Current Trends in Chemistry-Protein Structure and Stability
Ms. Catie Hallstrom and Mr. Youssef Fahri (Partner)
Submitted to: Ms. Charity Epley (TA)
CHEM 1046, Experiment No. 9, March 23, 2015
Laboratory Written Report
Honor Code Signature:______________________ Abstract:
Different situations affect the ability of a protein and may even denature the protein
completely so the protein can no longer carry out its specified function. Fluorescence, color, and
turbidity are specific characteristics that help determine when the protein is folded or unfolded.
Several solutions as well as change in temperature were tested to observe how the protein
changed if it did change. It was realized that a change in color or the absence of fluorescence
were signs of an unfolded protein.
I. Introduction:
Proteins are necessary components that perform certain functions in biological
systems. It is important to understand how molecules fold and what may affect the
stability and functionality of the protein because if the protein is not folded correctly,
the functions it is responsible for will not be carried out. Diseases such as
Alzheimer’s, Parkinson’s, and “Mad Cow” as well as others, result from misfolded
proteins.
A protein has a unique composition, number and arrangement of the amino acids
that create it. The amino acids are bonded by peptide bonds that fold in such a way as
to isolate the hydrophobic side chains from water. The function of each protein
depends on the overall tertiary and quaternary structure. For this experiment the
protein has twelve phycocyanins assembled to make one protein complex. The
phycocyanins bind tetrapyrrol pigment molecules in a linear fashion. The bonded pigments are necessary for this specific protein because the pigments collect light
photons for synthesis.
Intermolecular forces are also important concepts when determining why a
protein may be stable or unstable under certain conditions. Amino acids interact with
each other through dispersion forces, dipole-dipole interactions, hydrogen bonding,
and other intermolecular forces. Also, during folding there is also a change in
enthalpy as well as entropy. The Gibbs equation describes the relationship between
enthalpy and entropy, ΔGsys=ΔHsys-TΔSsys.
Temperature and other solutions have the potential to alter the folding of a
protein. During the lab several solutions will be added to the protein and buffer
solution being tested to observe if the protein is denatured. There will be a color
change and/or the solution will no longer be fluorescent. Changing the temperature
will also have an affect on the solution. Room temperature, freezing temperatures,
and boiling temperatures will be tested to determine if the protein structure is
II. affected. 1
Experimental:
Procedure
Part A: First, weigh 2.5 g of bacteria into a small beaker and add 30 mL of 0.1 M
sodium phosphate buffer at pH 7.00. Mix the two components together. Next, pour
samples into centrifuge tubes and centrifuge the solution for two minutes.
Meanwhile, take a piece of filter paper, fold it into quarters, and place the filter paper
into a funnel and wet it with water. Pour the centrifuged solution into the funnel and
catch the filtrate into and Erlenmeyer flask.
Part B: Use 1.0mL of the filtered solution in the Erlenmeyer flask and add the sample
to 5 mL of the solutions, 0.1 M Na3PO4 buffer pH 7.00, 0.1 M NaCl, distilled water, 6.0M Urea, soap solution, 1.0M HCl, 1.0M NaOH, and acetone. Take detailed
observations of each reaction specifically noting the color, turbidity, and fluorescence.
Part C: Prepare three test tubes with 1.0 mL of protein solution to 5.0mL of 0.1 M
sodium phosphate buffer pH 7.00. Place one test tube in boiling water, one in an ice
bath, and one in room temperature. Observe and record the fluorescence, turbidity,
and color of each test tube.
Data:
Table 1: Environmental Effects on Protein Stability Solution
0.1M Na3PO4 buffer
pH 7.00
0.1M NaCl Color
Clear blue/teal Fluorescence
Yes Turbidity
No Light, clear blue Yes Distilled Water
6.0M Urea
Soap Solution
1.0M HCl
1.0M NaOH
Acetone Light, clear blue
Clear blue/green
Clear green
Light blue
Yellow/green
Light blue Yes
Yes
Yes
No
No
No Very few particles
floating around
No
No
No
Yes
Yes
Yes Table 2:Temperature Effects on Protein Stability Temperature (with Color Fluorescence Turbidity Na3PO4 buffer)
Cold (Ice Bath)
Room Temperature
Hot (Boiling water) Light clear blue
Light clear blue
Very light green/blue Yes
Yes
No No
No
Yes Observations: During this experiment the protein was observed under certain conditions to determine if
the protein structure was denatured. First, different solutions were placed in test tubes with 1mL
of the algae buffer solution. 0.1M sodium phosphate buffer pH 7.00 was added to the already
clear blue algae solution. The color changed to a teal color, was not turbid, and turned red/purple
when a light was shined on the solution. If the color changed to a red/purple, it means that the
solution is fluorescent. When 0.1M sodium chloride was added the solution became a lighter,
clear blue with a few particles floating in the solution. Also, the solution is fluorescent since the
solution turned red. Distilled water, when added, also turned the solution to a light clear blue
that was not turbid but fluorescent. When 6.0M urea was added to the solution, the solution had
a few particles but was not fluorescent. The clear yellow soap solution changed the sample to
clear green and was not turbid. Also, the solution was not fluorescent. When 1.0M HCl was
combined with the algae solution, the color changed to a light, cloudy blue that was not
fluorescent. 1.0M of NaOH, when added to the solution, turned the algae solution to a
yellow/green color. It was also very turbid and not fluorescent. Finally, acetone changed the
solution to a light blue color but the solution became very turbid with particles floating in
solution. When a light was shined on the test tube, the solution was not fluorescent.
