Structural Biochemistry/Proteins/Purification/Salting Out

Introduction

The process of "salting out" is a purification method that relies on the basis of protein solubility. It relies on the principle that most proteins are less soluble in solutions of high salt concentrations because the addition of salt ions shield proteins with multi-ion charges. Those charges help protein molecules interact, aggregate, and precipitate. The exact concentration resulting in precipitation varies from protein to protein, allowing for the separation of different proteins (as proteins will precipitate at different points with increases in salt concentration). Salting out can also concentrate dilute solutions of proteins; once the protein precipitates, the remaining liquid can be removed. However, the salt can pose a problem to the purity of protein.

"Salting in" refers to the observation that at solutions of low salt concentrations, the solubility of a protein increases with the addition of salt. As the solubility of the salt is higher than that of the protein, it is more likely dissolve and take up space in the solution; therefore, proteins aggregate and precipitate. By contrast, "salting out" requires high salt concentration for the precipitation of the protein. There are two ways of "salting out". In one method, proteins are exposed to high concentrations of salt solutions, and in the other, the proteins are exposed to a series of low concentrated solutions.

Proteins contain various sequences and compositions of amino acids. Therefore, their solubility to water differs depending on the level of hydrophobic or hydrophilic properties of the surface. Proteins with surfaces that have greater hydrophobic properties will readily precipitate. The addition of ions creates an electron shielding effect that nullifies some activity between water particles and the protein, reducing solubility as the proteins bind with each other and begin to aggregate. Generally, larger proteins require less ionic input than do smaller proteins with lesser weight.

In the process of using low concentrations of salt solutions, the proteins are precipitated early in the process. In order to extract the proteins from the solution, cold solutions of ammonium sulfate at a series of decreasing concentrations are used on the precipitate. In order to recover the extracted protein, it is then recrystallized by warming the cold solution to room temperature. This process has many advantages because depending on the extracted protein, the efficiency rate can run anywhere from 30-90%, and rarely fails.

Ammonium sulfate is common substance used to precipitate proteins selectively since it is very soluble in water, it allows high concentration about 4M. At this state, harmful effects of proteins like irreversible denaturation are absent and NH_4⁺ and SO_4^2- are both favourable, non-denaturing, end of the Hofmeister series. Ammonium sulfate provides quantative precipitation of one protein from the mixture. This method is very useful to purify soluble proteins from the cell extracts.⁴

While proving itself to be an efficient method of protein separation, salting out requires that the solubility of the protein to be calculated or known initially. Proteins have differing amino acid chains and solubility. In trying to change the salt concentration to the point where the protein becomes insoluble, different ions can either increase or decrease the solubility of the protein. Hence, one must be careful in selecting the correct ions to alter salt concentration. A protein is typically least soluble near its isoelectric point, pI, or where it contains minimal net charge. The precipitation by salting out results in fractionation. An amount of precipitated protein is collected at one salt concentration and another amount from a different concentration. This is because some parts of the protein may be more soluble than another region.

Proteins with different pI values can be separated with salting out techniques via dynamic pH values in varying salt concentration. Since proteins are least soluble near their isoelectric point (pI), it is possible to cause them to precipitate them out of solution by increasing the salt concentration. This is possible since the hydration shell surrounding the protein structure is displaced by the increasing ionic concentration in the solvent. Thus by replacing the hydration shell with other ions, the water networks that solubilize proteins become destabilized and allow for aggregation at high salt concentration due to hydrophobic groups coming together. Ultimately proteins are precipitated with aggregation (or "crashed out"). This technique can be used to separate proteins that initially have similar precipitation points. By modifying the pH of the solution, one can increase or decrease the solubility of one protein without affecting the target protein. Furthermore, the solution can later be purified by using dialysis to remove the salt ions in solution.

Dialysis Process

Hofmeister Series

The effectiveness of the different ions was established by Franz Hofmeister in 1888. The first ion in the anion and cation series is the most effective in precipitating a protein out (dubbed "kosmotropes": ions that interact well with water, forming H-bonds and dehydrating proteins), and the ions at the end are the least ("chaotropes": ions that free up water by breaking H-bonds between water molecules, increasing protein solubility). ^

Cations: N(CH₃)₃⁺ > NH₄⁺ > K⁺ > Li⁺ > Mg²⁺ > Ca²⁺ > Al³⁺ > guanidinium

Anions: SO₄^2- > HPO₄^2- > CH₃COO^- > citrate > tartrate > F^- > Cl^- > Br^- > I^- > NO₃^- > ClO₄^- > SCN^-

The starting molecules strengthen hydrophobic interactions by decreasing solubility of the nonpolar molecules, thus salting out the system. However, the later molecules begin to denature the structure of the protein because of strong ionic interactions that disrupt hydrogen bonding. Although the later molecules can be salted out through solutions such as Ammonia Sulfate, certain molecules can also experience salting in, where the solubility of the protein increases through the later molecules of the list.

Dialysis

Dialysis is a protein purification process that separates proteins from other small molecules, such as salt, by using a semipermeable membrane. This membrane contain micro pores through which the small molecules will escape. Therefore, protein molecules having dimensions significantly greater than the pore diameter are retained inside the dialysis bag. The small molecules and salt will diffuse out through the membrane and into the dialysate outside of the bag. This technique is useful to remove salt ions and other small molecule but can not be used to distinguish proteins. To enhance the separation of the proteins in the bag from other impurities such as salt we can also take advantage of the equilibrium constants. In an aqueous environment the salt will flow through the plasma membrane until its concentration outside the dialysis bag is equal to the concentration inside the bag. At this point there is no net flow of salt through the membrane because equilibrium is reached. But if we add in a new solution of buffer, then the remaining amount of salt will then flow out of the dialysis bag until the concentration of salt in the new buffer equals the concentration in the dialysis bag. If we keep replacing the buffer solution this will enhance the purity of the proteins inside the dialysis bag because each time we replace the buffer the salt has to flow out inorder to attain its equilibrium constant. This principle can also be applied for other impurities that are able to escape through the membrane.

Dialysis in human body

In kidney-compromised patients, dialysis is often used as a procedure for removing undesirable solutes in the blood. For example, the calcium, potassium, and urea concentration of the dialysate is kept at low concentrations, enabling the target solutes in the blood to diffuse across the semi-permeable membrane. However, this entails the dialysate to be constantly cleaned in order to prevent concentration equilibrium, which would ultimately lead to a rising concentration of unwanted solutes in the blood. In another case, solutes can also be introduced into the blood. For example, bicarbonate ions are in high concentration in the dialysate, which diffuse across the membrane. This is done to prevent metabolic acidosis.

References

1. Berg, Jeremy M. 2007. Biochemistry. Sixth Ed. New York: W.H. Freeman. 68-69, 78.

2. Voet, Voet, Pratt (2004). - Fundamentals of Biochemistry

3. [[1]] Atlas of Diseases of the Kidney, Volume 5, Principles of Dialysis: Diffusion, Convection, and Dialysis Machines

4 "Chapter 9: Protein expression, purification and characterization", Proteins: Structure and Function, Whitford, 2005, John Wiley & Sons, Ltd