Materials Included In Kit

Additional Materials Required

Prelab Preparation

For best results, prepare protein and amino acid solutions within one week of use. Cap the bottles and shake gently to dissolve—vigorous shaking may cause foaming and denature the proteins.

• Albumin, 2%: Dissolve 2 g of albumin in 100 mL of distilled or deionized water.
• Ammonium sulfate, saturated: Dissolve 280 g of ammonium sulfate [(NH₄)₂SO₄] in 400 mL of distilled or deionized water. Stir the mixture for at least one hour to dissolve as much of the solid as possible. Decant or filter the solution and use the clear filtrate in the salting-out procedure.
• Arginine, 1%: Dissolve 1 g of arginine in 100 mL of distilled or deionized water.
• Casein, 2%: Casein is insoluble in water, but soluble in dilute base. Add 2 mL of 3 M NaOH solution to 98 mL of water, followed by 2 g of casein. Shake gently to dissolve.
• Copper(II) sulfate, 0.1 M: Add 2.5 g of copper(II) sulfate pentahydrate (CuSO₄•5H₂O) to 50 mL of distilled or deionized water. Stir to dissolve and then dilute to 100 mL. Mix again before dispensing.
• Cysteine, 1%: Dissolve 1 g of cysteine in 100 mL of distilled or deionized water.
• Gelatin, 2%: Dissolve 2 g of gelatin in 100 mL of distilled or deionized water.
• Hydrochloric acid, 3 M: Carefully add 25 mL of concentrated hydrochloric acid (12 M) to 60 mL of distilled or deionized water. Stir to mix, then dilute to 100 mL. Note: Always add acid to water!
• α-Naphthol, 0.1%: Dissolve 0.05 g of α-naphthol in 50 mL of ethyl alcohol (95%).
• Nitric acid, 3 M: Carefully add 29 mL of concentrated nitric acid (15.8 M) to 100 mL of distilled or deionized water. Stir to mix, then dilute to 150 mL. Note: Always add acid to water!
• Silver nitrate, 0.1 M: Add 0.85 g of silver nitrate (AgNO₃) to about 30 mL of distilled or deionized water. Stir to dissolve, and then dilute to 50 mL. Mix again before dispensing.
• Sodium hydroxide, 3 M: Cool 100 mL of distilled or deionized water in an ice-water bath. Add 24 g of sodium hydroxide pellets and stir to dissolve. Bring to room temperature and dilute to 200 mL with water.
• Sodium nitroferricyanide, 2%: Dissolve 2 g of sodium nitroferricyanide dihydrate [Na₂Fe(CN)₅NO•2H₂O] in 100 mL of distilled or deionized water.
• Tyrosine, 1%: Dissolve 1 g of tyrosine in 100 mL of distilled or deionized water.

Safety Precautions

Biuret solution contains copper sulfate, which is moderately toxic by ingestion, and sodium hydroxide, which is corrosive to eye and body tissue. α-Naphthol is slightly toxic by ingestion, inhalation and skin absorption and is a body tissue irritant. α-Naphthol solution contains ethyl alcohol and is a flammable liquid—avoid contact with flames or other sources of ignition. Hydrochloric acid, 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 allow this solution to come in contact with acids. Do not heat the solution. Dispense and use sodium nitroferricyanide in the hood or in a wellventilated lab only. Avoid exposure of all chemicals to eyes and skin. Wear chemical splash goggles, chemical-resistant gloves and a lab coat or chemical-resistant apron. A solution of sodium hypochlorite may be used for oxidizing sodium nitroferricyanide test solutions in Part A. Sodium hypochlorite and sodium nitroferricyanide will generate toxic gases upon reaction with concentrated strong acids. Please consult current Safety Data Sheets for additional safety, handling and disposal information. Remind students to wash their hands thoroughly with soap and water before leaving the laboratory.

