Post-laboratory Report on

Exercise 3
Protein Denaturation

Vikki Anne R. Cedo

CHEM 160.1 - 3L
2nd Semester 2014-2015

Groupmates:
Desiree Joy Cerico
Ma. Kriselle Ornales
Mary Ranzelle Pasang

Ms. Korina Vida G. Sinad

Laboratory Instructor

Exercise 3
Protein Denaturation
Proteins are large biological molecules made of numerous amino acids linked
by amide bonds. Proteins are by far the most important of all biological compounds.
The word's origin is a Greek word, proteios, meaning "of first importance". Proteins
can have four structures: primary, secondary, tertiary and quaternary.
The primary structure of a protein is the order of amino acids in the
polypeptide chain. This is stabilized by the peptide bond. The secondary structure of
a protein is the repetitive structural units; the local folding and the interactions
between adjacent amino acids. This is stabilized by hydrogen bonding in the peptide
backbone. The tertiary structure of a protein is the whole molecule folding or 3dimensional arrangement of every atom in the molecule. It includes interactions of
the side chains and not just the peptide backbone. This is stabilized by hydrophobic
interactions, disulfide bridges, hydrogen bonding, ionic or electrostatic interactions
and metal ion coordination (Campbell, 2007). The quaternary structure of a protein
determines how the different subunits of the protein fit into an organized whole. The
subunits are packed and held together by hydrogen bonds, salt bridges, and
hydrophobic interactions.
Protein denaturation is the loss of the secondary, tertiary and quaternary
structures of a protein by a chemical or physical agent that leaves the primary
structure intact. Various denaturation agents include acids and bases, intense heat,
heavy metals, and organic solvents. Protein denaturation can be reversible or
irreversible. It does not always cause the loss of a protein's biological function.
In this exercise, Phycocyanin was extracted from a Spirulina tablet. It was
mixed with silica, the same mass as the tablet, ground together using mortar and
pestle until smooth. 28 mL of phosphate buffer with pH 7 was added to the mixture
and was stirred using a glass stirring rod. After stirring the mixture, it was placed in
a centrifuge for two minutes at 10000 rpm. The supernatant was obtained and
placed in a beaker. The protein solution was observed to be dark blue with tinges of
red on top, near the surface. The class was divided into two groups; groups 1 and 2
merged, groups 3 and 4 did the same. 0.5 mL of the protein solution was placed in

each of the eight test tubes (eight test tubes per group). 1 mL of 6 M HCl was added
to test tube 1. 1 mL 6 M NaOH was added to test tube 2. 1 mL 0.2 M lead acetate
was added to test tube 3. 1 mL 10% acetone was added to test tube 4 and 1 mL
95% ethanol was added to test tube 5. Test tube 6 was placed in a hot water bath
while test tube 7 was placed in a cold water bath. Test tube 8 served as control.
After standing for a few minutes, the test tubes were observed. Table 3.1 and
3.2 contains the observations for each of the test tubes. Test tube 1 which contains
0.5 mL of protein solution and 1 mL of 6M HCl exhibited a faint blue green color and
no fluorescence. Test tube 2 which contains 0.5 mL of protein solution and 1 mL of
6M NaOH exhibited a clear yellowish color, and no fluorescence. Whitish mixture
with white precipitate and no fluorescence was observed from test tube 3 which
contains 0.5 mL of protein solution and 1 mL of 0.2 M lead acetate. Test tube 4
which contains 0.5 mL protein solution and 1 mL acetone, had exhibited faint blue
green color and showed no fluorescence. Test tube 5 which contains 0.5 mL protein
solution and 1 mL 95 % ethanol exhibited a whitish, cloudy color and no
fluorescence.
Table 3.1 Effect of known denaturing agents on phycocyanin from Spirulina.
Reagent
6 M HCl
6 M NaOH
0.2 M lead acetate

Observations
Faint blue green solution and no red fluorescence
Yellowish, clear solution and no red fluorescence
Whitish mixture with white precipitate and no red

Acetone
95% ethanol

fluorescence
Faint blue green solution and no red fluorescence
Whitish, no red fluorescence

Test tube 7 and 8 which contains 0.5 mL of the protein solution were
subjected to temperature treatments. Test tube 7 was placed in a hot water bath; it
exhibited faint blue green color afterwards and no fluorescence. Test tube 8 was
placed in a cold water bath and was observed to have red fluorescence together
with the original color of the protein solution (blue). The loss of red fluorescence
suggest the denaturation of protein, because it has lost its biological function. The
red fluorescence observed means that the native conformation of phycocyanin is
still intact. The loss of this red fluorescence simply means that the protein has been

denatured in some ways, destroying the native conformation together with its
biological function. Test tube 8, the control, was observed to exhibit red
fluorescence because it was not subjected to any denaturating agent.
Table 3.2. Effect of temperature on phycocyanin of Spirulina.
Water bath used
Hot
Cold

Observations
Faint blue green solution and no fluorescence
Blue solution with red fluorescence

Strong acids and bases destroy salt bridges and hydrogen bonds. Heavy
metal cations destroy disulfide bridges. Reducing agents, like heavy metal cations,
destroy disulfide bonds because of their high affinity and attraction for sulfur.
Alcohol, on the other hand, destroys hydration layers. Heat destroys hydrogen
bonds. Alkaloidal reagents combine with positively charged amino groups to disrupt
ionic bonds. Organic solvents engage in intermolecular hydrogen bonding with
protein molecules, disrupting intramolecular hydrogen bonding within the protein.
Clinical applications of protein denaturation include laser surgery and protein
profiling. In laser surgery, the laser beam is absorbed by tissues, and its energy is
converted to heat energy. This process can be used to cauterize incisions so that a
minimal amount of blood is lost during operation (Brown, 2005). Laser beams are
delivered by an instrument called fiberscope. The laser beam is guided through
many fibers, thousands which are fitted into a tube only 1 mm in diameter; this
way, the laser delivers energy for denaturation only where it is needed. It can seal
wounds or join blood vessels without the necessity of cutting through healthy
tissues. Another application is protein profiling for the production of specific
antibodies. This includes both intact and denatured proteins. Plasma samples
probed on denatured protein arrays produced autoantibody profiles distinct from
those probed on natively displayed proteins. This versatile protein microarray
platform allows the display of both natural and denatured proteins; it offers a new
dimension to search for disease-specific antibodies, broadens the repertoire of
potential biomarkers, and will potentially yield clinical diagnostics with greater
performance.

References:
Bettelheim, F. (2007). Introduction to General, Organic and Biochemistry.
Brooks/Cole by Thomson Learning.
Brown, C. (2005). Introduction to Biochemistry. McGraw-Hill International.
Ophardt, C. (2003). Virtual Chembook. Retrieved February 21, 2015, from Elmhurst
College: http://elmhurst.edu/~chm/vchembook/568denaturation.html
Wang, J., Barker, K., Steel, J., Park, J., Saul, J., Festa, F., Wallstrom, G., Yu, X., Bian, X.,
Anderson, K. S., Figueroa, J. D., LaBaer, J. and Qiu, J. (2013), A versatile protein
microarray platform enabling antibody profiling against denatured proteins. Prot.
Clin. Appl., 7: 378383. doi: 10.1002/prca.201200062