PROTEIN PURIFICATION AND FOLDING EXPERIMENT

EXPERIMENT 5


INTRODUCTION

Proteins, in nature, are like machines. They play an essential role in our body, such as
carrying oxygen to our cells or even duplicating our DNA. Proteins are composed of many
amino acids bonded together that form a relatively large molecule. A protein is a three-
dimensional structure formed by these amino acids. This 3-D structure is of great importance
since it is what determines the role of the protein.

The protein structure can be affected in various ways, some of which will be experienced
by us in this experiment.

This experiment is a simple protein chemistry experiment that will help to show the forces
involved in protein folding. The main objective of this experiment is to see what types of
molecules make the protein stable and what others make it unfold. This aspect will be easily
determined by the color of the solution.

We will isolate the protein, called phycocyanin which can be found in Spirulina cells,
sold in health stores in tablet form as a protein source. The function of this protein is to hold a
pigment molecule in a specific position. This pigment is necessary because it collects light
photons for photosynthesis. Indeed, when the pigment is curled up, it doesnt collect the right
color or as many photons as when it is held rigid. For this reason, the proteins role is to keep
it rigid.

Indeed, if the solution emits a red glow, we can say that the protein is properly folded
whereas if this red glow disappears, the protein is in an unfolded state. The solution should
also be dark blue when folded and more pale blue when unfolded. This is due to the proteins
initial function. As we discussed, phycocyanin is used to collect photons for photosynthesis.
Since we have, for the purpose of our experiment, isolated the protein, it is not in contact with
any other photosynthetic proteins that would continue the photosynthetic processes. For this
reason, the photons collected escape as light since they cannot be transferred to the next step
of the photosynthesis (i.e. red fluorescence).









APPARATUS AND CHEMICALS

Chemicals:

Spirulina capsule
Fine silica (1 g)
0.1 M Sodium Phosphate Buffer, pH 7.0 (60 mL)
0.1 M Sodium Chloride (10 mL)
Acetone (10 mL)
6 M Urea, pH 3.0 (10 mL)
Soap solution, 1:20 ratio soap to water (10 mL)
1 M Sucrose (10 mL)
1 M NaOH (10 mL)

Apparatus:

Pestle and mortar (x1)
Centrifuge (x1)
Flashlight (x1)
Filter papers (x2)
Funnels (x2)
Erlenmeyer Flask (x2)
Pasteur pipettes (x4)
10 mL test tubes (x8)
























PROCEDURE

PART I: Preparation

Preparation of solutions:
Before preparing the solution containing the protein, prepare all 8 solutions.
Using the electronic balance, weigh the Spirulina capsule and record the value. Place the
capsule in the mortar. Add an equal mass of silica to the mortar (record mass). Grind both the
silica and bacteria thoroughly (roughly four minutes). Grind the bacteria against the side and
bottom of the mortar to maximize the breaking of cell walls. Grinding harder will increase the
amount of protein separated from the cells. After four minutes, the mixture should be smooth.

Put the resultant mixture into 4 test tubes more or less equally. Add 7.0 mL sodium phosphate
buffer (pH 7) to each test tube. Using the flashlight if necessary, make sure the silica/Spirulina
mixture is suspended in the buffer in each of the 4 test tubes.

Centrifugation:
Using a glass rod, thoroughly mix each solution. Then, centrifuge all 4 solutions for ~2
minutes. Make sure to counterbalance each test tube with a test tube containing an equal
amount of liquid. Dont open the centrifuge while spinning!

Filtration:
Remove the tubes from the centrifuge and pour the liquid from the silica and cells into a dry,
clean test tube. Dont disturb the residue on the top of the solution -- try to leave as much of
that as possible in the test tube. Remove and discard the solid material left in the tube.

Fold a filtration paper into quarters, and place in a funnel. Wet the filter paper with distilled
water.

Pour test tubes into the filter paper, catching the filtrate in an Erlenmeyer flask. Most of the
larger particles should be removed in this process. Discard the solid left on the filter paper.
The protein solution left in the flask should be clear and dark blue, and around ~15mL. Keep
this solution for further study.
















PART II: Protein Folding/Unfolding

For each prepared solution, add 1.0 mL of protein solution and 5.0 mL of the solution in the
table to a test tube. Cap and shake. Record Results.

