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2013-14715
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more protein-protein interactions compared to protein-water interactions, decreasing protein solubility and salting
the proteins out. Ammonium sulphate was chosen due to the observation that alkali and magnesium salts of
sulphates and acetates are more powerful protein precipitants than others (Stenesh, 1984; Keller & Block, 1960).
By salting out, the equal volume of salt pushed the proteins out of the water. The differences among the
isoelectric points and solubilities of albumin and other substances present in egg white allowed albumin to remain
suspended in the solution despite the intended salting out effect. The resulting mixture was centrifuged for 10
minutes in order to separate the albumin from unwanted cell debris and contaminating proteins. As a result,
albumin stayed at the supernatant while the cell debris, which was then discarded, precipitated at the bottom of
the centrifuge tube. The solution was salted out again with equal volume of ammonium sulphate, allowed to
completely react for 10 minutes, and centrifuged again in a pre-weighed tube to separate the proteins from the
cytosol. This time, the resulting supernatant was discarded and the precipitate was collected.
3.6724 g of albumin was produced from the precipitate. This was dissolved in 10 mL 0.9% sodium
chloride to prevent denaturation and enzymatic activity, producing a 36.72 g/100 mL w/v (%) solution.
Casein extraction. For casein extraction, 15 mL of milk was treated with 0.1 M HCl to lower the pH to 4,
at about the isoelectric point of casein. This was performed in order to lower the solubility of casein in the
solution, as the isoelectric point is the pH at which protein solubility is lowest. This lowering of solubility is
caused by the maximisation of ionisable groups in the protein, making the different ionisable groups compete for
the water molecules that not enough remains to hydrate all these groups. In a process similar to salting in and
salting out, the competition results to more protein-protein interactions that decrease protein solubility (Keller &
Block, 1960; Stenesh, 1984). HCl treatment produced flocculent precipitation in the solution. Centrifugation was
performed to separate the casein from other substances in milk.
The precipitate of milk was washed twice with 1 mL 95% ethanol, and once with 1 mL acetone, in order
to remove any residues that remained with the precipitate. The separating action was caused by the low dielectric
constants of organic solvents (Keller & Block, 1960). The final precipitate was air-dried to remove any additional
fluids that may add to the casein weight measurement.
4.0 g casein was produced from the air-dried product. The crude casein extract was dissolved in 10 mL
0.01 M NaOH to prevent denaturation, producing 40 g/mL w/v (%).
Summary, Conclusions, and Recommendations
A 36.72 g/100 mL w/v (%) albumin solution and a 40 g/mL w/v (%) casein solution was produced from
the experiment. Physical methods, including centrifugation and homogenisation, were used to extract and isolate
the proteins in question. Chemical methods on reducing protein solubility were also applied, especially through
the addition of acid and inorganic solvents.
The methods used in the experiment are mainly rudimentary procedures. In order to improve the quality
and yield of the protein of interest, it is recommended to employ techniques that are more specific to the protein
of interest, and are more accurate in measuring the properties of the biomolecule in question. Some of these
techniques include, but are not limited to, gel filtration chromatography, high voltage electrophoresis and
polyacrylamide gel electrophoresis.
References
Campbell, M. (2009). Biochemistry (6th ed.). Philadelphia: Saunders College.
Doonan, S. (1996). Protein purification protocols. Totowa, N.J.: Humana Press.
Keller, S., & Block, R.J. (1960). Separation of proteins. In P. Alexander & R.J. Block, A laboratory manual of
analytical methods of protein chemistry (including polypeptides) (pp. 1-30). New York: MacMillan
Martin, D.W. Jr., Mayes, P.A., & Rodwell, V.W. (1983). Harper's review of biochemistry (19th ed.). Los Altos:
Lange Medical.
3.6724 g
4.0
g
albumin concentration=
Casein:
casein concentration=
3.6724 g 10
=36.72 g /100 mL w /v
10 mL
10
4 .0 g 10
=40 g /100 mL w /v
10 mL 10