The interaction of salt ions with water molecules removes the water barrier between protein molecules.
Protein precipitation is a method used to extract and purify proteins contained in a solution. Large, complex molecules, proteins often have negatively charged and positively charged parts, as well as hydrophilic and hydrophobic parts. There is a tendency for proteins in solution to clump together and precipitate due to the attraction between the negatively and positively charged parts of the molecules and the mutual attraction of the hydrophobic parts. Countering this tendency, however, is the fact that, in an aqueous solution, water molecules, which are polar, tend to organize around protein molecules due to the electrostatic attraction between oppositely charged parts of the water and the molecules. of protein. This results in the protein molecules being kept separate and remaining in solution,
Protein precipitation is a method used to extract and purify proteins contained in a solution.
The most commonly used method of protein precipitation is the addition of a salt solution, a technique often called “salting”. The most commonly used salt is ammonium sulfate. The interaction of the salt ions with the water molecules removes the water barrier between the protein molecules, allowing the hydrophobic parts of the protein to come into contact. This results in the aggregation of the protein molecules and precipitation from the solution. As a general rule, the higher the molecular weight of the protein, the lower the concentration of salt that is needed to cause the precipitation, therefore, it is possible to separate a mixture of different proteins in solution by gradually increasing the salt concentration, so that different proteins precipitate in solution. different stages, a process known as fractional precipitation.
The solubility of a protein in an aqueous medium can be reduced by the introduction of an organic solvent. This has the effect of reducing the dielectric constant, which in this context can be thought of as a measure of the polarity of a solvent. A reduction in polarity means that there is less tendency for solvent molecules to cluster around those of the protein, so there is less water barrier between the protein molecules and a greater tendency for protein precipitation. Many organic solvents interact with the hydrophobic parts of protein molecules, causing denaturization; however, some, such as ethanol and dimethyl sulfoxide (DMSO), do not.
Although proteins can have both negatively and positively charged parts, often in solution, they will have an overall positive or negative charge that varies with pH, and keeps them separate by electrostatic repulsion. Under acidic conditions, with low pH, proteins tend to have an overall positive charge, while at high pH, the charge is negative. Proteins have an intermediate point at which there is no overall charge – this is known as the isoelectric point, and for most proteins this is in the pH 4-6 range. The isoelectric point for a dissolved protein can be reached by adding an acid, usually hydrochloric or sulfuric acid, to reduce the pH to the proper level, allowing the protein molecules to clump together and precipitate. A disadvantage of this method is that acids tend to denature the protein, but it is often used to remove unwanted proteins.
Other protein precipitation methods include non-ionic hydrophilic polymers and metal ions. The former reduce the amount of water available to form a barrier between protein molecules, allowing them to clump together and precipitate. Positively charged metal ions can bind to negatively charged parts of the protein molecule, reducing the protein’s tendency to attract a layer of water molecules around it, again allowing the protein molecules to interact with each other and precipitate out of solution. Metal ions are effective even in very dilute solutions.