Name: Mary Ward

Class: AP Biology

Teacher: Mrs. Schulte

Experiment #: 1

Date of Experiment: November 16th and 17th 2017

Title
The effect of the acids and bases on protein denaturation.
 Abstract
The purpose of this lab was to test the physical properties of proteins. Testing the
physical properties will help to better understand the structures of proteins and how they fulfill
their vital biological function. Since the structure and the function of proteins are related, it is
important for the structure to remain stable. Protein folding is the term for the natural coiling and
folding a protein does in order to get its 3D structure. When a factor disrupts the native 3D
structure of a protein it is called denaturation. If the structure of a protein is disturbed the protein
will be unable to perform its function properly.

In this experiment, there were three protein solutions being tested. These were Albumin,
Casein, and Gelatin. To try to disrupt the structure of these proteins different acids and bases
were added to the protein solutions. In Part A drops of HCl and NaOH were added to each
protein solution. Also in Part A 2 mL of CuSO4, AgNO3, and isopropyl alcohol we each added
to a separate solution of Albumin. In Part B drops of NaOH and CuSO4 were added to an
albumin solution, filtrate from the gravity apparatus, and redissolved solid from the funnel used
in gravity apparatus. In Part C the temperature was recorded to look at the effect of heat on
protein structure.

Materials and Equipment Used

● Albumin, 2% aqueous solution, 22 mL
● Ammonium sulfate solution, (NH​4​)​2​SO​4​, saturated, 25 mL
● Casein, 2% aqueous solution, 2 mL
● Copper(II) sulfate solution, CuSO​4​, 0.1 M, 4 mL
● Gelatin, 2% aqueous solution, 2 mL
● Hydrochloric acid solution, HCl, 2.5 M, 6 mL
● Isopropyl alcohol, (CH​3​)​2​CHOH, 2 mL
● Silver nitrate solution, AgNO​3​, 0.1 M, 2 mL
● Sodium hydroxide solution, NaOH, 2.5 M, 5 mL
● Water, distilled or deionized
● Wash bottle
● Beakers, 50- and 250-mL
● Beral-type pipets, graduated, 9
● Erlenmeyer flask, 125-mL
● Filter paper and funnel
● Hot plate
● Stirring rod
● Test tubes, small, 3
● Test Tubes, medium, 1
● Test tube clamp
 ● Test tube rack
● Thermometer

Procedure and Methods

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 .
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 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

1. 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.
 2. 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.
3. Label three small tubes 1-3.
a. Add 2 mL of 2% albumin solution to test tube 1.
b. Add 2 mL of the filtrate from step 15 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.
4. To each test tube 1-3, add 10 drops of 2.5 M NaOH followed by 5 drops of 0.1 M CuSO​4
solution. Compare the appearance of the three solutions and record the observations in
Data Table B.

Part C: Effect of Heat

1. Prepare a hot water bath: Fill a 250-mL beaker half-full with water and heat it on a plate
at the lowest setting. Place a thermometer in the water bath to record the temperature of
the bath.
2. To a medium size test tube, add 5 mL of 2% albumin solution.
3. When the temperature of the hot water bath is 35-40 degrees C, place the test tube in the
bath. Record the initial temperature of 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.
4. 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.
5. 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).
6. When the temperature of the hot water bath reaches 85-90 degrees C, remove the test
tube. Record the final appearance of the protein sample in Data Table C.

