MALABAD, Krista Mae P.

22 February 2016
2014-40856 CHEM 160.1 1L

EXER 3: POST-LABORATORY REPORT

Protein Denaturation

I. RESULTS

Table 2.1. Effect of the known denaturing agents on phycocyanin from Spirulina

Reagents Observations
6 M HCl The protein solution became pale blue (to green) in color but it was still
in clear, liquid state.
6 M NaOH The solution became yellow (to gold) in color but it was still in clear,
liquid state.
0.2 M lead acetate The protein solution contained two layers illustrating the difference in
the densities of the phycocyanin and lead acetate. Visually, it formed a
whitish unclear liquid mixture.
10% trichloroacetic The solution became pale in color but it was still in clear, liquid state.
acid
95% ethanol The content became grayish in color and it seemed cloudy. It was also
in liquid state.

Table 2.2. Effect of temperature on phycocyanin of spirulina

Water bath used Observations

hot The protein solution became pale in color after placing it in a hot water
bath, but it was still in clear, liquid state.
cold The protein solution became darker in color after placing it in a cold
water bath, but it was still in clear, liquid state.

II. DISCUSSION

Proteins are the most abundant biomolecule that is present in the human body. These
nitrogenous organic compounds exist in four different structures namely: primary, secondary,
tertiary, and quaternary. Proteins, unlike simple and small molecules, are complex in formation
and contains various amino acids as subunits.

Particle Sciences (2009), an organization staffed by experienced industry experts who

offer drug product formulations, gave concise definitions for these structures. These definitions
include the bonds, linkages, and forces of attractions that maintain the structural composition of
proteins. They defined the primary structure as covalently-bonded long chains which form peptide
bonds, specifically amide bonds, between the -NH2 of a certain amino acid and the -COOH of
another. Next, the secondary structure is dependent on hydrogen bonding. There are two main
types of secondary structures which are the α-helix and the ß-sheet. Hydrogen bonds are
responsible for these helix structures and peptide bonds can also be identified. The other structure,
which is called the tertiary structure, is produced by many stabilizing forces due to bonding
interactions between the side-chain groups of the amino acids. Hydrogen bonds, disulfide bridges,
as well as ionic interactions (which are present in salt bridges), are also involved. Lastly,
quaternary structures of proteins are made up of multiple polypeptide chains which are arranged

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complexly. This structure is stabilized by various interactions such as: hydrogen-bonding, disulfide
bridges, and salt bridges (Particle Sciences, 2009).

In the exercise performed by the laboratory students of CHEM 160.1 1L, protein
denaturation was observed. From the definition by Ophardt (2003), the denaturation of proteins
can be characterized by the disturbance and possible destruction of the secondary, tertiary, or
quaternary structures of a certain protein. Because of denaturation that is caused by either
chemical or physical means, decrease or total damage of the biological activity of a certain protein
can be observed such as: precipitation and color change (Institute of Chemistry, University of the
Philippines - Los Baños, 2013). By observing the contents from 7 test tubes where 5 different
reagents and 2 varying temperatures were applied, it can be perceived how denaturation worked
within these containers due to the change in color, occurrence of precipitate, and variation in the
appearances of the solutions.

Comparing the first test tube with the control (8th test tube), where 1 mL of 6M HCl was
added, the protein solution became pale in color but it was still in clear, liquid state. There was a
color change due to the addition of a strong acid, in the presence of HCl. Adding strong acid to
the protein disrupts the intermolecular forces, and the tertiary structure becomes absent
(Chemical Education Digital Library, 2011).

In the second test tube where 1 mL of 6M NaOH was added, the solution became
yellowish in color and was in clear, liquid state. This is due to the loss of positive charges and
deprives protein of the salt bonds (Puri, 2011).

Combining the observations identified from the addition of HCl and NaOH, it can be
concluded that the addition of strong acids and bases disrupt salt bridges held together by ionic
charges (Ophardt, 2003). He also stated in his article that a type of double replacement reaction
occurs where the positive and negative ions in the salt change matches up with the positive and
negative ions in the new acid or base added.

In the third test tube, a pinch of 0.2 M lead acetate was added. After letting it stand for a
few seconds, it can be observed that the protein solution contained two layers illustrating the
difference in the densities of the phycocyanin and lead acetate. There was a formation of whitish
unclear liquid mixture with white precipitate staying at the bottom of the tube. The reagent added
falls under the category of heavy metal cation wherein, if it is added to a protein, the reaction
usually leads to an insoluble metal protein salt (Ophardt, 2003).