Disposal

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. Protein and amino acid solutions may be rinsed down the drain with excess water according to Flinn Suggested Disposal Method #26b. To avoid potential side reactions due to mixing incompatible chemicals in waste containers, test mixtures in Part A require segregation, neutralization or oxidation prior to waste collection and disposal. Sodium nitroferricyanide generates a toxic gas in contact with concentrated strong acids and is a characteristic hazardous waste. This may be prevented by oxidizing test mixtures containing sodium nitroferricyanide with bleach (sodium hypochlorite solution) as part of the laboratory procedure. Oxidation of sodium nitroferricyanide converts it to cyanate salts, which are generally considered nonhazardous. The pH of the sodium hypochlorite solution in the “waste beaker” must be kept basic (pH >10) during this process to avoid generating chlorine gas. This is accomplished in Part A by first adding the biuret test mixtures to the waste beaker—the pH of biuret test solution is 14 (it is extremely corrosive).

Lab Hints

• The experimental work for this lab can reasonably be completed within a typical 2-hour lab period.
• The classification tests in Part A 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 students rotate among the stations to complete the tests. Alternatively: Dispense smaller amounts 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 may be confusing to students and teachers. 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. The chemical called biuret is a derivative of urea that contains two amide (–CONH₂) groups in its structure and forms a purple coordination complex with copper ions. Biuret test solution itself does not contain any biuret!
• The xanthoproteic test was the subject of a famous public “Christmas” lecture in the 19th century by Michael Faraday, who demonstrated the reaction of silk and feathers with concentrated nitric acid. Consider performing this demonstration in class before the lab.
• 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 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 used as a pharmaceutical drug for 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.
• The ninhydrin reaction, a popular protein and amino acid test, has not been included in this activity. Ninhydrin is used to identify both amino acids and proteins and will give a positive test result with all of the samples included in this study. The ninhydrin test is most widely used to identify amino acids by chromatography.
• Albumin is the chief protein in egg white. It serves as a source of amino acids for the developing embryo. Casein is the principal protein in milk. It has a high concentration of phosphate groups attached to its amino acid residues and is also associated with the high calcium content in milk. Casein is readily precipitated from milk with dilute acid—it has its minimum solubility at a pH of 4.7. Gelatin is a mixture of proteins obtained by hydrolysis of collagen from animal skin, ligaments and tendons. Because of the way it is prepared, gelatin consists of shorter molecular weight protein fragments that are relatively insensitive to denaturation by acids.
• The effect of HCl on protein solubility and denaturation provides an excellent opportunity to reinforce safety rules concerning the dangerous corrosive effect of strong acids, especially on proteins in the eye.
• An alternative way to study the heat denaturation of proteins is to set up several different temperature baths in the classroom and have students measure the time it takes for albumin to coagulate and precipitate at different temperatures. Three hot water baths, at 40, 60 and 80 °C, should be enough to gather interesting data for students to compare. This experiment can also be extended to examine the “denaturation temperature” of different proteins, which vary in their sensitivity to heat. Albumin is one of the most heat-sensitive proteins. This could lead to a discussion of heat-resistant proteins in bacteria that thrive in hot springs.
• The “Protein Data Bank” (www.rcsb.org/pdb/) is a searchable database maintained by a collaboration of academic and government scientists. It offers access to thousands of protein structures.

Answers to Prelab Questions

1. The artificial sweetener aspartame is a dipeptide. Circle and label the following groups in the structure of aspartame: the peptide linkage, the “terminal” amino group and a hydrophobic amino acid side chain.
   {14049_PreLabAnswers_Figure_5}
2. Which amino acid side chain in aspartame would be expected to participate in hydrogen bonding?

   The carboxylic acid (COOH) side chain will form hydrogen bonds to other electronegative atoms or groups.

3. Would you expect aspartame to give a positive biuret test? Explain.

   Aspartame will not give a positive biuret test. The biuret reaction requires at least two peptide linkages in the reacting molecule.