Results
Data Table A: ​Solubility and Protein Denaturation
Effects of Strong Acid and Base

Test Tube 1 2 3

Protein Albumin Casein Gelatin

Solution

Initial Cloudy Slightly Cloudy Clear, Barely

Appearance Cloudy
 Effect of 2 Few bubbles Precipitate, cloudier Light, gooey
HCl drop
Addition s

5 Bubbles Precipitate, cloudy, became Coagulating

drop white
s

10 Elevated, cloudy More precipitate Precipitate,

drop clear,
s coagulate

10 Very cloudy More precipitate Coagulate

drop
s

Effect of 5 Clear Nothing Coagulation

NaOH drop
Addition s

10 Separates Cloudier Yellow tint

drop
s

Effect of Inorganic and Organic Additives

Text Tube 1 2 3

Additive CuSO​4 AgNO​3 Isopropyl Alcohol

Results Blue, fizzy, little Very cloudy and white Slightly cloudy, relatively
precipitate clear

In Data Table A the solubility and protein denaturation was tested. The independent
variable for this section is the HCl, NaOH, CuSO​4​, AgNO​3​, and isopropyl alcohol. The
dependent variable was how the appearance of the proteins (albumin, casein and gelatin)
changed from the initial appearance.

Data Table B: ​“Salting Out” with Ammonium Sulfate

Observations
 Effect of Ammonium Sulfate Very cloudy, white, slightly pink, milky tiny
precipitate

Test Tube 1 (Albumin + Light magenta color that is somewhat clear, tiny bit
CuSO​4​) of precipitate

Test Tube 2 (Filtrate + CuSO​4​) Purplish blue, thicker, very cloudy, no precipitation

Test Tube 3 (Redissolved solid Very clear with blue tint, no precipitate
+ CuSO​4​)

In Data Table B NaOH and CuSO​4​ were added to different kinds of the albumin and
ammonium sulfate mix solution. The NaOH and CuSO​4​ were the independent variables. The
dependent variable was the observations written down to describe the amount of reactivity
between solutions.

Data Table C: ​Effect of Heat

Temperature Additional Observations

Initial Temperature 38 Albumin is milky, cloudy white, bubbles

(water bath) on top

First signs of precipitate 38 Small amounts of white precipitate

appeared

Solution appeared milky 48 Milky white, more precipitate, bubbles,

white thicker

Final Observations 38 Boiling, bubbles at the bottom, very

cloudy

In Data Table C heat was used to determine if it was a factor of protein denaturation. The
independent variable was the heat added and the dependent variable was the temperature at
which the protein solution appeared cloudy and developed a precipitate.

Analysis/Conclusion
The outcome of this experiment showed that acids and bases do have an effect on protein
structure. This can be seen in the tables. Every time an acid or base was added to the protein
solution the appearance altered, expect once. The solution became cloudy or developed a
precipitate in Data Table A, the color of the solutions changed in Data Table B, and heat changed
the appearance of the solutions in Data Table C. The purpose of this experiment was fulfilled
because each part of this experiment tested the physical properties of proteins in a different ways.

However, there were errors in this experiment that could have influenced the results. The
sources of error came from equipment, procedural, and human error. In Part B there needs to be a
better explanation of how to set up the gravity filtration apparatus. During Part B our group
experienced difficulties with how to set this system up and the filter paper used. We barely had
any filtrate to scrape off which lead to a lack of filtrate in test tube 2. There also could have been
human error in Part B because the lack of knowledge of a gravity filtration apparatus.

For further investigation of this experiment perhaps another scientist could use a wider
range of solutions to take the investigation to the next level. They could use a greater amount and
more types of protein solutions, acids, and bases. If they used a wider variety of solutions they
would have more results that could lead to a more accurate outcome of how and what affects
protein structure.

Background Research and Bibliography

The structure and function of a protein often relate to one another. The biological activity
of a protein depends on its 3D shape. All proteins are made from amino acids joined by peptide
linkages. Amino acid side chains can interact through a variety of forces. When these forces are
disrupted it will cause the protein to fold and twist into different shapes. The natural process for
proteins to coil around/fold into themselves is known as protein folding. When the native
structure of a protein is disrupted it will cause the function to break down. This process is known
as denaturation. In this lab we tested what and how a protein can be denatured and if the
structure and function of a protein really do correlate with each other.

Physical Properties of Proteins. (2007). ​Flinn Scientific, Inc​, pp. 1-4.