Next, the protein sample was tested under different temperatures. The solution in room
temperature did not change, it remained a fluorescent clear, light blue solution. Likewise, when
the sample was placed in an ice bath, the solution became a lighter blue, but was still clear and
fluorescent. However, when placed in boiling water, the color changes to a pale green/blue that
is very turbid and not fluorescent.
III. Results and Discussion Table 3: Results and Observations Condition Solution Color Turbidity Red
Fluorescence 0.01 Sodium
Phosphate
Buffer
0.1M NaCl
Distilled
Water
Acetone
6.0M Urea
Soap Solution
1.0M HCl
1.0M NaOH Blue No Yes Protein:
Folded or
Unfolded
Folded Blue
Blue No
No Yes
Yes Folded
Folded Light blue
Blue/green
Green
Light blue
Yellow/green Yes
No
No
Yes
Yes No
No
Yes
No
No Unfolded
Unfolded
Folded
Unfolded
Unfolded Table 4:Energy Effect Condition
Buffer
NaCl
Distilled Water
Acetone
Soap Solution
Urea
1.0M HCl
1.0M NaOH None
X
X
X ΔH ΔS Both ΔH and ΔS X
X
X
X
X This experiment focused on what aspects cause a protein to denature and cease
performing its specified function. Table three illustrates the results collected after
several different solutions were added to the protein buffer solution. Before any
substances were added to the protein, the protein was weighed and then dissolved in a
buffer solution. The sodium phosphate buffer was added to the protein to help the protein maintain a pH in a certain buffer range. This way, not all solutions will cause
a drastic change in hydrogen ions and thus change the pH and denature the protein. 1
As seen in table three, when more of the buffer solution, sodium chloride, distilled
water, and soap solution were added, it was determined that the protein remained
folded. There was no turbidity, which means all solutions were clear/ transparent.
Also, all of the solutions remained fluorescent. A key observation during the
experiment was fluorescence. When the protein is properly folded, there are attached
pigments as side chains that absorb photons and cause a red glow when a light is
shined. It is also important to not that the solutions did not undergo a color change
which, is a sign that the protein was denatured. Only the soap solution changed from
blue to green. However, the soap solution was yellow prior to adding it to the blue
protein solution. Therefore, when the two solutions were combined, a green color
formed.
As for the addition of acetone, the color changed to a blue/green color. Also, the
solution was turbid and not fluorescent. All three factors indicate that the protein was
unfolded. Acetone has hydrogen bonding that most likely out competed the hydrogen
bonding between the proteins. This then caused the hydrophilic portions of the
protein to be exposed and cause clathrates, the aggregation of water molecules, to
form. With the formation of clathrates, there was a change of entropy as well since
this folding is favorable. Since the hydrogen bonds were broken, there was a change
in enthalpy, as noted in table four. Urea was an interesting case because the color did
change and the solution was not fluorescent, but the solution was not turbid. It can be
assumed that there was a change in enthalpy because bonds were broken to denature the protein, but since the protein did not clathrate and form particles, there was not
change in the entropy.
Hydrochloric acid did not cause a color change, but the solution was turbid and
not fluorescent which are key signs that the protein denatured. Hydrochloric acid is a
strong acid and a polar molecule that competed for the hydrogen and dipole-dipole
intermolecular forces with the protein which caused the protein to unfold. Also, as
seen in table four, there was an enthalpy and entropy change in the solution due to the
broken hydrogen bonds and formation of clalthrates. Finally, when sodium hydroxide
was added to the protein solution, the solution changed to a yellow/green, it was very
turbid and not fluorescent. Thus, there was an entropy and enthalpy change. With the
sodium hydroxide, there was an addition of sodium and hydroxide ions. The ions
would then affect the concentration of ions in the buffer solution and would cause the
solution to leave the buffer range. Without the buffer range, the hydrogen in the
hydroxide could combine with the proteins and overpower the intermolecular forces
between proteins.
