Biological Chemistry - Berezov, T. T., & Korovkin, B. F. 1998

Protein Chemistry
Methods for Protein Isolation and Purification
Protein Fractionation and Purification

Once complete Protein Extraction is achieved—meaning the Proteins are fully converted into a dissolved state—the next step involves Separation, namely the fractionation of the protein mixture into individual proteins. Various Methods are employed for this purpose, including salting-out, thermal Denaturation, precipitation with organic Solvents, chromatography, Electrophoresis, partitioning in two-phase systems, and crystallization, among others.

The dissolution of proteins in Water is driven by the Hydration of each molecule, leading to The formation of aqueous (hydration) shells around the protein globule, composed of water molecules arranged in a specific spatial orientation. In terms of their chemical and physical properties, the water molecules within the hydration shell differ from those of the pure solvent; specifically, its freezing point is -40°C. Sugars, salts, and other substances dissolve less readily in this water. Protein solutions are extremely unstable, and under METABOLISM/18.html">The Influence of various factors that disrupt hydration, proteins easily precipitate out of solution. Therefore, The addition of any dehydrating agents to a protein solution (such as alcohol, acetone, or concentrated solutions of neutral alkali metal salts), as well as The impact of physical factors (like heating or irradiation), results in the dehydration of protein molecules and their precipitation.

Salting-out. The addition of alkali and alkaline-earth metal salt solutions leads to the precipitation of proteins from solution. Typically, the protein retains its ability to redissolve in water once the salts are removed via dialysis or Gel chromatography. Salting-out is commonly used in clinical practice to analyze Serum proteins and other biological fluids, as well as in preparative enzymology for the preliminary precipitation and removal of ballast proteins or the ISOLATION OF A target enzyme. Different proteins precipitate from solution at varying concentrations of neutral ammonium sulfate solutions. Consequently, this method has found widespread clinical application for separating globulins (which precipitate at 50% saturation) and albumins (which precipitate at 100% saturation).

The extent of protein salting-out is influenced not only by The Nature and concentration of the salt, but also by the pH of the medium and the Temperature. It is widely accepted that ion valency plays a major role in this process. The effects of different ions are typically compared not by molar salt concentration, but by the so-called Ionic strength (μ), which is equal to half the sum of the products of the concentration of each ion (c) multiplied by the square of its valency (V):

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A finer fractionation of human Plasma Proteins is achieved by using varying concentrations of ethanol at low temperatures (-3 to -5°C) According to the Cohn method (Fig. 1.2). Under these conditions, proteins retain their native properties. This method is frequently used to obtain specific Blood fractions utilized as blood substitutes.

Fig. 1.2. Fractionation diagram of human Blood Plasma Proteins using ethanol (according to the Cohn method).

Recently, chromatographic and electrophoretic methods for protein separation have become the most widely adopted.

Chromatography. THE PRINCIPLE OF chromatography, developed in 1903 by the Russian scientist M. S. Tsvet, is based on the ability of pigments (or any other colored and colorless substances) to specifically adsorb onto an adsorbent packed within a Column*.

As a result, the analyzed substances are separated and concentrated in a strictly defined layer of the adsorbent. Suitable eluents are then passed through the column, weakening the adsorption forces and carrying individual substances out with the solvent flow. These substances are subsequently collected sequentially in a fraction collector (the sorption-desorption principle).

Column chromatography utilizing hydroxylapatite, various ion-exchange resins, and Cellulose derivatives as stationary phases has proven to be an extraordinarily effective means of fractionating protein mixtures. When isolating and purifying proteins, four MAIN TYPES OF chromatography are employed—adsorption, partition, ion-exchange, and Affinity Chromatography—based on the distinct Physical and Chemical mechanisms underlying each. Chromatography is widely used not only for Protein Isolation, but also for separating a multitude of other organic and inorganic substances found in living organisms.

Adsorption chromatography. The separation of mixture components (the sample) is based on their differing adsorbability on a solid adsorbent. Activated charcoal, calcium phosphate gel, and aluminum or silicon oxides are commonly used as adsorbents. The adsorbent, prepared as a suspension in a solvent (most often a buffer solution), is poured into a vertical Glass tube (column) and packed uniformly. The sample, dissolved in a small volume of solvent, is applied to the column, and the Components of the mixture are adsorbed onto the adsorbent. Next, the desorption stage begins, wherein the components are eluted from the column using appropriate eluents (Fig. 1.3). Fractions are collected using an automatic fraction collector.

