BIOCHEMISTRY - Textbook - Ostapchenko L. I. - 2012

Chapter 5. STRUCTURE, PROPERTIES, AND FUNCTIONS OF PROTEINS

5.7. Physicochemical Properties of Proteins and Methods for Their Isolation

5.7.2. Methods for Protein Isolation and Purification

Obtaining individual Proteins from biological Materials (Tissues, Organs, cellular structures) requires a sequence of operations that include:

✵ Homogenization of Biological material and disruption of Cell membranes;

✵ fractionation of Organelles containing specific proteins;

✵ Protein Extraction (bringing them into a soluble state);

✵ Separation of the protein mixture into individual proteins.

Methods for tissue disruption and protein extraction. To disrupt biological material, methods such as tissue homogenization, repeated freezing and thawing, and ultrasonic Cell Disruption are used.

Homogenization of biological material. The tissue in a buffer solution with a specific pH and salt concentration is placed into a homogenizer. During rotation, it is minced and ground by being forced between tightly fitted walls and a pestle.

As a result of repeated freezing and thawing, the formed ice crystals disrupt cell membranes.

Following tissue disruption, insoluble particles are pelleted by centrifugation. Subsequent centrifugation of the homogenate at various speeds yields individual fractions containing cell nuclei, Mitochondria, and other organelles, as well as a supernatant containing soluble cytosolic proteins. The target protein will be present in one of these fractions.

Extraction of membrane-bound proteins and dissociation of Oligomeric Proteins into protomers. A protein tightly bound to specific cell structures must be brought into solution. To disrupt hydrophobic interactions between proteins and Membrane Lipids, detergents are added to the solution; Triton X-100 or sodium dodecyl sulfate are most commonly used. The Mechanism of detergent action is described in the section "Protein Denaturation." Consequently, detergents typically disrupt hydrophobic interactions between protomers in oligomeric proteins as well.

Removal of Non-Protein Substances from the solution. Nucleic Acids, lipids, and other non-protein substances can be removed from the solution by exploiting their specific physicochemical properties. For instance, lipids are easily removed from the solution by adding organic Solvents, such as acetone. However, this Treatment must be brief, as acetone causes the denaturation of certain proteins. Nucleic acids are precipitated by adding streptomycin to the solution.

Protein Purification Methods. The most time-consuming step in obtaining individual proteins is purifying them from other proteins present in the solution derived from a given tissue. Frequently, the protein under study is present in small quantities, accounting for a fraction of a percent of all proteins in the solution.

Because proteins exhibit conformational lability, denaturing conditions must be avoided during handling; therefore, protein Isolation and Purification are performed at low temperatures.

In the initial Stages of Protein purification, it is advisable to use methods that exploit a specific characteristic of the protein in question, such as thermal stability or resistance to acidic solutions. The primary purification methods should remove the bulk of ballast proteins that differ significantly in their physicochemical properties from the target protein. Subsequently, finer purification methods are applied.

Protein purification via selective denaturation. Most proteins denature and precipitate upon brief heating of the solution to 50-70 °C or acidification to pH 5. If the target protein withstands these conditions, selective denaturation can be used to remove most extraneous proteins by filtering out the precipitated proteins or pelleting them via centrifugation.

Salting-out is a protein purification method based on differences in Protein solubility at varying salt concentrations in solution. Salts of alkali and alkaline earth metals cause reversible protein precipitation; that is, once the salts are removed, the proteins regain their solubility while retaining their native properties.

Ammonium sulfate - (NH₄)₂SO₄ at various concentrations is most frequently used for protein separation via salting-out. The higher the solubility of a protein, the greater the salt concentration required for its precipitation.

Chromatographic methods are widely used for protein separation, based on the distribution of substances between two phases: one mobile and the other stationary. Chromatographic techniques rely on various principles: Gel filtration, Ion Exchange, adsorption, and Affinity Chromatography.

The method of protein separation via gel filtration chromatography (or molecular exclusion) is based on the differential distribution of substances with varying molecular weights between the mobile and stationary phases. A chromatographic Column is packed with porous gel beads (Sephadex, agarose, etc.). Cross-links form within the polysaccharide Structure to create beads with "pores" through which Water and low-molecular-weight compounds easily pass. Depending on the conditions, beads with varying pore sizes can be produced.

The stationary phase is the liquid inside the beads, which low-molecular-weight substances and small proteins can penetrate. The protein mixture applied to the chromatographic column is eluted by passing a solvent through the column. The largest molecules move along with the solvent front.

Smaller molecules diffuse into the Sephadex beads and enter the stationary phase for some time, thereby delaying their migration. The pore size determines the size of the molecules capable of penetrating the interior of the beads (Fig. 5.54).

Since the gel structure of Sephadex is easily deformed under pressure, gels began to be replaced by more rigid matrices (Sephacryl, Toyopearl), which are spherical beads with varying pore sizes. The choice of pore size in the beads depends on the purpose of chromatography (other chromatographic methods will be described below).

