Vivien Coombs

Period 2

Schulte

Experiment #1

16 November 2017- 17 November 2017

The Effect of Acids, Bases, Inorganic Salts, Organic Solvents, and
Temperature on Proteins and Protein Synthesis
 Abstract
This experiment studies the effects of acids, bases, solvents, and temperature on
proteins and protein folding within the structures; the structures of the proteins
determines their functions and purpose. The denaturing of proteins is what is also being
studied in this experiment which means that the native structure of a protein is being
destroyed either chemically or physically; when the weak hydrogen bonds break and the
salt bridges are destroyed, the protein is then uncoiled and denatured. The most
common way to see the denaturing of the proteins was through the color changes seen
in the experiments, mainly in the second part of the experiment with the copper sulfate
solutions being mixed in with the filtrates causing ​salting out​. Salting out occurs in the
second part of the experiment when copper sulfate is mixed with inorganic salts or
ammonium sulfate; when the salting out occured, the solutions were all turned a
different shade of blue or purple which clearly showed the isolating of the proteins.

The first part of the experiment was the effects of two strong acids, hydrochloric
acid (HCl) and sodium hydroxide (NaOH), on protein base solutions, albumin,casein,
and gelatin.The results obtained from my experiment were not very different from the
original forms of the solutions. When heat was added into the experiment in part 3,
several more reactions occurred and were more profound; the albumin was exceedingly
more reactive as it turned thick and milky at initial temperature and then began to form a
precipitate and start boiling as it got thicker. As more factors, heat and filtrate, were
added, the proteins began to react and denature faster making it easier to see the
process of the proteins unfolding and weak hydrogen bonds breaking. The purpose of
this experiment is to more clearly understand and see the process of denaturing
proteins, the salting out process, protein folding, native structures and the different
effects of multiple additives, both chemical and physical, on the proteins themselves.
 ​Materials and Equipment Used

❖ Albumin, 2% aqueous solution, 22mL

❖ Ammonium sulfate solution, (NH​4​)​2​SO​4​, saturated, 25mL
❖ Casein, 2% aqueous solution, 2mL
❖ Copper(II) Sulfate solution, CuSO​4​, 0.1 M, 4 mL
❖ Gelatin, 2% aqueous solution, 2 mL
❖ Hydrochloric acid solution, HCl, 2.5M, 6 mL
❖ Isopropyl alcohol, (CH​3​)​2​CHOH, 2 mL
❖ Silver nitrate solution, AgNO​3​, 2 mL
❖ Sodium hydroxide solution, NaOH, 2.5M, 5 mL
❖ Water, distilled or deionized
❖ Wash bottle
❖ Beakers, 50-and 250mL
❖ Beral-type pipets, graduated, 9
❖ Erlenmeyer flask, 125-mL
❖ Filter Paper and funnel
❖ Hot plate
❖ Stirring rod
❖ Test tube, small 3
❖ Test tube, medium, 1
❖ Test tube clamp
❖ Test tube rack
❖ Thermometer
 Methods and Procedures
Part A:
1. The first step is to label three small test tubes 1-3.
2. Next, 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;
record the appearances of the solutions.
3. Add 2 drops of 2.5 M HCl to each test tube and gently stir the contents to mix
them then record the appearance.
4. Add 5 more drops of 2.5M HCl to each test tube 1-3, then stir the contents of
each test tube and record the appearances.
5. Add 10 more drops of 2.5M HCl to each test tube 1-3, then stir the contents and
record the appearance.
6. Add 10 more drops of 2.5M HCl to each test tube 1-3, stir the contents in each
test tube and record the appearances.
7. Wash the contents of each test tube down the drain with excess water and rinse
the tubes twice with distilled water from a wash bottle, relabel 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, 3,
respectively.
9. Add 5 drops of 2.5M NaOH to each test tube and gently stir the contents then
record the appearances.
10. Add 10 more drops of 2.5M NaOH to each test tube, stir the contents and record
the appearances of each.
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 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 0.1M
CuSO​4​ to test tube 1, 2 mL of 0.1M AgNO​3​ to test tube 2, and 2 mL of isopropyl
alcohol to test tube 3.
14. Stir each test tube gently to mix the contents, then record the appearances of
each test tube.

