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

Isolation and Purification of Proteins
Separation of Protein Mixtures
Methods Based on Differences in Protein Solubility

In most cases, Protein Extraction from tissue is accompanied by a certain degree of purification. However, the initial extract is always a complex mixture of Proteins, and their complete Separation requires a combination of various Methods. Let us examine the principal ones.

The previous section showed that differences in Protein solubility allow for preliminary purification even at the stage of extraction from Cells and Tissues. This same principle underlies A number of methods for further fractionation and purification. By utilizing various combinations of factors such as salt concentration, dielectric constant, and environmental pH, highly efficient Separation of proteins with similar properties can be achieved.

Salting-out. The classical method for separating serum albumins and globulins is based on the precipitation of the latter using 2 M ammonium sulfate (50% saturation). Albumins are precipitated only by 4 M ammonium sulfate (100% saturation). Other neutral salts—such as sodium sulfate, magnesium sulfate, and mixtures of mono- and disubstituted phosphates—can also be used instead of ammonium sulfate. Nevertheless, ammonium sulfate remains the most widely used precipitating agent for proteins. This is primarily due to the combination of its high Water solubility and the fact that its solubility is practically independent of Temperature. For instance, the concentration of a saturated ammonium sulfate solution drops only from 4.1 to 3.9 M when the temperature goes from 25 to 0°C. Conversely, precipitants such as sodium sulfate have a limited range of application due to their low solubility at temperatures close to 0°C. Ammonium sulfate is also very convenient because it exerts no denaturing effect on even the most labile proteins known.

Under strictly controlled conditions of temperature, precipitation duration, and pH, ammonium sulfate and other salt precipitants can achieve quite subtle fractionation. For example, stepwise changes in ammonium sulfate concentration from 0 to 1.34 M, then to 1.64 M, and finally to 2.05 M allow serum globulin to be fractionated into alpha-, beta-, and gamma-globulins, respectively. All of these noted advantages of ammonium sulfate continue to be widely utilized by researchers in the initial purification stages of various proteins, particularly those that are poorly studied and labile. It should be noted, however, that all salt precipitants share a common drawback: the difficulty of removing them from the resulting preparation. This necessitates either prolonged dialysis or reprecipitation by other methods.

Protein separation at low Ionic strength. Selective precipitation of certain proteins is also possible at relatively low ionic strengths. In particular, the Muscle Proteins Actomyosin and Myosin are precipitated when tissue extracts are diluted with water to an ionic strength of 0.1–0.3 and 0.05–0.1, respectively. The myosin purification Procedure proposed by Mommaerts and Parrish is a very illustrative example of using different ionic strengths to efficiently remove impurities. A muscle mince extract obtained at an ionic strength of 0.5 is dialyzed against water down to μ = 0.05. The resulting precipitate, containing myosin and actomyosin, is redissolved at μ = 0.5, after which the ionic strength is reduced to 0.28 by dilution with water. This causes actomyosin to precipitate, while Actin-free myosin remains in solution. Further dilution with water lowers the ionic strength to 0.05, resulting in the precipitation of highly purified myosin.

A classical example of protein separation at low ionic strength is also the fractionation of globulins into pseudo- and euglobulins. The latter are insoluble in salt-free aqueous solutions and precipitate upon dialysis against water.

Isoelectric precipitation. Many purification methods rely to some extent on the decrease in protein solubility at a pH close to the isoelectric point. For instance, the final step in Straub's purification method for actin—one of the muscle proteins—involves adjusting the pH of the aqueous extract to 4.7, which is the isoelectric point of actin. This proves sufficient for the complete precipitation of actin, virtually free of impurities. A number of proteins in crystalline state, including serum and egg albumin, are obtained by gradually increasing the salt concentration in protein solutions adjusted to their isoelectric point.

It is worth emphasizing once again, however, that certain proteins (such as myosin) tend to denature near their isoelectric point.

There are instances of relatively selective precipitation of certain proteins at pH values quite far from the isoelectric point, yet favorable for The formation of insoluble complexes with specific Biopolymers present in the mixture. For example, the primary purification of several Bacterial toxins and anatoxins (diphtheria, botulinum, tetanus) is achieved by precipitation at pH 3.5–4.0—an intermediate value relative to the isoelectric points of these proteins and the Nucleic Acids contained in the culture fluid. Under these conditions, the molecules of the proteins and nucleic acids carry opposite charges and readily form insoluble compounds.

The Effect of salts and hydrogen ion concentrations on protein solubility can be interpreted as the result of these Factors influencing the interaction of charged polar groups of the protein molecule with each other and with water molecules, as well as the interactions between entire protein molecules. During the salting-out process, salt ions intensively bind dipolar water molecules, thereby disrupting the Hydration "shells" of water molecules that surround the charged protein groups and stabilize the protein molecules In aqueous solutions. Reversible Binding of salt ions by charged groups may also be of certain significance. All of this agrees well with the profound effect that polyvalent ions have on protein solubility.

Obviously, the decrease in protein solubility near the isoelectric point is associated with the minimal net charge of the protein molecule under these conditions and, consequently, with the reduction in electrostatic forces that otherwise prevent the association of similarly charged protein molecules.

