Name: ANDAL, Daniel Seth Date Performed: Jan.

27, 2020
Group number: 2 Date Finished: Jan. 27, 2020

Exercise 2
PROTEIN DENATURATION
I. RESULTS
Table 2. Effect of known denaturing agents and conditions on phycocyanin from Spirulina.
Test tube
number Reagent/Condition Observations
1 6 M HCl Slightly cloudy blue-green solution, absence of red fluorescence.
2 6 M NaOH Clear yellow-green solution, absence of red fluorescence.
3 0.2 M lead acetate Cloudy complex with white precipitate, absence of red fluorescence.
4 10% trichloroacetic acid Turbid blue-green solution, absence of red fluorescence.
95% ethanol Yellow to clear solution with minute amount of white precipitate,
5
absence of red fluorescence.
Hot water bath Green solution with minute amount of white precipitate, absence of
6
red fluorescence.
7 Cold water bath Clear blue-green solution, absence of red fluorescence.
8 Control Blue-green solution, absence of red fluorescence.

II. DISCUSSION
Proteins are nitrogenous organic compounds that count as the most abundant biomolecule
in the human body. These occur in primary, secondary, tertiary, and quaternary structures.
Primary level of protein structure are stabilized by peptide bonds while secondary level are
maintained by intramolecular hydrogen bonds whereas the tertiary and quaternary levels are
bonded by covalent bonds, hydrogen bonding, salt bridges, hydrophobic interactions, and metal
ion coordination (Bettelheim, Brown, Campell, Farrell, 2007. pp. 315-321).

Figure 2.1. Effect of denaturing agents to phycocyanin. Figure 2.2. Phycocyanin extract from Spirulina.
 Denaturation occurs when a change in the protein structure results from the loss of
secondary, tertiary, and quaternary structures due to disruption in noncovalent interactions while
primary structure remains intact (Castellion and McMurry, 1999, p. 510). In this exercise, effects
of known protein denaturing agents and temperature conditions were demonstrated upon
phycocyanin, a pigment protein extracted from Spirulina as seen in Fig. 2.2, for observation and
analysis. As observed in Fig. 2.1, every test tube was recorded to exhibit the absence of red
fluorescence, including the control sample, which indicates that phycocyanin native conformation
was disrupted. Upon addition of 6M HCl, the sample exhibited a slightly cloudy blue-green solution
whereas addition of 10% trichloroacetic acid yielded a turbid blue-green solution. Effect of acids
to the pigment associate with positively charged amino groups in proteins to unsettle ionic bonds
(Bruice, 1996). On the other hand, addition of a strong base of 6M NaOH, the appearance of the
sample turned into clear yellow-green solution which is credited to the loss of positive charges
depriving the protein of its salt bonds (Puri, 2011). Another denaturing agent are the heavy metals
and is demonstrated in this exercise by subjecting 0.2M lead acetate to the sample. Observations
upon adding the reagent show that the sample formed a cloudy complex with white precipitate
which is due to insoluble metal protein salt yielded by reactions of heavy metal cations with
proteins (Ophardt, 2003). Ethanol as an organic solvent, specifically alcohol, results to a yellow
to clear solution with minute amount of white precipitate for its denaturing effect to phycocyanin.
Its ability to coagulate the surface of the pigment (Stoker, 2003, p. 456) and to disrupt hydrogen
bonds between amide groups in the secondary structure and between the side chains in the
tertiary structure (Ophardt, 2003) justifies the coloration slightly similar of the NaOH. Lastly,
temperature was also taken into account for the denaturing of phycocyanin. The effect of heat
to the sample yields a green solution with minute amount of white precipitate which is due to
high temperature causing the proteins to vibrate violently where their weakest bonds break as
their strings of amino acids begin to loosen (Norton, 2006). On the other hand, the subjection of
the sample to cold had little effect since it is closely similar to the control sample. Although cold
denaturation is rare, cooling the sample may also unfold the protein structure by subjecting to
lower temperature (Sanfelice and Temussi, 2016). The sample may have shown different results
if the temperature it is subjected is lower than of what is conducted during the experiment. Briefly,
extreme temperature promotes denaturation by unfolding the structures and breaking of weak
bonds between the linkages.
The denaturation of proteins is an important process for medical, commercial, industrial,
and other applicable purposes. It is important to understand the methods in order to utilize it
properly for future applications. An example of clinical application from protein denaturation is
from a study of Gaudet et.al (2010) regarding the utilization of protein denaturation for a
biophysical analysis of the interaction of FKBP-12 with rapamycin and to specifically determine
the energetic contribution of the interaction between its residue and the molecule which explains
the interaction of small molecule drugs with a protein and other advances that can be contributed
for biopharmaceutical formulations. Another example is the use of egg white as an antidote for
heavy metal poisoning as explained by Seager et. al (2018). The study explained that the egg
white has proteins whose structures consist of SH groups that can link itself to the ions of heavy
metals which is considered as an emergency protocol during heavy metal poisoning. These
advances with molecular biology and biochemistry not only help our scientists to understand the
concept even further but also to provide knowledge that is useful in the field of medicine and
physiology.
III. REFERENCES/LITERATURE CITED

BETTELHEIM, F.A., BROWN, W.H., CAMPBELL, M.K., FARRELL, S.O. 2007. Introduction to Organic
and Biochemistry, 6th edition. Belmont, CA: Thomson Brooks/Cole.

BRUICE, P.Y. 1996. Organic Chemistry. J. Chem. Educ. DOI: doi.org/10.1021/ed073pA92.2.

CASTELLION, M.E., MCMURRY, J. 1999. Fundamentals of General, Organic, and Biological

Chemistry, 3rd edition. Upper Saddle River, NJ: Prentice Hall.

GAUDET, M., REMTULLA, N., JACKSON, S.E., MAIN, E.R., BRACEWELL, D.G., AEPPLI, G., and
DALBY, P.A. 2010. Protein denaturation and Protein: Drugs interactions from intrinsic
protein fluorescence measurements at the nanolitre scale. Protein science: a publication
of the Protein Society, 19(8), 1544–1554. DOI:10.1002/pro.433.

NORTON, W.W. 2006. Heat Changes Protein Structure: Frying an Egg. Retrieved from Sumanas
Inc.:
http://www.sumanasinc.com/webcontent/animations/content/proteinstructure.html.

OPHARDT, C.E. 2003. Denaturation of Proteins. Retrieved from Virtual Chembook:

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

PURI, D. 2011. Textbook of Medical Biochemistry. Kundli, Haryana: Elsevier.

SANFELICE, D., & TEMUSSI, P. A. 2016. Cold denaturation as a tool to measure protein stability.
Biophysical chemistry, 208, 4–8. doi:10.1016/j.bpc.2015.05.007.

SEAGER, S.L., SLABAUGH, M.R., HANSEN, M.S. 2018. Chemistry for Today: General, Organic, and
Biochemistry, 9th edition. Chapter 19, Problem 19.56E. Boston, MA: Cengage Learning,
Inc.