ASI, Ranelle Janine L.

Chemistry 160.1 Section 3L

2015

Date performed: June 22, 2015

Date submitted: June 26,

EXERCISE 3. Protein Denaturation

Post-Laboratory Report
Proteins are produced as linear chains with different combinations of 20
amino acids, sequenced to form various classes that carry out diverse tasks once
functional. A proteins distinct sequence is its primary structure, formed by peptide
bonds between the amino and carboxyl groups of each amino acid (Lodish, et al.,
2007).
Depending on the primary structure, a protein chain folds into its secondary
structure stabilized by hydrogen bonds between certain amino acid residues
forming periodic arrangements called alpha helices, beta sheets and U-turns. An
alpha helix is formed through hydrogen bonds between the carbonyl oxygen of each
peptide bond and the amide hydrogen of an amino acid four residues away. A beta
sheet is formed by polypeptide chains that laterally bond to each other forming
pleated sheets that project residues on both surfaces. A U-turn is comprised of three
to four residues stabilized by hydrogen bonds between end residues (Lodish, et al.,
2007).
A protein achieves its functional form upon conforming into its tertiary
structure. Unlike in secondary structures, these shapes are formed by more than
one type of stabilizing force: hydrophobic interactions between nonpolar side
chains, hydrogen bonds between polar side chains, disulfide bonds between two
cysteine amino acids and ionic bonds between oppositely charged amino acid side
chains. These bonds hold helices, sheets and turns together in a compact but not
entirely rigid three-dimensional shape (Lodish, et al., 2007).
The last level of protein structural organization is quaternary structure, only
necessarily achieved by multimeric proteins. Quaternary structures are formed by
joining multiple oligomeric proteins and are stabilized by multiple forces similar to
those in tertiary structures (Lodish, et al., 2007).
A protein adapting its natural conformation based on these levels of
organization is said to be in its natural state, which is the most stably folded form of
the whole molecule. Denaturation of this state results from various treatments like
high heat that break stabilizing bonds, extreme pH that alter charges on amino acid
side chains, and chemicals like hydrochloride at high concentrations that can
disrupt weak noncovalent bonds. Once a protein loses its native conformation, its
chemical properties change and its biological activity is also lost (Cohen, 2004).
The protein studied in this exercise is phycocyanin extracted from the
cyanobacterium Spirulina sp. Phycocyanin is a chromophoric water-soluble protein
comprised of an alpha and beta subunit each containing a cysteine residue. It also
contains a prosthetic group called phycocyanobilin covalently attached to the

cysteine of the apoprotein through thioether linkage, and auxiliary proteins devoid
of chromophore called linker polypeptides (Britton, 1983).
To demonstrate protein denaturation, phycocyanin was mixed with different
denaturing reagents and exposed to different temperatures as shown in Table 3.1
and Table 3.2.
Table 3.1. Effects
Reagent
6 M HCl
6 M NaOH
0.2 M lead
acetate
10%
trichloroacetic
acid
95% ethanol

of known denaturing agents on phycocyanin from Spirulina.

Observations
Bubbling, solution turned clear light blue, no precipitate
Solution turned clear light yellow, no precipitate
Formation of white precipitate
Bubbling, solution turned clear light blue, no precipitate

Solution turned colorless

Table 3.2. Effect of temperature on phycocyanin from Spirulina.

Water bath used
Observations
hot
solution turned clear light blue, no precipitate
cold
no change
An isolated phycocyanin solution is colored dark blue with a tinge of red
signalling its functioning biological activity. As observed, all chemical reagents show
denaturing effects on phycocyanin as all resulted to loss of color.
It is known that reagents that cause denaturation target the abundant
hydrogen-bonds present in native proteins. Acidic reagents, in this case HCl, act
through protonation of carboxylic groups involved in hydrogen bonding, resulting to
loss of the said interactions, and through alteration of pH of the solution, which
decreases protein solubility (Lapanje, 1971). Basic reagents like NaOH have similar
effects on pH and protein solubility (Chawla, 2014).
Addition of organic solvents like alcohol, in this case ethanol, displaces water
molecules associated with proteins, causing a decrease in the concentration of
water in the solution which then lowers the solubility of the protein leading to
denaturation (Chawla, 2014).
Addition of heavy metal ions like lead acetate resulted to ionic bonding of the
lead cation with negatively charged groups on the proteins causing denaturation
and precipitation as metal proteinate (Chawla, 2014).
Theoretically, addition of high concentrations of trichloroacetic acid (TCA)
should lead to precipitation as negatively-charged TCA ions disrupt native
electrostatic interactions in protein, leading to formation of an intermediate form
with exposed nonpolar surfaces that promote intermolecular coalescence and
precipitation (Rajalingam, et al., 2009).

As most biological compounds are heat-sensitive, exposure to high

temperature caused protein denaturation as a result of peptide chain
disorganization and breakage of crosslinkages among chains due to increase of
conformational entropy (Wu and Wu, 1925). Low temperatures affect protein
structure at extremes (hence no change was observed in this experiment)
theoretically by promoting interactions of protein nonpolar groups with water
leading to unfolding (Privalov, 1990).
References:
Britton, G. 1983. The Biochemistry of Natural Pigments. Cambridge University Press,
Australia. p. 160.
Chawla, R. 2014. Practical Clinical Biochemistry: Methods and Interpretations.
Jaypee Brothers Medical Publishers, Ltd., India.
Cohen, G. N. 2004. Microbial Biochemistry. 1st ed. Springer Science & Business
Media, B. V. p. 285.
Lodish, H., Berk, A., Kaiser, C.A. and Krieger, M. 2007. Molecular Cell Biology. 6th ed.
W.H. Freeman & Co., New York. pp 60-66.
Lapanje, S., and Wadso, I. 1971. A Calorimetric Study of the Denaturation of
Lysozyme by Guanidine Hydrochloride and Hydrochloric Acid. Lund University,
Sweden.
Privalov, P. L. 1990. Critical Reviews in Biochemistry and Molecular Biology: Cold
Denaturation of Proteins. Volume 25, Issue 4. Retrieved from
http://informahealthcare.com/doi/pdf/
10.3109/10409239009090613
Rajalingam, D., et al. 2009. Trichloroacetic acid-induced protein precipitation
involves the reversible association of a stable partially structured
intermediate. University of Arkansas, Arkansas.
Wu, H., and Wu, D. Y. 1925. Nature of heat denaturation of proteins. Peking Union
Medical College, China.