* This remarkable discovery by the Russian scientist was duly appreciated by the global scientific community. For instance, the Swiss scientist P. Karrer wrote in 1948: "No other discovery has had such a profound impact or so vastly expanded the research capabilities of the organic chemist as Tsvet's chromatographic analysis. Research in the fields of Vitamins and Hormones, carotenoids, and numerous other natural compounds could never have developed so rapidly or yielded such great results had it not been founded on this new method, which revealed the vast diversity of related compounds present in nature."

Fig. 1.3. Adsorption chromatography (schematic). Separation of two different substances (A and B) moving through the column at different rates.

1 - Sample application to the column; 2 - midpoint of the experiment; 3 - end of the experiment.

Partition chromatography. Unlike adsorption chromatography, the solid phase serves merely as a support (matrix) for the stationary liquid phase. One type of partition chromatography, similar to adsorption chromatography, is performed in columns using moist starch or silica gel as the stationary phase. The sample is dissolved in a suitable solvent and then applied to the column; the separated substances undergo repeated partitioning between the stationary liquid phase (the aqueous layer) and the moving phase of the organic solvent, migrating toward the bottom of the column at different rates. Sample fractions collected by the fraction collector that contain a single substance are pooled to isolate that substance in pure form.

A variation of partition chromatography is paper chromatography, which is widely used in biochemical and clinical laboratories for separating Peptides, Amino Acids, and other substances (Fig. 1.4). In this method, water adsorbed by the cellulose fibers of the filter paper acts as the stationary phase. The sample is applied to one end of a paper strip, which is then immersed in an appropriate mixture of organic solvents (e.g., a specific ratio of butanol, acetic acid, and water). As the solvent moves up the paper via capillary action, the components of the mixture are separated. Once developed, the chromatogram is dried, and the Location of each separated substance is determined using chemical or Physicochemical methods.

Fig. 1.4. Paper chromatography (schematic).

A - ascending chromatography; B - descending chromatography (side view); C - chromatogram with separated and stained substances: 1 - solvent front, 2 - separated substances, 3 - sample application site.

Ion-exchange chromatography. Ion-exchange resins are polymeric Organic compounds containing functional groups capable of engaging in Ion Exchange. A distinction is made between positively charged anion exchangers, represented by organic bases and amines, and negatively charged cation exchangers, containing phenolic, sulfo-, or carboxyl groups. Among strongly and weakly basic anion exchangers, polystyrene and cellulose derivatives bearing functional groups are most frequently used:

Similar functional groups are found in triethylaminoethyl (TEAE)- and aminoethyl (AE)-celluloses.

Cation exchangers are represented by sulfonated polystyrenes (vinylbenzene or divinylbenzene derivatives) and carboxymethyl cellulose containing the following functional groups:

Depending on the charge of the proteins to be separated, a suitable ion-exchange resin is selected. Some proteins interact with its functional groups and are retained on the column, whereas others elute freely. The proteins "precipitated" on the column can be recovered by applying higher ionic strength salt solutions or by altering the pH of the eluent.

Cutting-edge ion-exchange chromatography techniques, notably High-Performance Liquid Chromatography (HPLC), are widely used in pharmacology (for drug design and assay), clinical biochemistry (for quantifying BIOLOGICALLY ACTIVE SUBSTANCES in physiological fluids), biotechnology, and other fields. They make it possible to detect substances in nano-, pico-, and femtomole quantities.

Affinity chromatography. This method is based on the principle of the selective interaction of proteins (or other macromolecules) with specific substances—ligands—immobilized on a solid support. These ligands can be substrates or Coenzymes (when isolating a specific enzyme), Antigens (or Antibodies), hormones, receptors, etc. Due to the high Specificity of proteins for the immobilized Ligand bound to the support (which packs the chromatographic column), only a single specific protein from a mixture binds to it. Elution of this protein is achieved by passing Buffer solutions with altered pH or ionic strength through the column, or by adding detergents to the eluent to weaken the bonds between proteins and ligands. A clear advantage of this method is The ability to isolate a target protein or other biopolymer of high purity in a single step. For instance, affinity chromatography has been successfully used to obtain purified preparations of Aminoacyl-tRNA synthetases on a polyacrylhydrazide-agarose gel coupled with specific tRNAs (Transfer RNAs) as ligands.

