BIOCHEMISTRY: A TEXTBOOK FOR MEDICAL UNIVERSITIES - E. S. Severin - 2004

SECTION 1. STRUCTURE, PROPERTIES, AND FUNCTIONS OF PROTEINS

VII. Physicochemical Properties of Proteins and Methods for Their Isolation

Isolation of individual Proteins from Organs and Tissues occupies an important place in biochemical research. Purified individual proteins are required to study their Primary Structure, obtain protein crystals for Spatial Structure analysis via X-ray crystallography, and establish the relationship between a protein's primary and spatial structure and its function.

Certain purified individual proteins are used in medicine as therapeutic agents; for instance, the hormone Insulin is used to treat Diabetes Mellitus, and pancreatic digestive Enzymes are prescribed as replacement therapy for exocrine pancreatic insufficiency. In addition, purified enzymes are frequently employed in biochemical research as chemical Reagents to assay substances in biological fluids.

Most Methods used to purify individual proteins are based on differences in their physicochemical properties, as well as their ability to specifically bind ligands.

A. PHYSICOCHEMICAL PROPERTIES OF Proteins

Individual proteins differ in their physicochemical properties, including molecular shape, molecular weight, net molecular charge, The ratio of polar to nonpolar groups On the surface of the native protein molecule, solubility, and resistance to Denaturing Agents.

1. Differences in Protein Molecular Shape

As noted above, based on _{molecular shape}, proteins are classified into globular and fibrous (fibrillar) types. Globular proteins possess a more compact structure with most of their hydrophobic residues buried within a Hydrophobic core. Consequently, they are significantly more soluble in Body Fluids than Fibrous proteins (with Membrane Proteins being an exception).

2. Differences in Molecular Weight

Proteins are macromolecular compounds that can vary widely in molecular weight, ranging from 6,000 to 1,000,000 Da and higher. The Molecular Weight of a protein depends on the number of amino acid residues in the polypeptide chain, and in Oligomeric Proteins, also on the number of constituent protomers (or subunits).

3. Net Charge of Proteins

Proteins contain Lysine, Arginine, Histidine, glutamic acid, and aspartic acid residues with functional groups capable of ionization (ionizable groups). Additionally, the N- and C-termini of polypeptide chains feature α-amino and α-carboxyl groups, which are also ionizable. The net charge of a protein molecule depends on the ratio of ionized anionic residues (Glu and Asp) to cationic residues (Lys, Arg, and His).

The degree of ionization of these functional groups depends on the pH of the medium. At a solution pH of approximately 7, all ionizable groups of the protein are in an ionized state. In an acidic environment, an increase in proton concentration (H⁺) suppresses the dissociation of carboxyl groups, thereby reducing the negative charge of proteins: -СОО^- + Н⁺ —> -СООН. In an alkaline environment, the binding of excess OH^- ions to protons released during the dissociation of NH₃⁺ (forming Water) leads to a decrease in the positive charge of proteins:

-NH₃⁺ +ОН^- —> -NH₂ + Н₂О.

The pH value at which a protein carries a net zero charge is termed the isoelectric point and is designated as pI. At the isoelectric point, the number of positively and negatively charged groups on the protein is equal, meaning the protein is in an isoelectric state.

Since the majority of cellular proteins contain a predominance of anionogenic groups (-СОО^-), their isoelectric point lies in a mildly acidic medium. Conversely, the isoelectric point of proteins rich in cationogenic groups is located in an alkaline medium. A prime example of such intracellular proteins, which contain high amounts of arginine and lysine, are Histones associated with Chromatin.

Proteins carrying a net positive or negative charge are more soluble than proteins at their isoelectric point. The net charge increases the number of water dipoles capable of binding to the protein molecule and prevents contact between molecules of the same charge, thereby enhancing Protein solubility. Charged proteins can migrate in an electric field: negatively charged anionic proteins move toward the positively charged anode (+), whereas cationic proteins move toward the negatively charged cathode (-). Proteins in the isoelectric state do not migrate in an electric field.

4. Ratio of Polar to Nonpolar Groups on The surface of Native Protein Molecules

The surface of most intracellular proteins is dominated by polar residues; however, the ratio of polar to nonpolar groups varies among individual proteins. For instance, the protomers of oligomeric proteins often contain hydrophobic residues at their contact interfaces. The surfaces of proteins that function within membranes or associate with them during their activity are similarly enriched in hydrophobic residues. Such proteins are more soluble in Lipids than in water.

