Electrofocusing and protein detection and isolation.

PROTEINS are generally least hydrated and least soluble at their respective isoelectric points. In this connection the process of electrofocusing of proteins (Svenson, 1962; Vesterberg & Svenson, 1966) is relevant. In this method several proteins are electrophoretically propelled-through an electrophoretically established pH gradient-until they reach the particular pH regions which equal their respective isoelectric points; there they remain stationary as their individual electrophoretic propulsive potentials disappear. In their electrofocused isoelectric pH regions, the individual proteins will be at their respectively least soluble conditions; and provided that the individual concentrations exceed their respective isoelectric point solubilities, precipitation should take place. (It should be noted here that the particular solubilities may differ somewhat depending upon the type ofmedium employed, e.g. ordinary aqueous solution or high density aqueous sucrose medium, or highly capillary medium such as acrylamide gel, where both buoyance and protein hydration may affect the solubility values. Also, the solubility, or suspendability, of a colloid should be affected when it is aligned in an electric field.) Protein separations and identification have been carried out using vertical columns of aqueous media in which the pH gradient is supported by a sucrose gradient; the presence of a protein can then be detected by using a fraction collector and appropriate tests, e.g. spectrophotometric. However, in this method, precipitation is undesirable because it interferes with separation and detection. Acrylamide gels have also been used for electrofocusing (Dale & Latner, 1968; Leaback & Rutter, 1968). However, such gel electrofocusing is normally followed by fixation of the protein on treatment with trichloracetic acid solutions followed by suitable staining. These procedures are necessary because the quantities of the proteins used, e.g. serum, are usually too low to result in the respective solubilities being exceeded at the particular isoelectric points. So far as the requirements for purification by precipitation are concerned, concentrations higher than those normally used would be necessary, when the proteins should appear as white precipitated bands at their respective isoelectric points. It must, however, be emphasized that the use of excessive quantities of proteins could well result in increase in respective band widths to an extent in which band fusion would result thereby making clean separation impracticable.