4. Define the term denaturation. What is the most common, visible change that indicates denaturation has occurred?

   Denaturation refers to the loss of biological activity that occurs when the three-dimensional structure of a protein is disrupted due to physical conditions or chemical treatment. Denaturation is usually identified when the protein precipitates out of solution.

5. Isopropyl alcohol is sold in drugstores as “rubbing alcohol,” a disinfectant. What effect might alcohols have on the structure of bacterial proteins?

   Alcohols disrupt hydrogen bonding in proteins and may denature them. When essential membrane proteins in bacteria are denatured, bacteria are killed because they lack proteins needed to stay alive.

6. Why is heat an effective form of sterilization for biological materials and equipment?

   Heat denatures protein structures and destroys their activity. Destroying the proteins in microorganisms kills them and sterilizes the equipment.

Sample Data

Laboratory Report

Part A. Classification Tests

Part B. Protein Denaturation and Salting-Out Effect of Strong Acid Effect of Inorganic and Organic Additives (Albumin) Effect of Heat (Albumin) Salting-Out with Ammonium Sulfate

Answers to Questions

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

   All the proteins gave positive test results (lavender or purple solutions) with the biuret test. All 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. Different proteins gave 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.

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 the proteins tested (albumin, casein, and gelatin) 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.

5. Compare and contrast the effect of strong acid (HCl) on albumin, casein and gelatin. Which protein was most sensitive to the action of strong acid? Least sensitive?

   Adding HCl to albumin and casein led to rapid coagulation and protein precipitation. Casein was the most sensitive to acid—only a few drops of HCl caused the casein to settle out of solution. Gelatin was the least affected by acid treatment; in fact, gelatin did not precipitate even after 20 drops of HCl had been added.

6. Which metal salts (CuSO₄ and AgNO₃) caused albumin denaturation? How does this observation relate to the toxicity of silver salts versus copper salts?

   AgNO₃ denatured albumin (a white precipitate was observed), whereas CuSO₄ did not affect the solubility. This may be related to the biological role of these metal ions. Although copper salts are slightly toxic, copper(II) ions are important enzyme cofactors and play an essential role in metabolism. Silver ion is toxic. The toxicity of heavy metal salts, such as Hg, Pb and Ag, is generally attributed to irreversible denaturation of proteins.

7. You have just been to the doctor’s office to receive an inoculation. Before administering the injection, the doctor wipes the area with an alcohol swab. Do the results for albumin denaturation support the use of isopropyl alcohol as a disinfectant? Explain. Alcohol is an effective disinfectant when applied to the skin.

   Alcohol denatures essential proteins in bacteria and kills them. Adding isopropyl alcohol to albumin caused the protein to denature and precipitate out of solution.

8. The biuret test is used to identify proteins. Compare the results obtained in the biuret test with albumin and the filtrate after the salting-out procedure in Part B. How effective is the “salting-out” procedure with ammonium sulfate?

   When biuret solution was added to albumin, a purple color was observed. This serves as a positive color test to identify the protein. The filtrate did not give a positive color test—it remained pale blue, the original color of the copper sulfate. This indicates that the filtrate did not contain any residual albumin and that all of the protein was “salted-out” by the addition of ammonium sulfate.

9. Is denaturation of albumin by ammonium sulfate reversible or irreversible? Explain on the basis of your observations for the biuret test with albumin and the redissolved precipitate, respectively.

   Denaturation of albumin by ammonium sulfate is reversible. This was demonstrated by two observations: the insoluble, denatured protein easily redissolved when water was added and the resulting protein solution gave a positive biuret test.

Introduction

What are the roles of amino acids in the structure and properties of proteins? Investigate the properties of proteins and amino acids and learn how these biological molecules can be identified. The effects of chemicals and environmental factors on the physical properties of proteins can help us understand their structures and how proteins fulfill their vital biological functions.

Concepts

• Proteins
• Amino acids
• Peptide linkage
• Biuret test
• Xanthoproteic test
• Protein folding
• Denaturation
• Salting-out

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 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 center of life itself—proteins catalyze our metabolic reactions, carry oxygen to our body tissues, protect the body from infection and maintain cell structure.