Part B:
1. Add 10 mL of 2% albumin to a 50-mL beaker, followed by approximately 25 mL
of saturated ammonium sulfate solution.
2. Stir the mixture thoroughly using a glass stirring rod. Describe the appearance.
3. Set up a gravity filtration apparatus and filter the mixture through a piece of
wetted filter paper.
4. Collect the liquid (filtrate) in a clean Erlenmeyer flask.
 5. 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 the gravity filtration device to test tube 2, then
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.
6. To each test tube 1-3, add 10 drops of 2.5M NaOH followed by 5 drops of 0.1M
CuSO​4​ solution.
7. Compare the appearances of the three solutions and record their appearances
in a data table.

Part C:
1. To 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.
2. Place a thermometer in the water bath to record the temperature of the bath.
3. To a medium size test tube, add 5 mL of 2% albumin solution.
4. When the temperature of the hot water is 35-40​o ​C, place the test tube in the
bath; record the initial temperature of the water bath in a separate data table,
then adjust the heat setting on the hot plate to a medium-high range to slowly
heat the protein solution.
5. Holding the test tube with a test tube clamp, gently stir the protein solution and
observe its appearance; be careful to watch the temperature of the bath at first
sights of protein precipitation are observed then record the appearances and
temperatures.
6. 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 or opaque.
7. When the temperature of the hot water bath reaches 85-90​o​ C, remove the test
tube and record the final appearance of the protein sample in the data table.

Be careful to handle these chemicals and be sure to wear protection in the form of
goggles, apron and gloves. Another precaution would be the use a hot plate, be careful
to use it with easiness so it will not cause destruction.
The dependent variables in Part A would be the bases, albumin, casein and
gelatin; the independent variables would be the strong acids, hydrochloric acid and
sodium hydroxide and then the other acids, copper sulfate, silver nitrate, and isopropyl
alcohol. The dependent variables in Part B would be the albumin, filtrate, and the
redissolved solid; the independent variable would be the copper sulfate. The dependent
variable in Part C is albumin; the independent variables are the temperature and the
water.
 Results

Effect of Strong Acid and Base

Test Tube 1 2 3

Protein Solution Albumin Casein Gelatin

Initial Appearance Very light and a little Yellow-ish, semi-cloudy Very clear
cloudy

2 drops of HCl Got a little more clear Cloudy chunks appeared No reaction
and got darker

5 drops of HCl Got slightly clearer Very cloudy, now whiteish No reaction

10 drops of HCl No reaction Thicker, more cloudy No reaction

10 drops of HCl Got slightly cloudy again Thickish, you can see Not mixing together
more chunks anymore

5 drops of NaOH No reaction A little bit foggy There is clear separation

between the solutions

10 drops of NaOH Tiny chunks have Slightly foggy Still clear separation
appeared

Effect of Inorganic and Organic Additives

Test Tube 1 2 3

Additive CuSO​4 AgNO​3 Isopropyl Alcohol

Results Turned blue Turned white and got Turned white and some
bubbly slight chunks appeared
 “Salting Out” with Ammonium Sulfate

Observations

Effect of Ammonium The bottom of the mixture turned yellow and then the top turned white.
Sulfate

Test Tube 1 The mixture turned a light purple and a small precipitate formed.
(Albumin + CuSO​4​)

Test Tube 2 The mixture turned a light blue, became thicker, but no precipitate
(Filtrate + CuSO​4​) formed.

Test Tube 3 The mixture turned a darkish blue purple and no precipitate formed.
(Redissolved Solid +
CuSO​4​)

Effect of Heat

Temperature Additional Observations

Initial Temperature 38​o Albumin is a milky, cloudy white, bubbles at the top, a
(water bath) little bit tan.

First Signs of 38​o There is a funky smell, a small amount of white

Precipitate Appeared precipitate.

Solution appeared 48​o The solution is milky white, more precipitate, bubbles, a
milky white little bit thicker.