Solution Color Cloudy or
Clear
Red
Fluorescence?
Protein Folded or
unfolded?
0.10 M Sodium
Phosphate (pH 7.0)
Light Blue Clear Yes Folded
0.10 M Sodium
Chloride
Light Blue Clear Yes Folded
Acetone Light
Green
Cloudy No Unfolded
Distilled Water Blue Clear Yes Folded
6.0 M Urea Yellow-
Green
Clear No Unfolded
Soap Solution Blue-
Green
Clear Yes Folded
1.0 M Sucrose Light Blue Clear Yes Folded
1.0 M NaOH Light
Yellow
Clear No Unfolded

Separate Tests
Combine 1.0 mL protein solution with 5.0 mL sodium phosphate buffer in a test tube. Place
the tube in a beaker of boiling water.

Record notes on reaction below. (Does the protein unfold or stabilize? Why?





CALCULATIONS
We observe that the solution gets much lighter; as the blue colors disappear we can see that
more greenish colors appear. Also, the red fluorescence that was present is gone. Indeed,
the boiling water seems to have denatured the protein.

The only calculations required for the lab were for the various molarities of the solutions, all
of which were done in advance.
Solution Molarity
(mol/L)
Solution
Volume
(mL)
Molecular
Mass
(g/mol)
Calculations Required
Mass of
solute (g)
Sodium
Phosphate
0.1 30 142.0 .1M*.03L*142 g/mol .43
Sodium
Chloride
0.1 5 58.44 .1M*.005L*58.44 g/mol .029
Urea 6.0 5 60.06 6M*.005L*60.06 g/mol 1.8
Sucrose 1.0 5 342.3 1M*.005L*342.3 g/mol 1.7
NaOH 1.0 5 40.00 1M*.005L*40 g/mol .20

DISCUSSION OF RESULTS


In acetone, the intermolecular forces that one would expect to see would be London
dispersion forces, which is the most common type of intermolecular force, as well as
Dipole-Dipole forces. These are both relatively weak intermolecular forces. However we
know that the weaker the forces, the lower surface tension we will have, for this reason
acetone has a lower surface tension than water, as discussed previously.



In urea, hydrogen bonding will be possible because of the N-H bonds. Hydrogen bonding is
the strongest of the intermolecular forces, therefore since stronger intermolecular forces lead
to a higher surface tension, we can conclude that from its structure, urea should have a higher
surface tension compared to water.

From our results, we can see that acetone has the effect of unfolding the protein. Indeed, the red
fluorescence is gone and the solution has gone from having a blue color to a greener color. This
can give us some indications concerning the surface tension of acetone. Since the protein is
unfolded we can deduce that it is relatively easy to make a cavity in the solution, which means
the surface tension is low. This can be easily understood by taking the reverse process: if the
surface tension is high, it is difficult to create a cavity in the solution, therefore the protein has to
be tightly folded, if the latter is unfolded then the surface tension has to be lower.



Urea denatures the protein, just as acetone does. However, ureas surface tension is higher
because of the hydrogen bonds, which is a contradiction since with a higher surface tension it
is harder to form a cavity in the solution, therefore the protein should stay folded in the small
cavity, which it is not. Possibly, the mechanisms occurring here are linked to the hydrogen
bonding that will tie up all the water. In this case, the amino acids on the outside of the protein
wont have their normal interactions with water, and they may move to interact with an amino
acid which they would normally ignore, which can contribute to the denaturation (i.e.
unfolding) of the protein.




CONCLUSION

In general, the experiment went relatively well and we did not encounter any major
problems. We understood the procedure quite well, which helped us conduct the experiment
correctly. We learned some interesting facts about proteins, and more specifically about
phycocyanin. This was a biochemistry experiment and therefore somewhat out of the scope of
the general chemistry course, but some aspects that we discussed such as surface tension and
intermolecular forces are quite relevant to the course.
The experience of using a centrifuge was very rewarding, as was following an
experiment which produced visually dramatic results. Despite a fairly inexact procedure, the
forgiving nature of organic chemistry made things turn out as expected. One thing which we
neglected to bring though and which would have been helpful was a flashlight for detecting
the whether or not the samples were colloidal. This functionality was easily replaced with a
lamp, but it would still have been easier.