Proteins are composed of amino acid molecules joined together in chain-like fashion via peptide linkages. Amino acids are thus often referred to as the “building blocks” of protein structure. The number of amino acids in a single protein 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 compounds are called polypeptides.

All amino acids have two structural groups in common—a carboxylic acid group (–COOH) on one end and an amino 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 made from about 20 different, naturally occurring amino acids, which can be arranged in an almost infinite number of ways. The primary structure of a protein is determined by the number and identity of amino acids and the order in which they are joined together. Higher levels of protein structure (called secondary, tertiary and quaternary structures) result as the polypeptide chains form ribbons, sheets and coils that then fold in on themselves to form more stable three-dimensional arrangements.

In addition to their reactive amino 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 side chains, such as large hydrocarbon groups. Protein molecules often fold in on themselves so that the hydrophobic amino acids are tucked away in the interior of the structure. This reduces unfavorable contact between the nonpolar side chains and polar water molecules within cells. Amino acids are classified as hydrophilic if they contain polar side chains that are able to form hydrogen bonds. Hydrophilic amino acids are often found at the “active” sites in enzymes, where they bind to small molecules and catalyze chemical reactions. 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 for Proteins and Amino Acids
Proteins can be identified using a simple color test based on the reaction of their polypeptide backbones with copper ions in basic solution. Compounds containing two or more peptide linkages react with copper sulfate to form a purple complex. This is called the biuret test. The purple product is due to coordination of peptide nitrogen atoms with copper ions. The amount of product that is formed and the intensity of the purple color depend on the nature of the protein and how much protein is present.

Specific amino acid residues in proteins can also be identified using chemical tests based on reactions of their different side chains. The amino acids that will be studied in this lab include tyrosine, an aromatic amino acid; arginine, which has a basic side chain; and cysteine, which has a sulfur-containing side chain (see Figure 2).

• 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 product.
• Arginine is identified by means of the Sakaguchi test, which involves reaction with α-naphthol and sodium hypochlorite to give a red solution.
• Cysteine can be identified by reaction with sodium nitroprusside (also called sodium nitroferricyanide) to give a purple or brown product.

Structure and Function
The relationship between structure and function is a key theme in biochemistry and is especially important in the properties of proteins. For example, the structure of hemoglobin allows it to bind and deliver oxygen to body tissues. The structure of a specific antibody protein allows it to recognize, bind and destroy a potentially harmful foreign substance. The structure of collagen makes skin both elastic and strong.

The biological activity of a protein depends on its three-dimensional shape. All proteins have a common structural “backbone” of peptide linkages connecting their amino acid building blocks. The side chains in the amino acid residues can be large or small, polar or nonpolar, acidic or basic, positively or negatively charged, and they can interact through a variety of forces. These forces include hydrogen bonding involving –OH groups, dipole interactions among polar amino acids, ionic bonds between positively and negatively charged side chains, and hydrophobic effects that stabilize large, nonpolar side chains. Intramolecular forces cause protein chains to twist and fold back on themselves into characteristic shapes. The forces that maintain the structure of proteins are illustrated schematically in Figure 3 for different types of amino acid side chains.

Protein folding is the name given to the process by which proteins naturally coil around or fold in on themselves in order to form stable, three-dimensional structures. Since every protein has a unique sequence of amino acids, every protein also has a unique shape—called its native structure—that makes the protein both stable and functional.

Denaturation
Any factor that disrupts the native structure of a protein will destroy its function. Destruction of the three-dimensional shape of a protein by physical or chemical means is called denaturation. Proteins become denatured by any action that breaks hydrogen bonds, destroys salt bridges, or interferes with hydrophobic interactions. Denaturation causes protein molecules to clump together and precipitate out of solution; the resulting loss of biological activity is often irreversible. Heating, freezing and agitation are physical processes that result in protein denaturation. Chemical agents that cause protein denaturation include strong acids and bases, organic solvents and heavy metal salts.

