PHYSICAL PROPERTIES OF PROTEINS
Purpose:
The purpose of this lab is to study the effects of acids and bases, inorganic salts, organic
solvents, and temperature on the physical properties of proteins.
Discussion:
The relationship between the structure of a molecule and its function is best exemplified
in proteins. The biological function of a protein depends on its three-dimensional shape.
Discuss the answers to the following questions:
1. What are the differences between the different levels of protein structure?
2. What forces hold proteins in their native structure?
3. Describe denaturation.
4. Describe the “salting out” of a protein.
What are the expected results of the following tests on solutions of albumin, casein and
gelatin?
1. Adding the strong acid hydrochloric acid.
2. Adding inorganic salts copper (II) sulfate and silver nitrate.
3. Adding the organic solvent isopropyl alcohol.
4. Adding a saturated solution of ammonium sulfate.
5. Heating the solution.
Objectives:
In this lab, you will analyze the results of adding …
1. acids and bases,
2. inorganic salts,
3. organic solvents,
4. and heat to proteins.
Materials:
Beakers, 50 mL and 250 mL
Plastic pipets
Erlenmeyer flask, 125 mL
Glass filter funnel
Filter paper, 12.5 cm
Hot plate
Glass stirring rod
Test tubes, 3 small
Test tube, 1 large
Test tube holder
Test tube rack
Thermometer
Spatula, small metal
Chemicals:
Protein solutions
Albumin, 2%
Casein, 2%
Gelatin, 2%
(NH4)2SO4 (aq), sat’d
CuSO4 (aq), 0.1 M
HCl (aq), 3 M
Isopropyl alcohol, (CH3)2CHOH (l)
AgNO3 (aq), 0.1 M
NaOH (aq), 3 M
Water, distilled
Safety:
 Hydrochloric acid and sodium hydroxide solutions are corrosive and can cause skin
burns.
 Silver nitrate solution is corrosive and toxic, and will stain skin and clothes.
 Isopropyl alcohol is flammable.
 Ammonium sulfate and copper (II) sulfate solutions are slightly toxic.
 Wear goggles!!!
Procedure:
Part A: Solubility and Protein Denaturation
1. Label three small test tubes 1-3.
2. Add ~1 mL of albumin, casein, and gelatin to test tubes 1, 2 and 3, respectively. Record
the initial appearance of each in Data Table A.
3. Add 2 drops of 3M 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. Repeat step 3 with 5 more drops of 3 M HCl.
5. Repeat step 3 with 10 more drops of 3 M HCl.
6. Repeat step 5.
7. Wash the contents of each test tube down the drain with excess water and clean the test
tubes. Relabel the test tubes 1-3, if necessary.
8. Add ~1 mL of albumin, casein, and gelatin to test tubes 1, 2 and 3, respectively.
9. Add 5 drops of 3 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 A.
10. Repeat step 9 with 10 more drops of 3 M NaOH.
11. Wash the contents of each test tube down the drain with excess water and clean the test
tubes. Relabel the test tubes 1-3, if necessary.
12. Add 1 mL of 2% albumin solution to each test tube.
13. Add 2 mL of 0.1 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. Gently swirl each test tube to mix the contents and
record the appearance of the solutions in Data Table A.
14. Wash the contents of each test tube down the drain with excess water and clean the test
tubes.
Part B: “Salting Out” with Ammonium Sulfate
15. Add 10 mL of 2 % albumin to a 50 mL beaker, followed by ~25 mL of saturated
(NH4)2SO4 solution. Stir the mixture thoroughly using a glass stirring rod. Describe the
appearance of the mixture in Data Table B.
16. Set up a gravity filtration apparatus and filter the mixture through a piece of dampened
filter paper. Collect the filtrate in a clean 125 mL Erlenmeyer flask.
17. Label three small test tubes 1-3.
a. Add 2 mL of the original 2% albumin solution to test tube 1.
b. Add 2 mL of the filtrate from step 16 to test tube 2.
c. 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.
18. To each test tube 1-3, add 10 drops of 3 M NaOH followed by 5 drops of 0.1 M CuSO4.
Compare the appearance of the three solutions and record the observations in Data Table
B.
Part C: Effect of Heat
19. Prepare a hot water bath by filling a 250 mL beaker half-full with tap water and heat it on
a hot plate at the lowest setting. Place a thermometer in the water bath to record the
temperature.
20. To a large test tube, add 5 mL of 2% albumin solution.
21. 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.
22. 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.
23. 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).
24. 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.
Cleanup:
 All protein mixtures and test solutions should be run through a paper towel filter to trap
solids. Run plenty of water while doing this. Put the paper towel with the solid residues
in the regular trash.
 Clean and return all equipment to its proper location.
Analysis:
 How did the hydrochloric acid affect each protein?
 How did the metal salts affect albumin?
 How did the isopropyl alcohol affect albumin?
 How effective was the “salting out” procedure on albumin?
 How did heat affect the albumin?