Post Lab Report on

Exercise No. 4

Protein Denaturation

BOHOLST, Elton John A.

CHEM 161.1 1L
Midyear 2017

ALVAREZ, Mark Louiegi

AMADO, Joel Magno
CAMO, Nestor Jr.
GARCIA, Angela Kaye

June 21, 2017

June 23, 2017

Ms. Rochelle P. Ibabao

 I. Data
Table 4.1. Observations on preparation of protein sample.
Reagents / Action Taken Observations
Spirulina tablet Black tablet
Fine silica White powder
Grounding of silica and tablet Smooth, greyish blue powder
Addition of phosphate buffer Creamy blue green mixture
Mixing of solution Creamy blue green mixture
Centrifugation
Supernatant liquid Blue solution with a tinge of red
Protein solution Turbid blue mixture
Residue Blue green powder

Table 4.2. Observations on the denaturation of phycocyanin from various denaturating agents.
Test Denaturating Agent Observations
tube No.
1 1 mL 6.0 M HCl Clear, yellow green solution with a tinge of
blue
2 1 mL 2.0 M NaOH Clear, yellow solution
3 1 mL 0.2 M lead acetate Heterogenous mixture with creamy white
precipitate and clear, colorless liquid
4 1 mL 10% trichloroacetic acid Clear, light blue green solution
5 1 mL acetone Cloudy, white solution
6 Hot water bath Clear yellow green solution
7 Cold water bath Clear turquoise solution
8 Control Clear turquoise solution

Table 4.3. Data on the Absorbance of Phycocyanin.

Wavelength, nm Absorbance
280 3.612
620 0.942
650 0.684

Table 4.4. Data on Calculation of Purity and Concentration of the Isolated Phycocyanin.
Parameter Values Remarks
CPC (mg/mL) 0.063
Purity of Phycocyanin 0.261

II. Results and Discussion

Amino acids are one of the four fundamental biomolecules together with carbohydrates,
lipids, and nucleic acids. When amino acids connect with each other via peptide bonds, it is
called a protein. Protein have four structures namely: primary, secondary, tertiary, and
quaternary. The primary structure of protein involves the linear chain of amino acids connected
via peptide bonds which dictates the structure, identity, and function of the protein. Then,
when the primary structure’s H-bonds and amide groups interact with each other and usually
form pleated sheet or helix structure, the protein achieved its secondary structure. Next, when
the amino acid side chains interact again, which can non-covalent such as H-bonding,
electrostatic interactions, and hydrophobic interactions or covalent such as disulfide linkage,
and forms a three-dimensional shape, the protein achieves its tertiary structure. This structure is
the highest biologically active architecture common for all proteins. Lastly, some proteins
achieved another structure known as the quaternary structure which is resulted from the
aggregation of individual polypeptide chains and stabilized by the same forces which stabilizes
the tertiary structure.

In this exercise, the concept of protein denaturation was studied. Protein denaturation is
defined as the disruption of the protein architecture except its primary structure. It can be
achieved physically via heating and other physical means or chemically via organic solvents,
strong acids and bases, and heavy metals. Denaturation also leads to loss of the protein’s
solubility, and biological activity (Nelson & Cox, 2005). The applications of this phenomenon
can be appreciated in the field of culinary and medicine.

The first part of the experiment was about the isolation of Phycocyanin from Spirulina. By
using a mortar and pestle, it was ensured that the maximum breaking of its multi-layered cell
wall was achieved. Various methods such as high pressure, freezing and thawing or lysozyme
digestion can also be used to achieve the breaking. Spirulina was also added to the grinding to
separate other components, via adsorption, of the Spirulina from the desired protein. After
grinding, the physiological pH of the cell was mimic by adding the phosphate buffer. Lastly, the
centrifugation was performed to separate the phycocyanin, as supernatant, from the residue.
The observations of the preparation of phycocyanin is tabulated in Table 4.1.

The second part of the experiment was performed to observe the effect of various
denaturating agents to the protein. The Table 4.2. tabulates the effect of various denaturating
agents.

Strong acids and bases affect the salt bridges in the protein. The salt bridges are the result of
the neutralization reaction between an acid and the amine found in the side chain of the protein.
These bridges will be disrupted of the strong electrostatic force of the charged ions (Ophardt,
2003). Therefore, the salt bridges will be broken and the protein’s structure will be disturbed.
The indication of this disruption in phycocyanin is the color change to light green to yellow.

Heavy metal salts behave similarly to acids and bases. It will also form an electrostatic
attraction with the charged side chains of the proteins. As a result, heavy metal ions will result
to insoluble metal salts. Based on the observations, addition of lead acetate formed a
white precipitate which is a lead salt.
 Organic solvents also interact with proteins since they can bond with the amine
group
through hydrogen bonding. In that case, the peptide chain will be broken leading to the
denaturation of protein. Color change to pale green and the formation of turbid mixture
were observed in the experiment.
Also, heat can affect the structure of the protein. Applying heat to the protein
molecule will result to the disruption of hydrogen bonding and non-polar hydrophobic
interactions. Addition energy is introduced to the molecules so the weakly bonds that
hold the protein together tend to unfold. Polar substances will likely react since the
hydrophobic region is exposed and unstable in aqueous solutions (Raven et.al, 2008).

The last part of the experiment was the calculation of concentration and purity of the
isolated protein. It was performed by determining the absorbance of the isolate under
280 nm, 620 nm, and 650 nm wavelengths. Table 4.3 tabulated the measured
absorbances of phycocyanin under these wavelengths. Using the formula number 1 in
sample calculations, the concentration of phycocyanin (CPC) expressed as mg/mL was
0.063 mg/mL. On the other hand, the purity of the sample was determined using
formula number 2 in sample calculations which was 0.261. With this value, it cannot be
concluded whether the sample is analytical grade or food grade since it was not within
the range indicated for the given grade.

III. Sample Calculations

1. Concentration of Phycocyanin:
mg [ A 620 −0.70 ( A650 ) ]
CPC ( )
mL
=
7.38
=0.0627642764 mg/mL
2. Purity of Phycocyanin:
A 620 0.942
Purity= = =0.2607973422
A 280 3.612

IV. References/ Literature Cited

Nelson, D. & Cox, M. (2005). Lehninger Principles of Biochemistry. 4 th Ed. New York: W.H.
Freeman

Ophardt, C. (2003). Virtual Chembook. Retrieved from http://chemistry.elmhurst.edu/

vchembook/568denaturation.html

Raven, P., Johnson, G., Mason, K., Losos, J. & Singer, S. (2008). Biology. 8 th Ed. USA:
McGraw- Hill Science