Most proteins are denatured by temperatures above 50 °C (normal body temperature is 37 °C). Cooking an egg provides an everyday example of the changes that occur when a protein solution—the egg white—is heated. Heat supplies excess energy that disrupts intramolecular forces in proteins. Strong acids or bases affect the charges on amino acid side chains and interfere with ionic “salt bridge” formation in proteins. Proteins have an optimal pH range where they are most soluble and most active. Small pH changes around the optimum pH may reduce the solubility of a protein, but are usually reversible.

High concentrations of strong acid or strong base, on the other hand, will precipitate proteins and lead to total loss of protein structure and function—irreversible denaturation. Proteins can also be denatured by the addition of polar organic solvents that interfere with hydrogen bonding, such as alcohols and acetone. High concentrations of inorganic salts, such as ammonium sulfate, are used to precipitate proteins without loss of protein activity. The solubility of a protein decreases as the concentration of ionic compounds increases, and the protein eventually precipitates out. This process—called salting-out—results from changes in hydrogen bonding between water molecules and the protein. Because salting out involves mild conditions, the process is generally reversible. Salting-out is used as a means of isolating and purifying proteins.

Materials

Prelab Questions

1. The artificial sweetener aspartame is a dipeptide. Circle and label the following groups in the structure of aspartame: the peptide linkage, the “terminal” amino group, and a hydrophobic amino acid side chain.
   {14049_PreLab_Figure_4}
2. Which amino acid side chain in aspartame would be expected to participate in hydrogen bonding?
3. Would you expect aspartame to give a positive biuret test? Explain.
4. Define the term denaturation. What is the most common, visible change that indicates denaturation has occurred?
5. Isopropyl alcohol is sold in drugstores as “rubbing alcohol,” a disinfectant. What effect might alcohols have on the structure of bacterial proteins?
6. Why is heat an effective form of sterilization for biological materials and equipment?

Safety Precautions

Biuret test solution contains copper sulfate, which is moderately toxic by ingestion and sodium hydroxide, which is corrosive to eye and body tissue. α-Naphthol is slightly toxic by ingestion, inhalation and skin absorption and is a body tissue irritant. α-Naphthol solution contains ethyl alcohol and is a flammable liquid—avoid contact with flames or other sources of ignition. Hydrochloric acid, 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 allow this solution to come in contact with acids. 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. Follow instructor guidelines for disposing of test solutions. A solution of sodium hypochlorite may be used for oxidizing sodium nitroferricyanide solutions in Part A. Sodium hypochlorite and sodium nitroferricyanide will generate toxic gases upon reaction with concentrated strong acids. Wear chemical splash goggles, chemical-resistant gloves and a lab coat or chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the lab.

Procedure

Prepare a boiling water bath for use in the xanthoproteic test and protein denaturation. Fill a 400-mL beaker half-full with water, add a boiling stone and heat to boiling on a hot plate at a medium setting.

Part A. Classification Tests

Biuret Test

1. Label a set of seven test tubes 1–7.
2. Use a graduated Beral-type pipet to add 1 mL of each solution to be tested to the appropriate test tube, as follows:
   {14049_Procedure_Table_1}
3. Add 1 mL of biuret test solution to each test tube.
4. Observe and record the color and appearance of each mixture.
5. Pour 25 mL of 5% sodium hypochlorite solution into a 600-mL beaker. Place this “waste beaker” in an operating fume hood for use in steps 6, 12, 17 and 21.
6. Rinse the contents of the test tubes with a large amount of water into the waste 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

7. Repeat step 2 to prepare a set of protein and amino acid samples to be tested.
8. Add 1 mL of 3 M nitric acid to each test tube.
9. Place the test tubes in the boiling water bath for 3–5 minutes.
10. Use a test tube clamp to remove the test tubes from the boiling water bath. Allow the solutions to cool and record observations.
11. Turn off the hot plate until the water bath will be used again in Part B.
12. Rinse the contents of the test tubes with a large amount of water into the waste beaker containing sodium hypochlorite and excess sodium hydroxide from steps 5 and 6.
13. Wash the test tubes and rinse well with distilled water. Relabel them 1–7, if necessary, for use in the next test.

