acids are thus often referred to as the “building blocks” of protein structure. The size of the amino acid chain in a single protein molecule can vary from around 50 amino acid residues in insulin to more than 500 in hemoglobin and more than 5,000 in some viruses. When fewer than 50 amino acids are joined together the resulting molecules are called polypeptides.

All amino acids have two structural features in common—they contain a carboxylic acid group (–COOH) on one end and an amine group (–NH₂) on the other end. Peptide linkages are created when the carboxyl group of one amino acid reacts with the amino group of the next amino acid in the sequence. As each amino acid is added to the growing polypeptide chain, a molecule of water is formed as a byproduct, as shown in Figure 1.

All proteins are derived from about 20 different, naturally occurring amino acids, which can be arranged in an almost infinite number of ways, giving rise to the thousands of unique proteins found in nature. The primary structure of a protein is determined by the number and identity of amino acids within the protein and the order in which they are joined together via peptide linkages. Higher levels of protein structure (called secondary, tertiary and quaternary structure) result as the polypeptide chains form ribbons, sheets and coils that ultimately fold in on themselves to form more compact and more stable three-dimensional arrangements.

In addition to their reactive amine and carboxylic acid functional groups, amino acids contain a third group of atoms, called the side chain (shown as “R” groups in Figure 1). Although not involved in peptide bond formation, the side chains may contain other functional groups that influence both the structure and function of proteins. Hydrophobic amino acids contain nonpolar, “water-fearing” side chains, such as large hydrocarbon groups. Protein molecules often fold in on themselves so that the hydrophobic amino acids are hidden or tucked away in the interior of the structure. This reduces unfavorable contact between the nonpolar side chains and the polar water molecules that make up the aqueous environment within cells. Amino acids are classified as hydrophilic if they contain polar or “water-loving” side chains that are able to form strong hydrogen bonds to each other. Hydrophilic amino acids are often found at the “active” sites in enzymes and receptors, where they bind to small molecules and catalyze chemical and physical changes. Finally, ionic amino acids contain extra acidic and basic groups in their side chains; at physiological pH these side chains exist in charged, ionic forms. Oppositely charged side chains form so-called “salt bridges” that stabilize the three-dimensional structure of proteins.

Classification Tests
Proteins can be identified using a simple color test based on the reaction of their polypeptide backbones with copper ions in basic solution. When molecules containing two or more peptide linkages react with copper sulfate in the presence of a strong base, a purple complex is formed. This is called the biuret test. The colored product is the result of coordination of peptide nitrogen atoms with copper ions. The amount of product that is formed and thus the intensity of the purple color depend on the nature of the protein and on how much protein is present.

The presence of specific amino acid residues in proteins can be identified using chemical tests that are geared to the reactivity of their different types of side chains. Among the groups that can be identified in this way are the aromatic ring in tyrosine, the basic side chain in arginine and the sulfur-containing side chain in cysteine. The aromatic amino acid tyrosine is identified by means of the xanthoproteic test (Greek for “yellow protein”). Reaction of tyrosine with nitric acid results in nitration of the aromatic ring to give a yellow-colored product. Arginine is identified by means of the Sakaguchi test, which involves reaction with α-naphthol and sodium hypochlorite (bleach) to give a deep red solution. Finally, the presence of the sulfur-containing amino acid cysteine is revealed using the nitroprusside test, which involves reaction with sodium nitroprusside (also called sodium nitroferricyanide) to give a purple or brown product. These amino acids will be identified in this lab activity; their structures are shown in Figure 2.

Materials

Prelab Questions

The popular commercial low-calorie sweetener NutraSweet^® (aspartame) is prepared from the amino acids phenylalanine and aspartic acid. The structure of aspartame is shown.

1. Circle and label the following parts of the structure of aspartame: the peptide linkage, the “terminal” amino group and the hydrophobic amino acid side chain.
2. Which amino acid side chain would be expected to participate in hydrogen bonding?
3. Would you expect aspartame to give a positive biuret test? Why or why not?

Safety Precautions

Biuret solution contains copper sulfate, which is moderately toxic by ingestion, and sodium hydroxide, which is severely corrosive to eye and body tissue. α-Naphthol solution is slightly toxic by ingestion, inhalation and skin absorption and is a body tissue irritant. The solution contains ethyl alcohol and is a flammable liquid. Avoid contact with flames or other sources of ignition. Nitric acid, sodium hydroxide and sodium hypochlorite solutions are corrosive liquids and can cause skin burns. Do not allow sodium hypochlorite (bleach) to come in contact with acids—toxic chlorine gas may be generated. Sodium nitroferricyanide solution is highly toxic by ingestion and inhalation. Do not allow this solution to come in contact with acids and do not heat the solution. Dispense and use sodium nitroferricyanide in the hood or in a well-ventilated lab only. Avoid exposure of all chemicals to eyes and skin. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron.

Procedure

1. Prepare a boiling water bath for use in the xanthoproteic test (steps 8–12). Fill a 400-mL beaker half-full with water and add a boiling stone. Heat the water bath to boiling using a hot plate or a Bunsen burner.

Biuret Test

2. Label a set of seven test tubes 1–7.
3. Use a graduated Beral-type pipet to add 1 mL of each solution to be tested to the appropriate test tube, as follows:
   {13377_Procedure_Table_1}
4. To each test tube add 1 mL of biuret test solution.
5. Observe the color and appearance of each solution and record the results in the data table.
6. Pour 25 mL of 5% sodium hypochlorite solution into a 600-mL beaker to be used for waste disposal. Set the beaker aside for use in steps 7, 12, 16 and 20.
7. Rinse the contents of the test tubes with a large amount of water into the waste disposal beaker. Wash the test tubes and rinse well with distilled water. Relabel them 1–7, if necessary, for use in the next test.

Xanthoproteic Test

8. Repeat step 3 to prepare a set of protein and amino acid samples to be tested.
9. To each test tube add 1 mL of 3 M nitric acid solution.
10. Place the test tubes in the boiling water bath for 3–5 minutes.
11. Use a test tube clamp 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.
12. Rinse the contents of the test tubes with a large amount of water into the waste disposal beaker. Wash the test tubes and rinse well with distilled water. Relabel them 1–7, if necessary, for use in the next test.

Sakaguchi Test

13. Repeat step 3 to prepare a set of protein and amino acid samples to be tested.
14. To each test tube add 3 drops of 3 M sodium hydroxide solution, followed by 5 drops of α-naphthol solution.
15. Add 10 drops of sodium hypochlorite solution (bleach) to each test tube and record the color and appearance of each solution in the data table.
16. Rinse the contents of the test tubes with a large amount of water into the waste disposal beaker. Wash the test tubes and rinse well with distilled water. Relabel them 1–7, if necessary, for use in the next test.

Nitroprusside Test

17. Repeat step 3 to prepare a set of protein and amino acid samples to be tested.
18. To each test tube add 20 drops of 3 M sodium hydroxide, followed by 10 drops of sodium nitroferricyanide solution.
19. Record the color and appearance of each solution in the data table.
20. Rinse the contents of the test tubes with a large amount of water into the waste disposal beaker. Wash and rinse the test tubes.
21. Consult your instructor regarding proper disposal of the solution in the waste beaker.

Student Worksheet PDF

13377_Student1.pdf