E1 Extraction and Isolation of Proteins

Student Nos. 2013-14715 2013-40963

Date Due: 3 February 2015 Date Submitted: 3 February 2015 EXPERIMENT NO. 1 EXTRACTION AND ISOLATION OF PROTEINS

Background of the Experiment The experiment is intended to extract and isolate the proteins casein and albumin from milk and egg white, respectively. The extracted proteins will be used in further experiments on protein properties. Results and Discussion Casein and albumin. Casein is a major protein in bovine milk, comprising about 80% of total protein content. The protein occurs in milk as a mixture of α-casein, β-casein, and γ-casein. α-casein and β-casein are known as phosphoproteins with phosphate groups present in the form of O-phosphoserine residues, while γcasein is known as the breakdown product of the β form. Casein has an isoelectric point (pI) of 4.7. (Stenesh, 1984). Egg albumin, also known as ovalbumin, has an almost similar isoelectric point of 4.6. Ovalbumin is known as a globular glycoprotein comprising of 2% neutral sugar and 1.2% acetyl hexosamine (Stenesh, 1984). Protein extraction and isolation. A series of processes known as protein extraction is used to obtain and isolate protein biomolecules. The broad range of processes can generally be divided in two categories: extraction, and isolation or purification. During extraction, the target protein is removed from its complex environment, with membranes, organelles and cellular structures broken down to free the protein of interest. This procedure is called homogenisation (Campbell, 2009). Extraction techniques greatly vary among different types of organisms. The extraction of animal proteins is relatively the most straightforward: the process only requires the application of sufficient stress for rupturing the plasma membrane, which is weakly supported by the cytoplasm. Compared to bacterial and plant cells, animal cells are very fragile and thus are more vulnerable to shear forces. Bacterial protein extraction is requires slightly more work, with the application of enzymatic methods such as lysozyme hydrolysis that cleave glycosidic linkages in the bacterial cell wall. The inner cell membrane is then disrupted through mechanical methods. For protein extraction in plants, the cells are either ground in the presence of acid-washed sand or flash frozen in liquid nitrogen to destroy its strong cell wall (Doonan, 1996). After extraction, several techniques are available for the isolation of the target protein. This includes the use of chromatography, high voltage electrophoresis (HVE), sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), the application of chemical separators, and mechanical methods such as centrifugation, with each dependent on different properties. SDS-PAGE depends on molecular size, while HVE depends on the protein’s pI, especially on its charge at a specific pH. On the other hand, chromatography and chemical separators typically depend on protein polarity (Martin et. al., 1983; Keller & Block, 1960) Albumin extraction. To extract albumin, 20 mL of egg white was stirred, while adding 2.0 mL of 1.0 M acetic acid. To further disrupt the cell membrane, the resulting solution was filtered through a damp cheesecloth and squeezed with a glass rod; the filtrate was collected in a 250 mL beaker. An equal volume of saturated ammonium sulphate was added to the filtrate to salt the proteins out. Salting in and salting out are isolation techniques that rely heavily upon the solubility of the proteins. When a salt is added to a mixture containing proteins, the added counterions surround the ionisable groups in protein, decreasing the protein-protein interactions and increasing the protein solubility. This effect is termed “salting in”. However, as more and more ions are added into the solution, the additional ions and protein molecules compete for the water molecules. At very high salt concentrations, the additional ions bind to the solvent molecules until there is significantly insufficient amount to hydrate the protein molecules. This results to

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.

Stenesh, J. (1984). Experimental biochemistry. New Jersey: Prentice Hall. Appendix Raw data: Albumin weight: Casein weight:

3.6724 g 4.0 g

Calculations for % w/v: Albumin:

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