Sakaguchi Test

14. Repeat step 2 to prepare a set of protein and amino acid samples to be tested.
15. Add 3 drops of 3 M sodium hydroxide, followed by 5 drops of α-naphthol solution to each test tube.
16. Add 10 drops of sodium hypochlorite to each test tube and record the color and appearance of each solution.
17. Rinse the contents of the test tubes with a large amount of water into the waste 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

18. Repeat step 2 to prepare a set of protein and amino acid samples to be tested.
19. Add 20 drops of 3 M sodium hydroxide, followed by 10 drops of sodium nitroferricyanide solution to each test tube.
20. Record the color and appearance of each solution.
21. Rinse the contents of the test tubes with a large amount of water into the waste beaker. Wash and rinse the test tubes.
22. Consult your instructor regarding proper disposal of the contents of the waste beaker.

Part B. Protein Denaturation and Salting-Out

1. Label three small test tubes 1–3.
2. Using a clean, graduated Beral-type pipet for each solution, add 1 mL of albumin, casein and gelatin to test tubes 1, 2 and 3, respectively. Record the initial appearance of each solution.
3. Add 2 drops of 3 M hydrochloric acid to each test tube. Gently swirl to mix the contents and record the appearance of the solutions.
4. Add 5 more drops of 3 M hydrochloric acid to each test tube. Swirl each sample mixture and record its appearance.
5. Add 10 more drops of 3 M hydrochloric acid to each test tube. Swirl each mixture and record observations.
6. Rinse the contents of the test tubes into a beaker for neutralization as directed by the instructor. Wash the test tubes with distilled water and relabel the test tubes 1–3, if necessary.
7. Add 1 mL of 2% albumin solution to each test tube.
8. Using a clean, graduated Beral-type pipet for each reagent, add 2 mL of 0.1 M copper(II) sulfate to test tube 1, 2 mL of 0.1 M silver nitrate to test tube 2, and 2 mL of isopropyl alcohol to test tube 3. Gently swirl each tube to mix the contents and record observations.
9. Wash and rinse the test tubes with distilled water.

Effect of Heat

10. Place a thermometer in the water bath used in Part A. Check the temperature and allow to cool to at least 40 °C.
11. Add 5 mL of 2% albumin solution to a medium-sized test tube.
12. When the temperature of the water bath is 35–40 °C, place the test tube containing 2% albumin in the bath. Adjust the heat setting on the hot plate to a medium-high range.
13. Holding the test tube with a test tube clamp, gently swirl the protein solution and observe its appearance. Note the temperature of the bath when the first signs of protein precipitation are observed. Record the temperature and additional observations.
14. Continue heating the protein solution. Observe and record the water temperature when the protein solution appears milky white (opaque).
15. When the temperature of the hot water bath reaches 85–90 °C, remove the test tube. Record the final appearance of the protein sample.
16. Dispose of the test tube contents as instructed.

Salting-Out

17. Add 10 mL of 2% albumin to a 50-mL beaker, followed by approximately 25 mL of saturated ammonium sulfate solution. Stir thoroughly using a glass stirring rod and observe the appearance of the mixture.
18. Filter the resulting mixture using gravity filtration. Collect the liquid (filtrate) in a clean Erlenmeyer flask.
19. Relabel three clean test tubes 1–3.
    • Add 2 mL of the original 2% albumin solution to test tube 1.
    • Add 2 mL of the filtrate to test tube 2.
    • Remove a small portion of the precipitate from the funnel with the tip of a spatula and dissolve the wet solid in 2 mL of distilled water in test tube 3.
20. Add 1 mL of biuret testing solution to each test tube 1–3. Compare the results and record observations

Student Worksheet PDF

14049_Student1.pdf