Biological Chemistry - Berezov T. T., Korovkin B. F. 1998

Protein Chemistry
Methods for Protein Isolation and Purification
Removal of Low-Molecular-Weight Impurities from Proteins

Applying the aforementioned Methods in a specific sequence yields a purified protein that, however, still contains some residual salts. To completely rid Proteins of low-molecular-weight impurities, modern laboratory practice employs dialysis, Gel Chromatography, crystallization, and ultrafiltration. Dialysis utilizes semipermeable membranes (such as cellophane or collodion film) with a wide range of pore diameters. As a rule, proteins do not diffuse through such membranes, whereas low-molecular-weight substances easily pass through them into the surrounding medium.

The protein crystallization method relies on reaching the critical threshold for protein precipitation from an ammonium sulfate solution by slowly raising the Temperature. Hundreds of crystalline proteins have already been obtained*. However, not every crystalline protein is homogeneous, since different proteins of similar size and mass may crystallize at the exact same concentration of the ammonium sulfate solution.

The best results in freeing proteins from low-molecular-weight impurities are achieved through gel chromatography and ultrafiltration. The latter is based on forcing protein solutions through special membranes that retain protein molecules, which not only removes low-molecular-weight impurities from protein solutions but also allows them to be concentrated.

* The first crystalline enzyme, urease, was obtained by J. Sumner in 1926.