Separation of the Protein Fraction from Chicken Liver

Proteins play many critical roles in the body and do most of their work in cells. Albumin, however,
is a protein made by the liver and is excreted into the blood, therefore present inside and outside the cell.
In the experiment, protein extraction was done by homogenizing a chicken liver in 7.4 pH phosphate
buffer solution using a cold blender. Due to the laminar flow and shear forces created by the rotation of
the blades (Source), the tissues were disrupted and albumin was extracted. A 7.4 pH phosphate buffer
was to simulate the physiological pH, preventing the denaturing of the proteins.

Isolation of Albumin
After the extraction, albumin was then isolated by means of fractionation using ammonium
sulfate. Albumin is a single chain protein that is ionized, containing a negative net charge, at the
physiological pH of 7.4. It is water soluble but consists of hydrophobic patches, and plays as a transport
protein for molecules through the plasma (Raoufinia, et al. 2016). By slowly increasing the concentration
of salt in the solution, impurities can be removed and albumin can be isolated. Less soluble components,
such as non-polar molecules, membranes and fat, precipitate at 25% saturation and was removed after
the solution was centrifuged. Albumin was precipitated out of the solution by further increasing the salt
saturation of the solution, decreasing the solubility of the protein.
Protein solubility is complex and varies depending on the pH, temperature and salt
concentration of the solution (Lehninger). The surface tension of water increases with the salt
concentration, increasing the hydrophobic interaction between water and protein. The protein
responds by decreasing its surface area to minimize the contact with the solvent, leading to
precipitation (Wingfield, 2001). Additionally, protein solubility is inversely related to the ionic
strength of a solution. With increasing salt concentration, the extra counter ions’ multiple ionic
charges shield the protein molecules more effectively, further supporting the salting out of the
protein (Nahar et al. 2017).
Ammonium sulfate is commonly used to increase the ionic strength of a solution because,
empirically, it is known that polyvalent anions are more effective at salting out univalent anions, while
polyvalent cations negate this effect. Anions can be arranged in terms of their relative effectiveness in
salting out, this is called the Hofmeister series (Dennison, 2003). In decreasing order: citrate > sulfate >
phosphate > chloride > nitrate > thiocyanate. Ammonium sulfate is commonly used, instead of citrate, due
to its availability and ability to stabilize the protein structure and not denature it (Wingfield, 2001).

Hydrolysis
By increasing the acidity of the solution, the proteins were hydrolyzed and were broken down into
its monomeric subunits, amino acids. The hydrolysis of a peptide, though an exergonic reaction, occurs
slowly due to its high activation energy (Lehninger). To speed up this process, the solutions were heated
to boiling for thirty minutes. A marble was placed on top of the test tube to prevent the vapor from
escaping. Upon dissociation of amino acids, the solutions were neutralized so that the amino acids return
to their form at neutral pH.

Qualitative Tests
There are numerous available qualitative tests developed to identify or classify proteins and
amino acids and they commonly involve precipitation or a color change. Four tests were to be preformed
for the four solutions, but only three was feasible due to lacking reagents. The four test tubes (P1, P2,
HP1, HP2) were subjected to the biuret test, ninhydrin test and xanthoproteic test. Though Sakaguchi test
was not possible it will still be discussed.

The biuret test is a chemical test to detect the presence of two or more peptide bonds, thus
dipeptides and free amino acids will test negative. Peptide bonds are indicated by the purple-violet color
of the solution. This color is caused by the coordination of the cupric ions with the electron pairs of the
nitrogen in the peptide to form a complex (Chatterhaa, 2013). Table 1 shows that both HP1 and HP2
showed negative results while P1 and P2 were positive. HP1 and HP2 contain the hydrolyzed albumin
and, therefore, gave the negative result.

Ninhydrin detects the presence of amino acids. Ninhydrin is reduced by an α-amino acid and,
depending on the amino acid, forms dyes varying in color: pink, purple or blue (Ruhemann’s purple),
yellow or brown. Proline gives a yellow color while amide containing amino acids form a brown color
(Vasudevan, 2013). As seen in table 1, P1 and P2 turned purple, which was caused by the protein. HP1
and HP2, however, turned golden brown which indicates the presence of amide containing amino acids.

Xanthoproteic test is used to detect the presence of aromatic amino acids. Yellow precipitate is
formed when heated with concentrated HNO3 due to the nitration of the aromatic rings. The color is
intensified by turning the solution basic (Chatterhaa, 2013). Table 1 shows that P1 and P2 confirms the
presence of aromatic residue in the protein while results are negative for the hydrolyzed test tubes.

Lastly, Sakaguchi test is a specific test for arginine of a protein. Free arginine or arginyl residue
reacts to give a bright-red color, which is due to the guanidine group (Vasudevan, 2013). Theoretically, all
four test tubes should produce this bright-red color.

Raoufinia, R., Mota, A., Keyhanvar, N., Safari, F., Shamekhi, S., & Abdolalizadeh, J. (2016). Overview of
Albumin and Its Purification Methods. Advanced pharmaceutical bulletin, 6(4), 495–507.
doi:10.15171/apb.2016.063

Nahar, M. K., Zakaria, Z., Hashim, U., & Bari, M. F. (2017). Effect of pH and Salt Concentration on Protein
Solubility of Slaughtered and Non-Slaughtered Broiler Chicken Meat. Sains Malaysiana, 46(5), 719–724.
doi: 10.17576/jsm-2017-4605-06

Dennison, C. (2003). A Guide to Protein Isolation. Focus on Structural Biology. doi: 10.1007/978-94-017-
0269-0

Wingfield P. (2001). Protein precipitation using ammonium sulfate. Current protocols in protein
science, Appendix 3, Appendix–3F. doi:10.1002/0471140864.psa03fs13

Chatterhaa, M. N., & Shinde, R. (2013). Textbook of medical biochemistry. New Delhi: Jaypee Brothers
Medical Publishers (P) Ltd.

Vasudevan, D. M., Sreekumari, S., & Vaidyanathan, K. (2013). Textbook of biochemistry for medical
students. New Delhi: Jaypee Brothers Medical Publishers (P) LTD.