Molecular Biology: Protein Structure and Functions - Stepanov V.M. 2005

Protein Isolation
Characteristic properties of proteins underlying their separation

What PHYSICOCHEMICAL PROPERTIES OF Proteins can their Separation Methods be based on? First, it is the size and geometry of the molecule. Methods such as Gel filtration, ultrafiltration, and partly gel Electrophoresis rely on this feature.

Second, the distribution of charged groups on the protein's surface is characteristic of a given protein. The ratio of cationic to anionic groups in a protein varies depending on the pH. The isoelectric points of proteins—pI (the pH value at which positive and negative charges of the protein are completely balanced and the net charge is zero)—vary significantly among different proteins. Some proteins are cationic under physiological conditions, others are anionic, and some are molecules without any noticeable predominance of either charge. The differences in protein charge at varying pH values form The basis of their separation by electrophoresis, isoelectric focusing, as well as isoelectric and Ion-exchange Chromatography. Importantly, however, it is not just the ratio of charged groups that determines the pI value. Proteins with similar isoelectric points may differ in the surface distribution of their charged functional groups. These groups can be arranged more or less uniformly or, conversely, form local clusters and patches of identically charged groups, which significantly affects protein ion-exchange chromatography.

Third, proteins differ in the number and nature of Structure/109.html">Hydrophobic surface regions, which is exploited in Hydrophobic Interaction Chromatography.

Note that none of the features discussed above can, on its own, ensure the isolation of an individual protein from a complex mixture—they are not sufficiently specific and do not guarantee selective purification. Utilizing the Functional Properties of proteins for their isolation is much more promising in this regard. Indeed, among the multitude of proteins in the starting material, there will be many with similar molecular weights or close isoelectric points; however, the number of, say, Phosphatases or amylases will be inherently small. Obviously, an isolation method based on the ability of these Enzymes to interact with their specific substrate is incomparably more selective than any technique relying on differences in physicochemical properties.

Protein Purification schemes that rely on a single principle alone are rare; typically, various fractionation approaches are combined and Complement one another.