Molecular Biology: Protein Structure and Functions - Stepanov V.M. 2005

Protein Isolation
Separation of proteins by molecular weight. Gel chromatography (gel filtration)

This method employs granular gels made of cross-linked hydrophilic Materials, such as dextran (Sephadex, Sepharose, and their analogues), polyacrylamide (Biogels and their analogues), or polyvinyl alcohol (Toyopearl). The beads are formed by a three-dimensional polymer network that is impermeable to large macromolecules, partially permeable to molecules of intermediate size, and readily permeable to small molecules, salts, and Water. Depending on the average pore size of the polymer gel and the geometry of the molecule, a greater or lesser fraction of the total gel bead volume is accessible to the latter.

As a solution containing Proteins and other molecules flows down a Column packed with swollen gel beads, the mixture components that penetrate the gel are temporarily retained within it. Consequently, they lag behind larger molecules that cannot enter the beads and remain exclusively in the surrounding Mobile phase. Being excluded from the gel, large molecules emerge in the eluate as soon as the "void" volume of the column, $V_0$—which corresponds to the solution volume trapped between the gel beads—has passed through. This volume is determined by the packing density and geometry of the beads. For spherical particles, which are the standard physical form of Gel Chromatography media, the void volume accounts for 30–35% of the total column volume, $V_t$.

If the protein molecules are of a size that allows them to penetrate the pores comprising a certain fraction of the bead volume, elution is delayed, and the protein emerges at an elution volume $V_e$ related to the partition coefficient $K_{av}$ (the fraction of the gel volume accessible to a given molecular species) by the equation

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where $V_t$ is the total column volume minus the volume occupied by the gel-forming polymer matrix itself.

Each protein corresponds to a specific $K_{av}$ value determined by its Molecular dimensions, which forms The basis of Separation in gel chromatography. Naturally, if the elution volume $V_e$ is close to the void volume $V_0$, $K_{av}$ approaches zero, and no separation will occur for proteins whose molecules are virtually excluded from the gel pores. Similarly, small molecules for which the entire gel volume is accessible ($V_e$ is close to $V_t$, and $K_{av}$ approaches unity) will not be resolved on a gel with such characteristics. Optimal resolution is achieved when $K_{av}$ falls within the range of 0.4–0.6. Of course, the separation range can be extended by using large-pore gels for high-molecular-weight proteins and small-pore gels for smaller ones.

Strictly speaking, separation in gel chromatography is governed not by molecular weight, but by the physical dimensions of the molecule in solution. Accordingly, extended or elongated molecules encounter greater steric hindrance and penetrate the gel less readily than spherical molecules of the same molecular weight due to their tumbling motion in solution. This explains the early elution of denatured proteins, which behave as disordered, random coils rather than compact globular structures.

The straightforward relationship between elution volume and molecular weight (which holds true, of course, only for compact, spherical molecules) and the operational simplicity of the technique have made gel chromatography a preferred METHOD FOR DETERMINING protein molecular weights. For this purpose, a column packed with an appropriate gel is calibrated using a set of standard proteins with known molecular weights; the elution volume of the protein of interest is then measured, and its molecular weight is determined by interpolation (Fig. 3.1). Although the precision of this method is moderate, it is entirely adequate for the vast majority of Structure/179.html">Practical Applications.

When employing this method, one must account for limitations arising from the fact that the gel matrix is not entirely inert, as theoretical assumptions might suggest; rather, it can interact with the solutes being separated, thereby distorting the relationship between elution volume and molecular size. This effect is particularly pronounced when separating small quantities of protein, because the sorption capacity of the gel matrix is relatively low, whereas in large-scale experiments, matrix-Protein Interactions have a negligible impact on the overall process.

Protein binding to gel-forming materials can be driven by ion-exchange interactions, notably due to the presence of negatively charged groups within polysaccharide matrices (such as Sepharose or Sephadex) as well as polyacrylamide media. In the latter, carboxyl groups arise from the spontaneous Hydrolysis of amide groups, whereas in Polysaccharides they can be generated through oxidation. Retention caused by ion-exchange interactions with the matrix is particularly characteristic of cationic proteins, such as Lysozyme and certain subtilisins. This effect is often quite substantial and may even interfere with the Desalting of protein solutions. In analytical applications, such unwanted retention can usually be suppressed by significantly increasing the Ionic strength of the buffer.

Another source of anomalous solute retention in gel chromatography—especially noticeable during the isolation of small molecules such as Peptides—is hydrophobic binding to the gel matrix. Hydrophobic sites are introduced into hydrophilic polysaccharide matrices during their synthesis when they are treated with cross-linking agents such as epichlorohydrin, as seen in the preparation of Sephadex. Peptides containing hydrophobic residues, particularly aromatic ones (such as phenylalanine or Tryptophan), are sometimes retained by the matrix to such an extent that they emerge in the eluate even later than inorganic salts.

Fig. 3.1. Plots of protein molecular weight versus partition coefficient for Sephadex G-series (“superfine”) gels (A) and Sepharose 2B, 4B, and 6B (B). Optimal results are obtained when $K_{av}$ for the target protein lies between 0.4 and 0.6.

While the resolving power of the method is moderate, its operational simplicity and mild experimental conditions are undeniable advantages. The applicability of the technique in the Cytology/cytology/16.html">Early stages of purification is limited by the requirement that, for satisfactory fractionation, the Sample application volume must not exceed 3–5% of the total column volume. Consequently, gel chromatography is typically employed in the intermediate or final stages of Protein Purification. Naturally, when removing low-molecular-weight contaminants—such as during desalting—the sample volume can be considerably larger, as high resolution is not required. Gel filtration is particularly frequently utilized in this simplified operational mode.

Despite these limitations, gel chromatography remains a convenient and powerful tool for protein fractionation. It is widely used both for separating proteins from low-molecular-weight impurities and for removing salts.

Recently, alongside traditional gel media, macroporous inorganic Supports—such as controlled-pore Glass and porous silica—have begun to be utilized for size-based protein separation. The surfaces of these materials are typically coated with hydrophilic organic polymers to eliminate irreversible protein adsorption. The mechanical rigidity of these supports allows size-exclusion chromatography to be performed at elevated pressures, which accelerates the separation process and minimizes diffusional broadening.