Protein precipitation is a method to extract and purify proteins. Adding salt, organic solvents, or acid can reduce solubility and cause precipitation. Metal ions and nonionic hydrophilic polymers can also be used.
Protein precipitation is a method used to extract and purify proteins contained in a solution. Large and complex molecules, proteins generally have parts that have a negative electrical charge and parts that have a positive charge, as well as hydrophilic and hydrophobic parts. There is a tendency for proteins in solution to aggregate and precipitate due to the attraction between the negatively and positively charged parts of the molecules and the mutual attraction of the hydrophobic parts. Counteracting this tendency, however, is the fact that in an aqueous solution the water molecules, which are polar, will tend to arrange themselves around the protein molecules due to the electrostatic attraction between oppositely charged parts of the water and the protein molecules. This causes the protein molecules to be kept separate and remain in solution, but there are various methods of achieving protein precipitation.
The most commonly used method for protein precipitation is by adding a solution of a salt, a technique often referred to as “salting”. The most commonly used salt is ammonium sulphate. The interaction of salt ions with water molecules removes the water barrier between protein molecules, allowing the hydrophobic parts of the protein to come into contact. This results in the protein molecules clumping together and precipitating out of solution. As a general rule, the higher the molecular weight of the protein, the lower the salt concentration needed to cause precipitation, so it is possible to separate a mixture of different proteins in solution by gradually increasing the salt concentration, so that protein different precipitate in different phases, a process known as fractional precipitation.
The solubility of a protein in an aqueous medium can be reduced by introducing an organic solvent. This has the effect of reducing the dielectric constant, which in this context can be considered 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 that there is less water barrier between protein molecules and a greater tendency for proteins to precipitate. Many organic solvents interact with the hydrophobic parts of protein molecules, causing their denaturation; however, some, such as ethanol and dimethyl sulfoxide (DMSO), do not.
Although proteins can have negatively and positively charged parts, often, in solution, they will have an overall positive or negative charge that varies with pH, and will keep them apart by electrostatic repulsion. Under acidic conditions, with a low pH, proteins tend to have an overall positive charge, while at a high pH, the charge is negative. Proteins have a midpoint where there is no overall charge – this is known as the isoelectric point, and for most proteins it is in the pH range of 4-6. 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 appropriate level, allowing the protein molecules to clump together and precipitate. A disadvantage of this method is that the acids tend to denature the protein, but it is often used to remove unwanted proteins.
Other protein precipitation methods include nonionic hydrophilic polymers and metal ions. The former reduce the amount of water available to form a barrier between protein molecules, allowing them to aggregate and precipitate. Positively charged metal ions can bind with 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.
Protect your devices with Threat Protection by NordVPN
