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Laraib Awan

AP Biology

Schulte

Experiment # 1

November 16th-17th, 2017

The effects of acids and bases, inorganic salts, organic solvents, and temperature on the physical

properties of proteins

Abstract
In part A of this lab, the experimenters were testing the effect of strong acids and bases.
First, three test tubes were prepared which contained a different protein solution in each one:
albumin, casein, and gelatin. It was then tested the effect of the acid’s, hydrochloric acid,
addition to these three solutions. Out of the three solutions, casein was the most reactive to the
hydrochloric acid and gelatine had been the least reactive due to the lack of change in the
gelatine as it had only became slightly cloudy while the casein obtained an oily texture and much
precipitate. Albumin became slightly cloudy but not as much as casein had. NaOH was also
added to these three solutions, after being rid of the hydrochloric acid, in increasing amounts.
Most of the reactions remained the same but albumin had become slightly more reactive. This is
because the enzymes and substrates are more reactive between albumin and sodium hydroxide.
Copper sulfate, silver nitrate and isopropyl alcohol was added to three different test tubes which
contained 2% albumin. They all had different reactions but silver nitrate seemed to have the
most reaction which caused albumin denaturation.
In part B, 10-mL of 2% albumin was added to a 50-mL beaker and made a mixture with
25-mL of saturated ammonium sulfate. The mixture was then filtered and the filtrate was
collected. Three test tubes were created: albumin solution, the filtrate, and a small portion of the
precipitate from the funnel with distilled water. The experiment called for the addition of
ammonium sulfate to each of the test tubes. The results varied greatly as the colors and textures
changed for each. Test tube 2 was the most reactive as it had changed color to a periwinkle blue
and became thicker and cloudier. This is because the substances were ‘salted out’ or purified
and the precipitate was removed.
In part C, a test tube was placed in a water bath and increased the temperature while
recording observations of the albumin solution. Data was recorded at 38, 48 and 88 degrees
Fahrenheit during times when precipitate first appeared, the solution appeared milky white, and
at the very end. There was more of a reaction when the water both had reached 88 degrees
which might be due to enzyme denaturation.
The purpose of this lab is to observe the reactions of the enzymes when affected by
different factors such as acids, bases, and extreme temperature.

Materials and Equipment Used


Albumin, 2% aqueous solution, 22mL Ammonium sulfate solution, (NH4)2SO4,
saturated, 25mL
Casein, 2% aqueous solution, 2mL Erlenmeyer flask, 125-mL
Copper(II) sulfate solution, CuSO4, 0.1 M, Filter paper and funnel
4mL Hot plate
Gelatin, 2% aqueous solution, 2mL Stirring rod
Hydrochloric acid solution, HCL, 2.5 M, Test tubes, small, 3
5mL Test tube, medium, 1
Water, distilled or deionized Test tube clamp
Wash Bottle Test tube rack
Beakers, 50- and 250-mL Thermometer
Beral-type pipets, graduated, 9 Procedure
Procedures
Part A
1. Label three test tubes 1-3.
2. Using a graduated Beral-type pipet for each solution, add 1 mL of albumin, casein, and
gelatin to test tubes 1,2, and 3. Record the initial appearance.
3. Add 2 drops of 2.5 M HCL to each test tube 1-3. Mix the mixture and record the
appearance.
4. Add 5 more drops of 2.5 M HCL to each test tube 1-3. Mix the mixture and record the
appearance.
5. Add 10 more drops of 2.5 M HCL to each test tube 1-3. Mix the mixture and record the
appearance.
6. Add 10 more drops of 2.5 M HCL to each test tube 1-3. Mix the mixture and record the
appearance.
7. Wash the test tubes. Relabel the test tubes 1-3.
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.
9. Add 5 drops of 2.5 M NaOH to each test tube 1-3. Gently mix each tube and record the
appearances.
10. Add 10 more drops of 2.5 M NaOH to each test tube 1-3. Mix the mixture and record its
appearance.
11. Wash the test tubes. Relabel if necessary.
12. Add 1 mL of 2% albumin solution of 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 0.2 M AgNO3 to test 2, and 2 mL of isopropyl alcohol to test tube 3.
Mix the contents in the test tube and record appearance.
Part B.
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. Record the appearance of the mixture.
2. Set up a gravity filtration apparatus and filter the mixture through a piece of wetted filter
paper. Collect the filtrate in a clean Erlenmeyer flask.
3. Label three small test 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 and dissolve the wet
solid in 2 mL 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 CuSO4
solution. Compare the appearance of the three solutions and record your observations.
Part C.
1. 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.
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 celsius, place the test tube in
the bath. Record the initial temperature of the water bath. 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, mix the protein solution and observe its
appearance.
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.
6. When the temperature of the hot water bath reaches 85-90 degrees celsius, remove the
test tube. Record the final appearance of the protein sample.
HAZARDS: Beware of glass that might be broken throughout the lab
Wear eye goggles and do not get chemicals near the face area
Gloves so chemical does not have skin contact
The independent variable of this experiment was the amount of HCL in part A, the
contents in the test tubes for part B, and the temperature of the hot water bath in part C.
The dependent variable is how much the independent variable had affected it.
The control is is the contents in the test tubes for Part A, the content being added into the test
tube for part B, and the substance in the hot water bath in Part C.