During the second portion of the lab, temperature was changed to determine how
the solution would react. In table two the data shows that a room temperature and at
freezing temperatures, the solution was light blue (the color of the filtrated solution
before any conditions were changed), not turbid, and fluorescent. It can then be
concluded that the protein remained folded since there were no observations that
coincided with unfolded proteins. However, when the protein solution was placed in
boiling water, the solution became very turbid, a pale greenish blue, and was not
fluorescent. Since the temperature was very hot, it caused the atoms in the protein to
vibrate, like most molecules do when they are heated. The proteins were affected by the vibrating and thus the hydrogen bonds between proteins were broken and the
protein unfolded. This coincides with the observations relating to unfolded proteins.
As said in the lab and A Simple Protein Purification and Folding Experiment,
color change, no fluorescence, and turbidity, are signs that they phycolbiliproteins
were denatured. Solutions such as hydrochloric acid, sodium hydroxide, urea, and
acetone caused observations related to the denaturing of the protein. Similarly, these
observations occurred when the solution was heated. Calculation:
Post Lab:
1. Ka=7.5x10-3 pKa=-log(7.5x10-3)=2.12
2.12-1=1.12 2.12+1=3.12
Buffer Region: 1.12-3.12
Uncertainty:
During the experiment the algae and sodium phosphate buffer is filtered from a green
mixture to a clear blue solution. If the solution is not properly filtered, then the turbidity will be
affected. In turn, the fluorescence and color may be altered providing inaccurate observations.
Also, if not enough of the protein is placed in the tube to react with the co-solvent, then the
observations will be skewed because change may not be clear in the test tube. If a solution is not
mixed together, then the solution added to the protein solution may not react with all of the
protein and cause only a portion of the proteins to denature.
IV. Conclusion After completing this experiment, it can be determined that color change, turbidity, and no fluorescence are good indicators that the protein was denatured. When urea, acetone,
sodium chloride, or hydrochloric acid were added to the solution the previously listed
characteristics were made. They were also apparent when the solution was heated. Therfore, in
order for the protein to function properly, high temperatures and the addition of certain solutions
that would outcompete intermolecular forces should be avoided. Whereas the other conditions
tested allowed the protein to function normally and remain folded which is why the protein could
still absorb photons and fluoresce.
References
1.
Patricia Amateis, M. D., Victoria Long, General Chemistry 1046 Laboratory Manual.
Hayden-McNeil Publishing: p 105-110.
2.
Buffers for Biomedical Reactions. https://www.promega.com/resources/product-guidesand-selectors/protocols-and-applications-guide/buffers-for-biochemical-reactions/
(accessed 3/23/15).
3.
Brewton-Parker College.
http://www.bpc.edu/mathscience/chemistry/table_of_polyprotic_acids.html (accessed 3/23/15).
4.
BioLabs. https://www.neb.com/tools-and-resources/usage-guidelines/amino-acidstructures (accessed 3/23/15). V. Post-Lab Questions
1. The addition of the 0.1M sodium phosphate buffer in the protein was necessary to
create a buffer range for the protein. With the added buffer, the solution will resist
a pH change by resisting changes in hydrogen ion concentration. The general
“rule of thumb” when determining a buffer range is to use the dissociation
constant of the acid in the buffer solution to determine the pKa.2 Then, the pKa
that was determined is the middle of the buffer range; subtract and add one pH
unit to the pKa to calculate the range. The Ka equals 7.5x10-3 therefor the pKa is
equal to 2.12, therefore the buffer region is 1.12-3.12.3 2. Phycocyanin binds tetrapyrrol pigment molecules in a linear fashion when
properly folded. These pigments absorb photons and in turn allow the solution to
be fluorescent under light. If the protein was not present, then the protein would
lose its linear conformation and curl into a lock-washer conformation. In this
form, the pigment does not collect the right color or as many photons. If there are
not as many photons to be absorbed, then the fluorescence is lost.1 3. Acetone Urea Acetone and urea are both polar solutions. Since both are polar, there will be
dipole-dipole intermolecular forces. Also, urea exhibits hydrogen bonding
because there are two nitrogen atoms bonded to two hydrogen atoms each. Since
there are hydrogen atoms on the acetone, the hydrogens from the acetone will
most likely bond with the nitrogens from the urea. This will create a hydrogen
bond since a hydrogen bond consists of hydrogen atoms bonded to nitrogen,
oxygen, or fluorine. The stronger force between these species will be the
hydrogen bonds.
4. Amino Acids with positive or aromatic side chains4
Phenylalanine
Tyrosine Lysine Histidine Tryptophan Arginine 5. In an amino acid the two functional groups that are almost always in the structure
is an amine group and a carboxylic acid group. Proline is the only amino acid out
of the twenty that does not have both. It has a carboxylic acid group, but it does
not have an amine group. Instead, the nitrogen atom in the structure of the
molecule only has one hydrogen atom attached. Amine groups have two
hydrogen atoms attached to the nitrogen atom. In proline, there is a ring that has a
nitrogen atom instead of a carbon.4

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