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Wednesday, May 4, 2011

Protein Separation Based on Different Solubility Characteristics

Proteins can be separated by exploiting differences in their solubility in aqueous solutions. The solubility of a protein molecule is determined by its amino acid sequence because this determines its size, shape, hydrophobicity and electrical charge. Proteins can be selectively precipitated or solubilized by altering the pH, ionic strength, dielectric constant or temperature of a solution. They are often used as the first step in any separation procedure because the majority of the contaminating materials can be easily removed.

Salting out

Proteins are precipitated from aqueous solutions when the salt concentration exceeds a critical level, which is known as salting-out, because all the water is "bound" to the salts, and is therefore not available to hydrate the proteins. Ammonium sulfate [(NH4)2SO4] is commonly used because it has a high water-solubility, although other neutral salts may also be used, e.g., NaCl or KCl. Generally a two-step procedure is used to maximize the separation efficiency. In the first step, the salt is added at a concentration just below that necessary to precipitate out the protein of interest. The solution is then centrifuged to remove any proteins that are less soluble than the protein of interest. The salt concentration is then increased to a point just above that required to cause precipitation of the protein. This precipitates out the protein of interest (which can be separated by centrifugation), but leaves more soluble proteins in solution. The main problem with this method is that large concentrations of salt contaminate the solution, which must be removed before the protein can be resolubilzed, e.g., by dialysis or ultrafiltration.

Isoelectric Precipitation

The isoelectric point (pI) of a protein is the pH where the net charge on the protein is zero. Proteins tend to aggregate and precipitate at their pI because there is no electrostatic repulsion keeping them apart. Proteins have different isoelectric points because of their different amino acid sequences (i.e., relative numbers of anionic and cationic groups), and thus they can be separated by adjusting the pH of a solution. When the pH is adjusted to the pI of a particular protein it precipitates leaving the other proteins in solution.

Solvent Fractionation

The solubility of a protein depends on the dielectric constant of the solution that surrounds it because this alters the magnitude of the electrostatic interactions between charged groups. As the dielectric constant of a solution decreases the magnitude of the electrostatic interactions between charged species increases. This tends to decrease the solubility of proteins in solution because they are less ionized, and therefore the electrostatic repulsion between them is not sufficient to prevent them from aggregating. The dielectric constant of aqueous solutions can be lowered by adding water-soluble organic solvents, such as ethanol or acetone. The amount of organic solvent required to cause precipitation depends on the protein and therefore proteins can be separated on this basis. The optimum quantity of organic solvent required to precipitate a protein varies from about 5 to 60%. Solvent fractionation is usually performed at 0oC or below to prevent protein denaturation caused by temperature increases that occur when organic solvents are mixed with water.

Denaturation of Contaminating Proteins

Many proteins are denatured and precipitate from solution when heated above a certain temperature or by adjusting a solution to highly acid or basic pHs. Proteins that are stable at high temperature or at extremes of pH are most easily separated by this technique because contaminating proteins can be precipitated while the protein of interest remains in solution.


References

http://www-unix.oit.umass.edu/~mcclemen/581Proteins.html

http://www.biochem.arizona.edu/classes/bioc462/462a/NOTES/Protein_Properties/protein_purification.htm