5. Protein Solubility

The Solubility of proteins in water depends on all the aforementioned properties: shape, molecular weight, magnitude of charge, and the ratio of polar to nonpolar functional groups on the protein surface. Furthermore, protein solubility is determined by solvent composition, i.e., the presence of other solutes in the solution. For example, certain proteins dissolve more readily in dilute saline solutions than in distilled water. Conversely, increasing the concentration of neutral salts can induce the precipitation of specific proteins. Denaturing agents present in the solution also decrease protein solubility.

B. Methods for Protein Isolation and Purification

Obtaining individual proteins from biological material (tissues, organs, Cell cultures) requires a series of sequential Procedures, including:

✵ mechanical disruption of biological material and destruction of cell membranes;

✵ fractionation of Organelles containing specific proteins;

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

✵ Separation of a protein mixture into individual proteins.

1. Methods of Tissue Disruption and Protein Extraction

To disrupt biological material, several methods are used: tissue homogenization, repeated freezing and thawing, and ultrasonic Cell Disruption.

Homogenization of Biological Material

The tissue, suspended in a buffer solution with a specific pH and salt concentration, is placed in a Glass vessel (homogenizer) with a pestle. The rotating pestle grinds and crushes the tissue against the tightly fitted walls of the vessel.

Tissue Freezing and Thawing Method

As a result of alternating freezing and thawing, the resulting ice crystals disrupt cell membranes.

Following tissue disruption, insoluble particles are pelleted by centrifugation. Subsequent differential centrifugation of the homogenate at various speeds yields individual fractions containing cell nuclei, Mitochondria, and other organelles, as well as the 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

If the target protein is tightly bound to cellular structures, it must be brought into solution. For example, 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" (see Fig. 1-15). Detergents typically also disrupt the hydrophobic interactions between protomers in oligomeric proteins.

Removal of Non-Protein Substances from Solution

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

2. Protein Purification Methods

The most laborious stage in obtaining individual proteins is their purification from other proteins present in the solution derived from the tissue. Often, the protein of interest is present in small quantities, accounting for only a fraction of a percent of all proteins in the solution.

Because proteins exhibit conformational lability, denaturing conditions should be avoided during handling; therefore, Protein Isolation and purification are carried out at low temperatures.

In the early Stages of Protein purification, it is advisable to use methods that target a specific characteristic of the protein, such as thermostability or resistance to acidic solutions. The initial purification methods must remove the bulk of contaminating proteins that differ significantly in physicochemical properties from the protein being isolated. Subsequently, increasingly refined purification techniques are applied.

Protein Purification by Selective Denaturation

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

Salting-Out

A protein purification method based on differences in solubility at varying salt concentrations in solution. Alkali and alkaline-earth metal salts cause reversible protein precipitation; that is, upon salt removal, proteins regain their solubility while retaining their native properties.

Most commonly, varying concentrations of ammonium sulfate—(NН₄)₂SO₄—are used for protein separation via salting-out. The higher the solubility of a protein, the greater the salt concentration required for its precipitation.

Gel filtration, or the Molecular Sieve Method

Proteins are frequently separated using chromatographic Methods based on the distribution of substances between two phases: a Mobile phase and a stationary phase. These methods rely on various underlying principles, including gel filtration, Ion Exchange, adsorption, and biological affinity.

The protein separation method using gel filtration chromatography relies on the differential distribution of molecules of varying molecular weights between the stationary and mobile phases. A chromatographic Column is packed with porous gel beads (such as Sephadex, agarose, etc.). Cross-linking within the polysaccharide structure forms beads perforated with "pores" that readily allow the passage of water and low-molecular-weight substances. Depending on the conditions, beads with varying pore sizes can be produced.

The stationary phase consists of the liquid trapped inside the gel beads, which low-molecular-weight substances and small proteins can readily penetrate. A protein mixture applied to the chromatographic column is washed out (eluted) by passing a solvent through the column. The largest molecules travel alongside the solvent front.

Smaller molecules diffuse into the Sephadex beads and enter the stationary phase for a brief period, which delays their migration. The pore size determines the upper limit of Molecular dimensions capable of penetrating the beads (Fig. 1-55).

Class="center">Fig. 1-55. Separation of a protein mixture by gel filtration.

Because the gel structure of Sephadex is easily deformed under pressure, gels have increasingly been replaced by more rigid matrices (such as Sephacryl and Toyopearl), which consist of spherical beads with various pore sizes. The choice of bead pore size depends on the specific goals of the chromatography (other chromatographic methods are discussed below).

