Chemistry and Biology of Proteins - F. Haurowitz 1953

Methods for Protein Isolation, Purification, and Characterization
Fractionation of Protein Mixtures

Extracts derived from Cells or Organs contain mixtures of various Proteins. Blood serum and other Body Fluids are also heterogeneous in their protein composition. By carefully increasing or decreasing the Ionic strength of the solution (or its dielectric constant) and gradually adjusting the pH to the isoelectric point of various proteins, one can isolate pure proteins or fractions containing predominantly a single protein from protein mixtures. The classical method for obtaining albumins and globulins is based on the fractional addition of ammonium sulfate (see ch. VIII, p. 172).

In many studies, the concentration of ammonium sulfate used for Protein Isolation is expressed as a percentage of saturation. For example, a solution containing equal parts of Water and a saturated solution of ammonium sulfate is considered half-saturated (50% saturation). This designation method is not entirely precise, because saturation depends to some extent on the solution's Temperature. It is therefore preferable to express the concentration of ammonium sulfate in a manner more standard in chemistry, such as in moles. Cohn and his coworkers successfully applied the method of fractional precipitation with ammonium sulfate and ethanol to separate Serum proteins. Using this method, they obtained A large number of fractions containing biologically active proteins from serum (see ch. VIII). When fractionating large quantities of protein, the alcohol method has advantages over other Methods, since most of the alcohol is easily removed by evaporation, whereas salt removal via dialysis is time-consuming and laborious. An essential feature of the new method is that during protein precipitation with alcohol, the ionic strength, hydrogen ion concentration, temperature, and Dielectric Constant of the solution are systematically varied. By varying the aforementioned factors, it was possible to isolate a large number of new proteins from blood serum.

In some cases, the ISOLATION OF A pure protein from protein mixtures is facilitated by the selective Denaturation of accompanying proteins. For example, a crude Trypsin preparation can be purified by heating its solution in 0.5 N $ ext{HCl}$ to 90° [18], since under these conditions all inert proteins are denatured.