Ultracentrifugation. This separation method is also based on differences in protein molecular weights. The Sedimentation Rate of substances during centrifugation in an ultracentrifuge, where centrifugal acceleration reaches 100,000–500,000 g, is proportional to their molecular weight. A thin layer of the protein mixture is applied to The surface of a buffer solution placed in a cuvette. The cuvette is then placed in the ultracentrifuge rotor. Due to rotor rotation for 10–12 hours, larger molecules (with higher molecular weights) settle in the buffer solution at a faster rate. As a result, the protein mixture separates into distinct fractions with different molecular weights within the cuvette (Fig. 5.55). After the separation of protein fractions, the bottom of the cuvette is punctured with a needle, and the contents are collected drop by drop in small portions into test tubes.

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Fig. 5.54. Separation of a protein mixture by gel filtration

Fig. 5.55. Cuvette filled with buffer solution containing separated protein fractions

Protein Electrophoresis. This method is based on the principle that, at a given pH and Ionic strength of the solution, proteins move in an electric field at a speed proportional to their net charge. Proteins with a net negative charge move toward the anode (+), while positively charged proteins move toward the cathode (-).

Electrophoresis is performed using various supporting media: paper, starch gel, polyacrylamide gel, etc. Unlike paper electrophoresis, where the migration rate of proteins is proportional solely to their net charge, in Polyacrylamide gel electrophoresis, the migration rate is proportional to their molecular weights.

The resolution of polyacrylamide gel electrophoresis is higher than that of paper electrophoresis. For example, paper electrophoresis of human Serum proteins reveals only five main fractions: albumins, α1-globulins, α2-globulins, β-globulins, and γ-globulins (Fig. 5.56). Electrophoresis of the same proteins in polyacrylamide gel allows for the separation of up to 18 distinct fractions. To visualize the protein fractions, the paper strips or gel columns are treated with a dye (most commonly bromophenol blue or amido black). The stained protein-dye complex makes it possible to detect the positions of various fractions on the support.

Fig. 5.56. Electrophoresis of a healthy human serum protein (on paper)

Ion-exchange chromatography, much like electrophoresis, is based on the Separation of proteins that differ in net charge at specific pH values and ionic strengths of the solution. When a protein solution is passed through a chromatographic column packed with a solid porous charged material, some proteins are retained on it due to Electrostatic Interactions. Ion exchangers—polymeric organic substances containing charged functional groups—are used as the stationary phase.

A distinction is made between positively charged anion exchangers, the most common being diethylaminoethyl Cellulose (DEAE-cellulose) containing cationic groups, and negatively charged cation exchangers, such as carboxymethyl cellulose (CM-cellulose) containing anionic groups:

The choice of ion exchanger is determined by the charge of the protein under study. For instance, an anion exchanger is used to isolate a negatively charged protein. When the protein solution is passed through the column, the strength of the protein's binding to the anion exchanger depends on the number of negatively charged carboxyl groups in the molecule. Proteins adsorbed on the anion exchanger can be washed off (eluted) using Buffer solutions with varying salt concentrations (most commonly NaCl) and different pH values. Chloride ions bind to the positively charged Functional groups of the anion exchanger and displace the protein carboxyl groups. Proteins weakly bound to the anion exchanger will elute at low salt concentrations. A gradual increase in salt concentration or A change in pH, which alters the charge of the protein molecule, leads to the elution of protein fractions, one of which contains the target protein.

Affinity chromatography is the most specific method for isolating individual proteins; it is based on the selective interaction of proteins with ligands attached (immobilized) to a solid support. A substrate or coenzyme can be used as a Ligand when isolating a specific enzyme, Antigens for isolating Antibodies, and so on. A solution containing a mixture of proteins is passed through a column packed with the immobilized ligand. Only the protein that specifically interacts with the ligand binds to it; all other proteins emerge in the eluate (Fig. 5.57). The protein adsorbed on the column can be recovered by washing the column with a solution of altered pH or ionic strength. In some cases, a detergent solution is used to disrupt the Hydrophobic bonds between the protein and the ligand.

Affinity chromatography is characterized by high selectivity and makes it possible to purify the isolated protein thousands of times.

Fig. 5.57. Affinity chromatography

Dialysis is used to remove low-molecular-weight compounds, such as ammonium sulfate after salting out. This method is based on the fact that a semipermeable membrane allows low-molecular-weight compounds to pass through while retaining larger proteins. A semipermeable bag filled with the salt-protein solution is immersed in a large beaker (about 1 L) containing a buffer solution.

The rate at which salt diffuses out of the bag into the buffer solution is proportional to the concentration gradient across the membrane. As the salt leaves the bag, the buffer solution in the beaker is replaced.

Gel filtration is also employed to purify proteins from low-molecular-weight impurities.

High-resolution methods—such as polyacrylamide gel electrophoresis and High-Performance Liquid Chromatography—are used to determine the purity (homogeneity) of the isolated protein. The biological efficacy and allergenicity (i.e., The ability to provoke allergic reactions) of a therapeutic protein preparation depend on its purity. The higher the quality of purification, the lower the likelihood of adverse reactions during its administration.