Fundamentals of Biochemistry - Filippovych, Yu. B. 1999

Proteins
Protein Purification

Proteins isolated using the described Methods invariably contain certain amounts of low-molecular-weight impurities, particularly salt ions. To completely remove these impurities, proteins undergo further purification via dialysis, electrodialysis, ultrafiltration, recrystallization, Gel filtration, and other techniques.

Protein purification by dialysis involves the prolonged (several days) Circulation of Water through a vessel enclosing a dialysis bag. The bag is made of Materials highly permeable to low-molecular-weight compounds and ions while being impermeable to large protein molecules. Cellophane serves as a common semipermeable membrane material, the pore size of which can be adjusted over a wide range by Treatment with a ZnCl₂ solution. The protein solution is placed inside the dialysis bag (chamber), which is typically equipped with a capillary tube. During the initial hours of dialysis, a portion of the protein solution enters this tube as the volume increases due to water flowing into the chamber (Fig. 12.1).

Even after prolonged protein purification via dialysis, some ions adsorbed by protein particles remain. To completely remove these contaminating ions, electrodialysis is employed: electrodes connected to a voltage source are placed near the semipermeable membranes of the dialysis chamber, causing residual ions to migrate into the water washing the membranes and thereby be flushed out of the apparatus (Fig. 12.II).

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Fig. 12. Apparatus for protein purification by dialysis (I) and electrodialysis (II):

1 — water inlet tube; 2 — dialyzer body; 3 — dialysis bag; 4 — water outlet and level-regulation tube; 5 — capillary tube; A — dialysis chamber; B — electrodialyzer body; a and a₁ — semipermeable membranes; b and b₁ — electrodes; arrows indicate the direction of water flow

High-strength semipermeable membranes made of non-cellulosic materials with calibrated pores—which allow low-molecular-weight substances to pass while blocking macromolecules—are utilized for protein purification via ultrafiltration. The protein solution is forced through such a membrane using compressed gas or centrifugal force. The protein retained on the membrane during ultrafiltration is not only purified from low-molecular-weight compounds and ions but is also concentrated. By selecting membrane pore sizes that allow smaller proteins to pass while retaining larger protein molecules, ultrafiltration can also serve as a method for protein fractionation.

For a long time, it was believed that protein substances existed solely in an amorphous state. However, in 1906, A. D. Rozenfeld first crystallized oxidase isolated from radish, and in 1926, the American scientist J. Sumner obtained another crystalline protein—the enzyme urease (from Latin urea). In the subsequent years, dozens of proteins were successfully obtained in crystalline form.

It is now established that any protein can exist in a crystalline state. The various techniques for protein crystallization are all based on THE PRINCIPLE OF very slowly approaching the critical point at which a protein transitions from a dissolved state to a precipitate. Under these conditions, protein molecules have sufficient time to assemble into supramolecular aggregates in an orderly manner, forming crystalline structures. In practice, such slow protein precipitation is achieved by introducing salt through a semipermeable membrane or by slowly evaporating water from a salt-containing protein solution until the salting-out point is reached. Recently, protein crystallization using Organic compounds—such as 2-methyl-2,4-pentanediol and polyethylene glycol—has become widespread, enabling the crystallization of A number of proteins that resisted Traditional Methods.

Several types of protein crystals are shown in Fig. 13. Crystalline proteins, especially those obtained through recrystallization, exhibit a high degree of purity.

Gel filtration yields excellent results in purifying proteins from low-molecular-weight impurities. Sephadex beads retain low-molecular-weight substances for a sufficient duration, allowing the protein fraction to elute from the Column in a pure form within that timeframe. Filtration through Sephadex gel is widely applied for Desalting protein solutions.

Fig. 13. Types of protein crystals: Chymotrypsin (A), Pepsin (B), Yeast protein (C), Ribonuclease (I)