Results

Effect of Strong Acid and Base

Test Tube 1 2 3

Protein Solution Albumin Casein Gelatin

Initial Appearance Cloudy Slightly cloudy Clear

Effect of 2 drops Few bubbles Cloudy, white Coagulating


HCL
Addition 5 drops Few more A bit oily More coagulation
bubbles looking

10 drops Much more Increased The same


cloudy, amounts of
elevated coagulation
10 drops Really cloudy More separated Gooey looking
with precipitate substance

Effect of 5 drops Bubbles made Nothing Coagulation


NaOH
Addition
10 drops Separation Cloudy, Yellow tint
separating

Effect of Inorganic and Organic Additives

Test Tube 1 2 3

Additive CuSO4 AgNO3 Isopropyl Alcohol

Results Blue, fizzy, little bit White, bubbly, foamy A little cloudy, there is
of precipitant a yellow tiny

Part A is shown in the graph above. The graph shows the results of both parts and how the
solutions reacted to the additives. The first chart reveals the effects of strong acids and bases and
the second chart shows the effect of inorganic and organic additives.

Part B
Observations

Effect of Ammonium Sulfate Cloudy, milky, white with the formation of


some bubbles which gather at the surface

Test Tube 1 A light magenta color that is relatively


(Albumin + CuSO4) clear, a tiny bit of precipitant
Test Tube 2 Periwinkle blue, slightly thicker than
(Filtrate + CuSO4) before, cloudier

Test tube 3 Clear, medium blue tiny, seems to be no


(Dissolved solid + CuSO4) precipitate

The chart above represents the data which was recorded in part B of this experiment. It is to
show how the solution had filtered or ‘salted out’ the contents in the different test tubes.

Part C
Temperature Additional Observations

Initial Temperature (water 38 degrees Celsius Albumin is milky, kind of


bath) tan

First signs of precipitate 38 degrees Celsius Funky shells, small amount


appeared of white precipitate

Solution appeared milky 48 degrees Celsius Solution is milky, white,


white more precipitate, bubbles,
thicker

Final observations 88 degrees Celsius Boiling, bubbles at bottom


of the bath, very cloudy,
water boiling

The chart above represents part C of the lab. It describes what was observed as the temperature
in the hot water bath increased.
Analysis/Conclusion
In part A, the purpose of finding the effects of strong acids and bases was fulfilled as
noted that the more solution that was added would result in increased reactions such as a cloudy
color, precipitate being formed, and coagulation, and results of the test tube contents. My
hypothesis was if there was an increased addition of the acids and bases in the solution, then the
reaction will be greater; this was proven correct because there was more of a reaction in each of
the test tubes as HCL, the acid, and NaOH, the base, was added in increasing amounts. The
experiment was also to find the effect of inorganic and organic additives. My hypothesis was
fulfilled because I said if an inorganic additive is added to an organic solution then there would
be more of a reaction rather than if an organic additive was added to an organic solution. This
was showing in the results because the two inorganic additives, copper sulfate and silver nitrate,
had more of a reaction then the organic additive, Isopropyl alcohol. I believe there was some
error in this part of the lab. When AgNO3, an inorganic substance, mixes with an organic
substance, which in this case is albumin, it should turn into a darker color rather than the cloudy
white it had turned into. I believe this is because the experimenters put in an insufficient amount
of the AgNO3 or had not cleaned out the test tubes enough.
In part B, our purpose was to find whether or not the proteins were precipitated and
purified. Our purpose of this part of the lab seemed to be fulfilled as there was some purifying
done. Our solutions were salted out as solutions which had precipitate no longer did after the
sodium hydroxide and copper sulfate were added and the solutions were more clear than before
they were added. The public often see salting out in aqueous solutions that have high ionic
strength which are able to make the molecule unable to precipitate proteins. My hypothesis was
proven to be incorrect as I had predicted that if the ammonium sulfate was added, the solutions
would become cloudier and more precipitate would be added; however, I was proven wrong as
the solutions had become clearer.
In part C, the effect of heat on the various solutions was found. My hypothesis was
validated by the data as my hypothesis was that if there was an increase of temperature, then
there would be an increased reaction. It was validated because when the temperature increased,
these reactions listed were heightened. Extreme temperatures are able to denature an enzyme,
whether that be high or low. In the case of this experiment, an increasing temperature is seen.
Although it was not high enough to completely denature the enzyme, a reaction had still taken
place as the temperature increased. The experiment has achieved the slight denaturation of the
enzymes in this protein solution.
To continue this investigation, the method of how extreme cold temperatures affect the
solutions just as was done in part C will be tested. When enzymes denature, the cell is unable to
function due to the enzyme not being able to react with its substrate. This can cause the cell to
shut down, resulting in organs to shut down, harming a human and damaging their system.
Background research: The Nuffield Foundation has tested the reaction of enzymes in various
conditions. It was tested in hydrochloric acid with the catalase along with the effect of pH.
Background Research and Bibliography

Factors affecting enzyme activity. (n.d.). Retrieved December 07, 2017, from
http://www.nuffieldfoundation.org/practical-biology/factors-affecting-enzyme-activity

L. (2016, July 29). Salting Out. Retrieved December 04, 2017, from
https://chem.libretexts.org/Core/Physical_and_Theoretical_Chemistry/Thermodynamics/
Non-Ideal_Systems/Salting_Out

Urry, L. A. (2017). Campbell biology in focus. Boston: Pearson.