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
280
620
650
Absorbance
3.612
0.942
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:
[𝐴620 − 0.70(𝐴650 )]
𝑚𝑔
𝐶𝑃𝐶 ( ) =
= 0.0627642764 mg/mL
𝑚𝐿
7.38
2. Purity of Phycocyanin:
𝐴620
0.942
𝑃𝑢𝑟𝑖𝑡𝑦 =
=
= 0.2607973422
𝐴280
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