Gel filtration chromatography. For preparative purposes, especially in Protein Purification, the molecular sieve method, or gel filtration, is widely used. Treating the polysaccharide dextran* with epichlorohydrin introduces cross-links of varying degrees, resulting in the formation of large hydrophilic, water-insoluble grains known as Sephadex. Due to their high affinity for water, these grains swell significantly in aqueous media to form a gel used to pack the chromatographic column. Separation by this method is based on the fact that large molecules are excluded from the internal water phase of the gel (which acts as the stationary phase) and remain in the interstitial space, moving down the column alongside the Mobile phase. Conversely, small molecules freely diffuse into the gel beads, establishing an equilibrium between the mobile and stationary phases, and thus migrate down the column at a slower rate (Fig. 1.5). Typically, the elution volume of a substance from a Sephadex column is expressed by the formula:

where V is the elution volume of the substance with a given K, mL; V₀ is the void volume of the column, or the total volume of the external solvent (outside the gel beads), mL; V_i is the volume of the solvent inside the gel, mL; and K is the partition coefficient for the solute between the internal solvent of the gel beads and the surrounding solvent. If a sample containing one solute with K = 1 and another with K = 0 is applied to a Sephadex column, the second substance will emerge in the eluate immediately after the void volume V₀ has passed, whereas the first will elute only after a volume of V₀ + V_i.

Because protein molecules with large molecular weights and sizes cannot diffuse into the Sephadex beads, they are washed out of the column first, right after the void volume V₀, while all other substances (including low-molecular-weight impurities) elute after a volume equal to V₀ + K ∙ V_i has passed.

This method has found widespread application in preparative enzymology, as Sephadex enables the fractionation of proteins with different molecular weights.

Electrophoresis. The free electrophoresis method, detailed by Nobel laureate A. Tiselius, is based on differences in the migration rates (mobilities) of proteins in an electric field, which

* Dextran is a polysaccharide synthesized by microorganisms. Sephadex is a commercial product whose name is derived from the first syllables of three words: separation, Pharmacia (the name of a Swedish pharmaceutical company), and dextran. Along with Sephadex, other matrices such as Sepharose, Sephacryl, Bio-Gel, Molselect, and Acrylex are also used to separate proteins and other Biomolecules.

Fig. 1.5. Gel filtration Chromatography on a Sephadex column (schematic diagram).

Large open circles with crosses represent Sephadex beads; small black and red circles and triangles represent proteins of various molecular weights; A is the column at THE START OF the process; B, C, and D show the column at successive time points. The elution profile clearly demonstrates the separation of the protein components.

are determined by the net charge of the protein at specific pH values and ionic strengths of the solution. Recently, zonal electrophoresis methods using various solid support media—such as starch gels, polyacrylamide gels, and cellulose—have gained broader acceptance. Their advantages over free electrophoresis include the elimination of boundary blurring caused by diffusion and convection, the absence of complex equipment required to track moving boundaries, and the small Sample size needed for analysis (these methods and their corresponding apparatus are detailed in practical biochemistry manuals).

One of the most common techniques for protein fractionation (as well as for assessing homogeneity) is polyacrylamide gel Disc electrophoresis (where "disc" stands for discontinuous), which employs buffer systems with different pH values and gels of varying porosity. Gel electrophoresis is notable for its exceptionally high resolving power. For example, while paper electrophoresis of human serum proteins reveals only 6 fractions, Starch gel electrophoresis resolves 10, and Polyacrylamide gel electrophoresis can separate up to 18 distinct protein fractions.

To visualize proteins after gel electrophoresis, they are stained with Dyes such as bromophenol blue, amido black 10B, acid blue 83, or Coomassie brilliant blue R-250. Staining intensity, and consequently the relative Abundance of each protein fraction, is usually quantified densitometrically by direct scanning. In recent years, gradient gel electrophoresis has been introduced, significantly enhancing resolution, particularly when fractionating high-molecular-weight proteins exceeding 50,000–100,000.

Various modalities of isoelectric focusing and isotachophoresis—electrophoretic techniques conducted in supporting media (such as columns or thin layers) featuring a pH gradient—have proven to be highly promising for protein separation and the determination of physicochemical properties. The precise position of each protein in the mixture is dictated by its isoelectric point, the state at which the net electrical charge of the protein particle is zero at a given pH. Isoelectric focusing utilizes mixtures of synthetic polyaminopolycarboxylic acids (ampholytes) to establish a pH gradient ranging from 3.0 to 10.0.

In recent years, two-dimensional electrophoresis, which combines isoelectric focusing and polyacrylamide gel disc electrophoresis, has become widely adopted for protein fractionation, allowing hundreds or even thousands of protein fractions to be analyzed simultaneously.