The Dielectric Constant of the medium also exerts a significant influence on the electrostatic interaction forces between the charged groups of a protein molecule. Water has a high dielectric constant. Therefore, all agents that substantially lower the dielectric constant act as effective protein precipitants. Such agents include a number of water-miscible organic Solvents.

Fractionation using organic solvents. Organic solvents such as ethanol and acetone are commonly used for protein fractionation. Because they exert a denaturing effect on many proteins, two crucial conditions for their application are, first, working at low temperatures (−5 to −10°C) and, second, strictly limiting the duration of exposure. Compliance with the latter condition is facilitated by the fact that, unlike salt precipitants, organic solvents can be rapidly and completely removed from protein preparations by freeze-drying. Efficient fractionation using organic solvents is achieved not only by reaching specific concentrations of these agents, but also by selecting combinations of ionic strength, pH, and temperature optimal for isolating each fraction.

A widespread method of this type is Cohn's plasma protein fractionation. The high efficiency, reproducibility, and practicality of this method allow it to be used not only in laboratory settings but also for the industrial production of highly purified plasma protein preparations. However, the purification scheme using this method is complex, and its description goes beyond The Scope of this Manual. An example of a simpler method utilizing organic solvents is Jones's fractionation of core Nuclear Proteins—Histones—from Thymus tissue. The tissue homogenate is extracted with a mixture of 4 parts ethanol and 1 part 1.25 N Hydrochloric acid. An Arginine-rich histone fraction is precipitated from the extract by dialysis against absolute ethanol. Simultaneously, the bulk of the hydrochloric acid is removed from the extract. The remaining histones in solution, which are moderately rich in arginine, are precipitated by adding 3 parts of acetone. Further extraction of the tissue homogenate residue is then performed using a 0.25 N hydrochloric acid solution to obtain Lysine-rich histones. In turn, very lysine-rich histones (75% acetone by volume) and moderately lysine-rich histones (83% acetone by volume) are precipitated from this extract using increasing concentrations of acetone. All histone precipitates obtained by this method are washed with acetone to remove traces of water and ethanol, after which the acetone is easily removed by vacuum drying.

Selective precipitation of proteins by Other Reagents.

In special cases, powerful precipitants such as metaphosphoric acid, certain heavy metal salts, trichloroacetic acid, and several others can be useful in Protein Purification. Obviously, The Use of such precipitants—known as Denaturing Agents—is feasible only when purifying proteins that are exceptionally resistant to Denaturation, or it requires The Development of very stringent conditions to protect the proteins from denaturation. At the same time, the possibility of using relatively low concentrations of such reagents and the high completeness of precipitation make them attractive when developing industrial production schemes for certain proteins. For instance, the method of primary purification of bacterial anatoxins via precipitation with low concentrations of metaphosphoric acid has proven to be highly efficient and practical. One variant of the method for isolating lysine-rich histones involves their precipitation with trichloroacetic acid, during which a number of accompanying proteins are intentionally denatured. The exceptionally high resistance to denaturation exhibited by Enzymes such as arginase and hyaluronidase permits the use of manganese and copper sulfates for their selective precipitation. Finally, under strict temperature and pH control, coupled with the rapid and complete removal of the precipitant by special reagents, lead, cadmium, copper, and mercury salts can even be used to fractionate Serum proteins. Naturally, however, methods of this type have not gained widespread application for serum protein purification due to the existence of milder and more reproducible methods.

Protein crystallization. Many Proteins can be obtained in crystalline form. To achieve this, the protein to be crystallized must first be purified to the extent that it is undeniably the predominant component in the mother liquor. The crystallization process itself generally boils down to the following. A protein solution is prepared at a concentration close to saturation in a given solvent. A precipitating agent is gradually introduced to lower the protein solubility to the saturation level or slight supersaturation. As a rule, neutral salts—such as ammonium sulfate, magnesium sulfate, etc.—or organic solvents (ethanol, acetone) are used as precipitants during crystallization. The gradual Introduction of the precipitating agent is a key condition for success. This can be accomplished, for example, by dialyzing the mother liquor against a solution with an excess concentration of the precipitant. This is followed by prolonged, sometimes multi-day, incubation of the solution, during which crystals gradually form and grow. Crystal size is usually small, requiring observation under a Microscope at low magnification. Crystallization is significantly accelerated by seeding—adding a small quantity of crystals of the same protein. Crystalline preparations of egg and serum albumin, Hemoglobin, Myoglobin, Trypsin, Chymotrypsin, and many others were obtained in this manner. Photomicrographs of certain crystalline proteins are presented in Fig. 1.

Crystallization, as well as multiple recrystallization, serves as a good method for the additional purification of proteins at the final stages of isolation. It should not be assumed, however, that obtaining a protein in crystalline form indicates the achievement of the highest degree of purification—i.e., protein homogeneity. There are numerous known instances where crystalline protein preparations contain some (usually small) amount of impurities. It should also be borne in mind that many proteins currently defy crystallization.