Protein Chemistry - Part 1 - General Protein Chemistry - Ashmarin I. P. 1968

Isolation and Purification of Proteins
Removal of Non-Protein Substances

Generally speaking, this task is considerably simpler than the fractionation of Proteins themselves. Removing a significant portion of low-molecular-weight impurities can be achieved through repeated reprecipitation of the target protein, followed by redissolving it in media free of the unwanted compounds. Various precipitation Methods are described below. However, achieving complete Removal of Low-molecular-weight contaminants this way remains challenging. Furthermore, reprecipitation often necessitates introducing specific substances into the preparation, such as precipitants. Therefore, complete elimination of low-molecular-weight impurities requires dialysis (or electrodialysis), as well as molecular filtration using Sephadexes. The latter technique will be discussed later in the context of using Sephadexes for protein fractionation.

As for dialysis, A wide variety of techniques and devices have been developed to facilitate and accelerate this process. Selecting appropriate membranes is crucial; they must be sufficiently robust while remaining permeable to low-molecular-weight compounds. Cellophane is generally preferred, although collodion membranes can also be used in certain cases, provided they are rigorously tested for cracks. Dialysis is accelerated by periodically or continuously replacing the external solution, as well as by stirring both the inner and outer solutions. Haurowitz's recommendation is particularly noteworthy: he suggested positioning the dialysis bag containing the protein salt solution near the top of the vessel holding the external liquid. As the outer liquid layers near the bag rapidly enrich with salts, they become denser and sink to the bottom of the vessel, making way for fresh portions of the external liquid.

Significant challenges arise when high-molecular-weight impurities, such as Nucleic Acids and Polysaccharides, must be removed. Generally speaking, all protein fractionation techniques described below can also be applied to separate proteins from these Biopolymers. Additionally, nucleic acids and polysaccharides can be degraded using appropriate hydrolytic Enzymes. During the Isolation and Purification of Water-soluble proteins, Lipids typically do not co-purify, especially in multi-step purification schemes. However, removing lipids becomes extremely difficult when they form stable complexes with the protein, as lipid extractants—organic Solvents—can denature the protein.