This is followed by the effect of 1 mL of 10% trichloroacetic acid which is known to be an

alkaloidal reagent. After adding this substance, the solution became pale in color but it was still
in clear, liquid state. These reagents associate with positively charged amino groups in proteins
to unsettle ionic bonds (UC Davis Chem Wiki, n.d.).

The next test tube with phycocyanin, was added with 1 mL of 95% ethanol. Ethanol is an
organic solvent and is also classified as an alcohol. After placing ethanol inside the container, the
content became grayish in color and it seemed cloudy. This is due to the disruption of hydrogen
bonds between amide groups in the secondary structure of the protein and also hydrogen bonds
between the side chains occurring in its tertiary structure (Ophardt, 2003). The author also added
that new hydrogen bonds are formed between the new alcohol molecule and the protein side
chains.

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 Temperature also denatures proteins. This can be evidently seen by observing test tubes
6 and 7. Test tube 6 was subjected to heat while test tube 7 was placed in a cold bath. When
protein is exposed to high temperature, proteins vibrate violently, their weakest bonds break, and
their strings of amino acids begin to loosen (Norton & Sumanas, 2006). On the other hand, if a
protein is exposed to low temperature, the process is enthalpically driven, leads to partial
unfolding of the polypeptide chain, and is a result of change in interaction between water and
hydrophobic groups (Ganesan, 2015). She also added in her article that with the decrease in
temperature, the unfavorable interaction of nonpolar residues with water decreases, thus
increasing their hydration.

In general, temperature promotes denaturation by breaking weak bonds, loosening amino

acids, and increasing their hydration.

Protein denaturation, negative as it seems, is a process which benefits the field of clinical
applications. An example would be microbial control by sterilization. Alcohol denature proteins
and dissolve membrane lipids as an effective means of skin and instrument disinfection for
microbial control (Czura, n.d.). Also, medical supplies and instruments are sterilized by heating to
denature proteins in bacteria and thus destroy the bacteria (Ophardt, 2003). Another case is by
using the method of protein denaturation as an antidote. This process can be used as a remedy
for lead or mercury poisoning. Das (2015) stated that egg whites can be given to an individual
who has ingested a heavy metal for egg whites are denaturated by the heavy metals and a
precipitate is formed; thus, vomiting is induced to eliminate the precipitate.

Overall, the denaturation of proteins is an important process for medical, commercial,

industrial, and other applicable purposes. It is significant to understand and be familiar with this
method in order to utilize it properly for future applications.

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 III. REFERENCES

Chemical Education Digital Library. (2011). Denaturation of Protein by Strong Acid. Retrieved
from Chemical Education Digital Library:
http://www.chemeddl.org/alfresco/service/org/chemeddl/nsdl/groups?id=chemeddl_0682
&guest=true
Czura, A. (n.d.). Physical Methods Of Microbial Control. Retrieved from Suffolk County
Community College: www2.sunysuffolk.edu/czuraa/.../BIO244Chapter6&7Handout.pdf
Das, K. (2015, May 16). Denaturation of Proteins. Retrieved from SlideShare:
http://www.slideshare.net/suvham/denaturation-of-proteins-48216503
Ganesan, S. (2015, February 12). How does cold denaturation of proteins happen? Retrieved
from Quora: https://www.quora.com/How-does-cold-denaturation-of-proteins-happen
Institute of Chemistry, University of the Philippines - Los Baños. (2013). Laboratory Instruction
Manual for CHEM 160.1: Introductory Biochemistry. Los Baños, Laguna.
Norton, W. W., & Sumanas. (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
Particle Sciences. (2009). Protein Structure. Retrieved from Particle Sciences:
http://www.particlesciences.com/news/technical-briefs/2009/protein-structure.html
Puri, D. (2011). Textbook of Medical Biochemistry. Kundli, Haryana: Elsevier.
UC Davis Chem Wiki. (n.d.). 18.4 Proteins. Retrieved from UC Davis Chem Wiki :
http://chemwiki.ucdavis.edu/Textbook_Maps/General_Chemistry_Textbook_Maps/Map
%3A_The_Basics_of_GOB_Chemistry_(Ball_et_al.)/18%3A_Amino_Acids,_Proteins,_a
nd_Enzymes/18.04_Proteins

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