Materials Included In Kit

Additional Materials Required

Prelab Preparation

1. Protein solutions should be freshly prepared. For best results, prepare the solutions no more than one week prior to lab. The correct amount of protein has been provided in each sample bottle to prepare the concentrations and sample volumes required for a class of 30 students working in pairs. Cap the samples and shake them gently to dissolve. More extreme shaking or agitation of the protein solutions may cause the proteins to denature and precipitate out of solution.
2. To prepare 2% albumin: Add 400 mL distilled or deionized water directly to the sample bottle.
3. To prepare 2% gelatin: Add 50 mL distilled or deionized water directly to the sample bottle.
4. To prepare 2% casein: Add 49 mL of distilled or deionized water, followed by 1 mL of 2.5 M NaOH directly to the sample bottle. The protein casein is insoluble in water, but soluble in dilute base.

Safety Precautions

Hydrochloric acid and sodium hydroxide solutions are highly corrosive liquids and can cause skin burns. Silver nitrate solution is a corrosive liquid and toxic by ingestion; it will stain skin and clothes. Isopropyl alcohol is a flammable organic solvent; do not use near flames or other sources of ignition. Ammonium sulfate and copper sulfate solutions are slightly toxic by ingestion. 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 solutions and test mixtures from Parts A, B, and C can be flushed down the drain with excess water according to Flinn Suggested Disposal Method #26b.

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 experiments in this activity can be performed in any order. To avoid congestion, consider staggering the starting points for different student groups. Another suggestion—dispense beforehand smaller amounts of all of the solutions for each lab table or bench to use separately. Encourage students to label their Beral-type pipets to prevent contamination and waste. The kit includes nine pipets for each lab pair, enough for a separate, clean pipet for each chemical.
• The experiments in this kit are complementary to those described in a second Student Laboratory Kit from Flinn Scientific. In AP1769, Identifying Proteins and Amino Acids, students perform qualitative tests to analyze proteins and identify specific amino acids in different proteins. They learn about the chemical structures of amino acids and proteins.
• Albumin is the chief protein occurring 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 molecules that are relatively insensitive to denaturation by acids.
• The effect of 2.5 M HCl on protein solubility and denaturation provides an excellent opportunity to reinforce safety rules concerning the corrosive nature of strong acids. Consider leading into this lab with a safety demonstration that shows how strong acid affects cow-eye tissue. Call or write us at Flinn Scientific and we will fax you a free handout of our Publication No. 10062, Cow Eye Demonstration. Your students will never again forget to wear their safety goggles!
• The reaction of copper sulfate with proteins in the presence of sodium hydroxide (Part B) is called the biuret test and is the most general color test to identify the presence of proteins. Copper ions coordinate with the amide functional groups in the polypeptide backbone to form highly colored, purple complex ions.
• 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 an interesting discussion of heat-resistant proteins that are present in unusual bacteria, such as those that thrive in hot springs.

Answers to Prelab Questions

1. 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.

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

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

Sample Data

Data Table A. Solubility and Protein Denaturation

Data Table B. “Salting Out” with Ammonium Sulfate Data Table C. Effect of Heat

Answers to Questions

1. Compare and contrast the effect of strong acid (2.5 M HCl) on albumin, casein and gelatin. Which protein was most sensitive to the action of strong acid? Least sensitive?

   Adding 2.5 M 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 more than 25 drops of HCl had been added.

2. Do strong acid and strong base have similar effects on protein solubility and denaturation? Explain.

   No, strong acid and strong base do not have the same effect on protein solubility. Protein denaturation was not observed even after 15 drops of 2.5 M NaOH were added. Actually, addition of base seemed to increase the solubility of proteins, since the initial cloudy protein solutions turned clear.

3. Which metal salts (CuSO₄ and AgNO₃) caused albumin denaturation? Relate this observation to the fact that silver salts are more toxic than 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 highly toxic (although it does not pose as harmful an environmental hazard as mercury and lead). The toxicity of heavy metal salts is generally attributed to irreversible denaturation of proteins.