Ultracentrifugation

This separation method also relies on differences in the molecular weights of proteins. The Sedimentation Rate of substances during ultracentrifugation—where centrifugal acceleration reaches 100,000 to 500,000 g—is proportional to their molecular weight. A thin layer of a protein mixture is carefully layered onto the surface of a buffer solution contained within a cuvette, and the cuvette is placed into the ultracentrifuge rotor. When the rotor spins for 10–12 hours, larger molecules (with higher molecular weights) settle through the buffer solution at a faster rate. As a result, the protein mixture fractionates in the cuvette according to molecular weight (Fig. 1-56). Following fractionation, the bottom of the cuvette is punctured with a needle, and the contents are collected dropwise into small test tubes.

Fig. 1-56. A cuvette filled with buffer solution containing separated protein fractions.

Protein Electrophoresis

This method is based on the principle that at a specific pH and Ionic strength, proteins migrate in an electric field at a velocity proportional to their net charge. Proteins with a net negative charge move toward the anode (+), whereas positively charged proteins migrate toward the cathode (-).

Electrophoresis is performed using various supporting media, such as paper, starch gel, polyacrylamide gel, and others. Unlike paper electrophoresis—where protein migration velocity is proportional solely to their net charge—in Polyacrylamide gel electrophoresis, migration velocity is also dependent on molecular weight.

The resolving power of polyacrylamide gel electrophoresis is significantly higher than that of paper electrophoresis. For example, paper electrophoresis of human Serum proteins reveals only 5 major fractions: albumins, α₁-globulins, β₂-globulins, β-globulins, and γ-globulins (Fig. 1-57). Electrophoresis of the same proteins in polyacrylamide gel resolves 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 resulting stained protein-dye complexes reveal the positions of the various fractions on the support medium.

Fig. 1-57. Paper electrophoresis of serum proteins from a healthy individual.

Ion-exchange chromatography

Much like electrophoresis, this method relies on separating proteins that differ in net charge at a given pH and ionic strength. When a protein solution is passed through a chromatographic column packed with a solid, porous, charged material, a portion of the proteins is retained via Electrostatic Interactions.

Ion exchangers—polymeric Organic compounds containing charged functional groups—serve as the stationary phase.

A distinction is made between positively charged anion exchangers (the most common being diethylaminoethylcellulose, or DEAE-Cellulose, which contains cationic groups) and negatively charged cation exchangers (such as carboxymethylcellulose, or CM-cellulose, which contains anionic groups).

The choice of ion exchanger is determined by the charge of the target protein. For instance, an anion exchanger is used to isolate a negatively charged protein. As the protein solution passes 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 onto the anion exchanger can be washed off (eluted) using Buffer solutions with varying salt concentrations (most frequently NaCl) and different pH values. Chloride ions bind to the positively charged Functional groups of the anion exchanger, displacing the protein's carboxyl groups. Proteins that are weakly bound to the anion exchanger elute at low salt concentrations. A gradual increase in salt concentration or A change in pH alters the net charge of the protein molecules, leading to the successive elution of protein fractions, one of which contains the protein of interest.

Affinity Chromatography

This is the most specific method for isolating individual proteins, based on the selective interaction of proteins with ligands attached (immobilized) to a solid support. The Ligand can be a substrate or a coenzyme (when isolating an enzyme), Antigens for isolating Antibodies, etc. A solution containing a protein mixture 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 pass through with the eluate (Fig. 1-58). The protein adsorbed on the column can be recovered by washing the column with a solution of altered pH or changed ionic strength. In some cases, a detergent solution is used to disrupt the Hydrophobic bonds between the protein and the ligand.

Fig. 1-58. Affinity chromatography.

Affinity chromatography is characterized by high selectivity and allows the target protein to be purified thousands of times.

3. Removal of low-molecular-weight impurities from proteins

Dialysis is used to remove low-molecular-weight compounds, particularly ammonium sulfate after salting out. The method is based on the fact that semipermeable membranes allow low-molecular-weight substances to pass through while retaining high-molecular-weight proteins. A semipermeable bag filled with a protein solution containing salt is placed 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 its concentration gradient across the membrane. As the salt leaves the bag, the buffer solution in the beaker is replaced.

Gel filtration (see above) is also used to purify proteins from low-molecular-weight impurities.

High-resolution techniques, 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 cause allergic reactions) of a therapeutic protein preparation depend on its purity. The higher the quality of purification, the lower the likelihood of complications during its use.