Principles of Biochemistry

8.2.4.1.2 Precipitation

Enzymes and other proteins are highly charged molecules, and can be precipitated with appropriate charge neutralizing chemicals. Once their charges are broken, they form aggregates and settle down as precipitate.

 

Acids and Bases

When, an acid or base is added, the enzyme protein can be brought to its isoelectric pH. At this pH, there is no net charge on enzyme molecules and electrostatic repulsion between them is low so that they tend to aggregate. Therefore, adjusting the pH to the isoelectric point of a protein causes its precipitation. Acids and bases, however, often inactivate the enzyme, so that their use for precipitation is not recommended in most cases.

 

Salts

Salts are used for precipitation in a process called 'salting out'. Salts can change the structure of the solvent, which can lead to large changes in protein conformation by altering the electrostatic interactions between charged groups on the protein surface. The salt also competes with the protein for solvent molecules and thereby lowers its solution.

 

Organic salvents

Some organic solvents like acetone, methanol and ethanol are also used for enzyme precipitation, since water miscible solvents decrease the solubility of proteins, leading to precipitation. They are cooled upto 40 → 60°C before their use, and precipitation is carried out at O°C, because precipitation at room temperature causes denaturation of the enzyme, in most cases. Organic solvents are added drop by drop to avoid local concentration.

 

Water soluble non ionic polymers

Water soluble non ionic polymers such as polyethylene glycol, alginate, pectate, carboxymethyl cellulose, polyacrylic and polymeta acrylic acids, etc. also cause enzyme precipitation. Polyethyleneimine is also widely used as protein precipitant at large scale. They primarily act through the removal of solvent sphere of the enzyme protein.