4. 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 your results for the effect of alcohol on 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 the bacteria. In our experiment, adding isopropyl alcohol to albumin caused the protein to denature and precipitate out of solution.

5. The reaction of CuSO₄ with proteins in strong base is used as a color test to identify proteins. What do the results obtained in Part B for the reaction of CuSO₄ and NaOH with albumin and the filtrate tell you about the effectiveness of the “salting out” procedure with ammonium sulfate?

   When copper sulfate was added to albumin (test tube 1) in the presence of sodium hydroxide, a purple color was observed. This serves as a positive color test to identify the protein. The filtrate (test tube 2) did not give a positive color test—it remained pale blue, the original color of copper sulfate by itself. This indicates that the filtrate did not contain any residual albumin, that in fact all of the protein was “salted out” by the addition of ammonium sulfate.

6. Is the denaturation of albumin by ammonium sulfate reversible or irreversible? Explain on the basis of your observations for the reaction of CuSO₄ with albumin (test tube 1) and the redissolved precipitate (test tube 3), respectively, in Part B.

   Denaturation of albumin by ammonium sulfate is reversible. This is demonstrated by two observations: the insoluble, denatured protein easily redissolved when water was added and the resulting protein solution (test tube 3) gave a positive test with copper sulfate and sodium hydroxide, just like the albumin control (test tube 1).

7. Based on the results of Part C, suggest a reason why heat is an effective form of sterilization for biological materials and equipment.

   Heat is an effective form of sterilization because it destroys essential bacteria in many microbial life forms, including fungi, bacteria and viruses.

Introduction

The effects of acids and bases, inorganic salts, organic solvents and temperature on the physical properties of proteins can help us understand the structures of proteins and how they fulfill their vital biological functions.

Concepts

• Protein folding
• Native structure
• Denaturation
• Salting out

Background

Structure often relates to function—nowhere is this relationship more evident than in the description of the structures, physical properties and biological roles of proteins. The structure of hemoglobin allows it to bind to oxygen 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”— amino acid building blocks joined by chemical bonds called peptide linkages. There are more than 20 different, naturally occurring amino acids; they differ in the types of atoms that are attached to the main polypeptide backbone. These amino acid side chains—which can be large or small, polar or nonpolar, acidic or basic, positively or negatively charged—interact through a variety of forces. These forces include hydrogen bonding involving side chain –OH groups, dipole interactions among polar amino acids, ionic “salt bridges” between positively and negatively charged side chains and hydrophobic effects that stabilize large, nonpolar side chains. All of these forces cause protein chains to twist and fold back on themselves into globular or spherical shapes.

Protein folding is the name given to the process by which proteins naturally coil around or fold in on themselves in order to form a stable three-dimensional structure. 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.

The forces that maintain the structure of proteins are illustrated schematically in Figure 1.

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 which 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 generally 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 and destabilizes all of the major forces that hold a protein together. Addition of strong acids or bases affects the number of charges on amino acid side chains and interferes with ionic “salt bridge” formation in proteins. Strong acids increase the concentration of H+ ions in solution, which neutralize negatively charged side chains. Strong bases increase the concentration of OH– ions that in turn neutralize positively charged side chains. Proteins have an optimal pH range at which they are most stable, most soluble, and most active. Small pH changes around the optimum pH may reduce the solubility of a protein, but these changes are usually reversible. High concentrations of strong acid and strong base, on the other hand, coagulate 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, such as alcohols and acetone, that interfere with hydrogen bonding. The poisonous nature of heavy metal salts containing Ag⁺, Hg²⁺ and Pb²⁺ ions is due to protein denaturation as well.

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 completely. 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 and thus ideally suited as a means of isolating proteins and purifying them to remove contaminants.

Materials

Prelab Questions

1. Define the term denaturation. What is the most common, visible change that indicates denaturation has occurred?
2. Isopropyl alcohol is sold in drugstores as “rubbing alcohol,” a disinfectant. What effect might alcohols have on bacterial proteins?