Final Observations 88​o The solution is boiling, bubbles at the bottom of the test
tube, protein is very cloudy, water is boiling.
 Analysis
The outcome of Part A did not result in a lot of strong reactions between the
bases and the strong acids; the strongest reactions were between the casein and the
hydrochloric acid in which a slight precipitate began to form and the solution got thicker
and a whitish color meaning that the proteins had indeed denatured, making the
experiment a success. In addition, the additives of the copper sulfate, silver nitrate, and
isopropyl alcohol caused further reactions in the solutions; the copper sulfate turned the
solution a light blue, the silver nitrate turned the solution white and cause it to start
bubbling, and the isopropyl alcohol caused the solution to turn white and form a
precipitate. In Part B, all of the solutions turned a different color in some way; in the first
test tube, albumin and copper sulfate, the solution turned a light purple color and a
small precipitate formed; in test tube 2, the filtrate and copper sulfate, the solution
turned a light blue periwinkle color and got thicker although there was no precipitate;
finally in test tube 3, the solution turns a darkish blue color and there was no precipitate.
All of the color changes in Part B proved that the proteins were being salted out; the
solubility decreased and the concentration of ionic bonds increased but the protein
activity was not lost. With the addition of heat in Part C the reactions within the solutions
happened quicker; at the initial temperature of the water bath (38​o​) the albumin turned a
milky white then a little later, at the same temperature, the solution began to have a
strange smell and a precipitate to form. At 48​o​ C the solution appeared to turn thick and
more precipitate formed as well as bubbles, and finally, at 88​o​ C the solution was
boiling, the proteins became very cloudy and appeared very thick; the fact that the
solution was white and formed a precipitate reveals that the proteins in the solution
were denatured very quickly due to the heat.
I believe that the measurements in Part A would need to be exact to get more of
a reaction from the bases; the casein might have reacted better had the solution been
properly stirred or if the measurement of the casein was more precise, and the same
with the gelatin. Having not completely exact measurements might have unbalanced the
ratio of bases and strong acids causing the reaction to not work to its full extent. Not
properly cleaning the test tubes after each part, not using proper measurements, and
mislabeling could all be factors in affecting the results and compromising the outcomes.
For further investigation, another experiment could be performed in which instead
of heating the solutions like in Part C, the solutions would be placed in ice water to
make the temperature drop and note the reactions; if this test was performed, the
solution would most likely stay in its mixed state for a longer time and the proteins would
not denature nearly as fast due to the cold temperatures keeping the proteins from
unfolding and unraveling. This would be a success in the fact that cold temperatures do
not affect the protein denaturing process as much as heat would and at a much slower
rate.
 Background Information

Protein denaturing is the destruction of the secondary and tertiary structures due
to the small circle of tolerance at which a protein can perform its normal functions and
responsibilities. The proteins are most sensitive to temperature and pH; heat in
particular causes the weak hydrogen bonds in the protein structure to break making the
protein uncoil. Occasionally the proteins can be renatured and brought back to their
original state; once the denaturing factor is removed from the environment, the protein
can sometimes recoil itself and build back the hydrogen bonds and disulfide bridges
letting the protein resume its original function. In the “salting out” part of the experiment,
the process of putting the ammonium sulfate could be reversed and the protein could
then be purified to be brought back to its original state. If someone were to work to
remove the ammonium sulfate from the solution in Part B, the proteins would be able to
be build back up the weak hydrogen bonds and put themselves back together.
However, in the other parts of the experiment the proteins would not be able to put itself
back together and would remain denatured.
 Bibliography

Ophardt, C. E. (2003). Denaturation Protein. Retrieved December 08, 2017, from

http://chemistry.elmhurst.edu/vchembook/568denaturation.html

Man, P. (2017, September 6). The Protein Man's Blog | A Discussion of Protein
Research. Retrieved December 08, 2017, from
https://info.gbiosciences.com/blog/ammonium-sulfate-protein-precipitation-the-key-to-sa
lting-out

L. (2016, July 21). Protein Folding. Retrieved December 08, 2017, from
https://chem.libretexts.org/Core/Biological_Chemistry/Proteins/Protein_Structure/Protein
_Folding