Safety Precautions

Hydrochloric acid and sodium hydroxide solutions are highly corrosive liquids and can cause skin burns. Silver nitrate solution is a corrosive liquid and toxic by ingestion; it will stain skin and clothes. Isopropyl alcohol is a flammable organic solvent; do not use near flames or other sources of ignition. Ammonium sulfate and copper sulfate solutions are slightly toxic by ingestion. Avoid exposure of all chemicals to eyes and skin. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory.

Procedure

Part A. Solubility and Protein Denaturation

1. Label three small test tubes 1–3.
2. Using a clean, graduated Beral-type pipet for each solution, add approximately 1 mL of albumin, casein and gelatin to test tubes 1, 2 and 3, respectively. Record the initial appearance of each solution in Data Table A.
3. Add 2 drops of 2.5 M HCl to each test tube 1–3. Gently swirl each tube to mix the contents, then record the appearance of the solutions in Data Table A.
4. Add 5 more drops of 2.5 M HCl to each test tube 1–3. Swirl each sample mixture and record its appearance in Data Table A.
5. Add 10 more drops of 2.5 M HCl to each test tube 1–3. Swirl each sample mixture and record its appearance in Data Table A.
6. Add 10 more drops of 2.5 M HCl to each test tube 1–3. Swirl each sample mixture and record its appearance in Data Table A.
7. Wash the contents of each test tube down the drain with excess water and rinse the test tubes twice with distilled water from a wash bottle. Relabel the test tubes 1–3, if necessary.
8. Using the appropriate graduated Beral-type pipet for each solution, add approximately 1 mL of albumin, casein and gelatin to test tubes 1, 2 and 3, respectively.
9. Add 5 drops of 2.5 M NaOH to each test tube 1–3. Gently swirl each tube to mix the contents and record the appearance of the solutions in Data Table 1.
10. Add 10 more drops of 2.5 M NaOH to each test tube 1–3. Swirl each sample mixture and record its appearance in Data Table A.
11. Wash the test tube contents down the drain with excess water and rinse the test tubes twice with distilled water. Relabel the test tubes 1–3, if necessary.
12. Add 1 mL of 2% albumin solution to each tube.
13. Using a clean, graduated Beral-type pipet for each reagent, add 2 mL of 0.1 M CuSO₄ to test tube 1, 2 mL of 0.1 M AgNO₃ to test tube 2 and 2 mL of isopropyl alcohol to test tube 3. Swirl each tube gently to mix the contents, then record the appearance of each sample in Data Table A.

Part B. “Salting Out” with Ammonium Sulfate

14. Add 10 mL of 2% albumin to a 50-mL beaker, followed by approximately 25 mL of saturated ammonium sulfate solution. Stir the mixture thoroughly using a glass stirring rod. Describe the appearance of the mixture in Data Table B.
15. Set up a gravity filtration apparatus and filter the mixture through a piece of wetted filter paper. Collect the liquid (filtrate) in a clean Erlenmeyer flask.
16. Label three small test tubes 1–3.
    • Add 2 mL of 2% albumin solution to test tube 1.
    • Add 2 mL of the filtrate from step 15 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.
17. To each test tube 1–3, add 10 drops of 2.5 M NaOH followed by 5 drops of 0.1 M CuSO₄ solution. Compare the appearance of the three solutions and record the observations in Data Table B.

Part C. Effect of Heat

18. Prepare a hot water bath: Fill a 250-mL beaker half-full with water and heat it on a hot plate at the lowest setting. Place a thermometer in the water bath to record the temperature of the bath.
19. To a medium size test tube, add 5 mL of 2% albumin solution.
20. When the temperature of the hot water bath is 35–40 °C, place the test tube in the bath. Record the initial temperature of the water bath in Data Table C. Adjust the heat setting on the hot plate to a medium-high range to slowly heat the protein solution.
21. 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 make observations in Data Table C.
22. Continue heating the protein solution. Record the temperature of the hot water bath and make observations of the protein solution when it first appears milky white (opaque).
23. When the temperature of the hot water bath reaches 85–90 °C, remove the test tube. Record the final appearance of the protein sample in Data Table C.
24. Consult your instructor for appropriate disposal procedures.

Student Worksheet PDF

13375_Student1.pdf