Protein chromatography

Gel filtration: separation by molecular weight

This method is based on the molecular sieve effect. The essence of the process is sieving protein molecules through a specialized carrier — swollen sephadex beads.

Sephadex itself is a complex spatial structure. These are three-dimensional polysaccharide chains of dextran with pores of a defined diameter formed inside them. When passing a mixture through the column, the rate of movement of various proteins directly depends on their molecular weight.

Separation occurs according to the following principle:

• Molecules with a lower mass freely penetrate into the inner space of the sephadex bead pores. Because of this, their path is extended, they are retained in the column, and they emerge from it significantly later.
• Molecules with a high mass, conversely, are physically unable to enter the pores of dextran. They bypass the beads on the outside and, consequently, elute (wash out) from the chromatographic column much faster.

Ion-exchange chromatography: the role of electrical charge

This type of fractionation is based on electrostatic interaction. The process proceeds due to the binding of ionized groups of the proteins themselves with oppositely charged functional groups of the carrier. Ion-exchange resins — special insoluble polymeric materials — act as such carriers.

The key rule of the method: the strength of adsorption (binding of the protein to the resin) is strictly proportional to the magnitude of the electrical charge of the protein molecule. The more strongly charged the protein is, the more firmly it is fixed on the polymer.

To remove the isolated protein from the column, the elution (washing) procedure is used. The adsorbed proteins are washed out using sodium chloride (NaCl) solutions. The salt concentration is gradually increased in the process. The pattern of elution is as follows: the lower the charge of a particular protein, the lower the NaCl concentration required to break its bond with the ionogenic groups of the resin and wash it into the test tube.

Affinity chromatography: absolute specificity

Among all the approaches considered, affinity chromatography stands out as the most specific method for isolating individual proteins. It allows for the targeted "fishing out" of the desired macromolecule from highly complex mixtures.

The mechanism of this method includes several sequential stages:

1. Carrier preparation: a ligand — a molecule with which the target protein interacts under natural conditions — is firmly attached to a chemically inert polymer via covalent bonding.
2. Passing the mixture: the protein solution is applied to the column. At this moment, strictly complementary binding of the target protein to the immobilized ligand occurs.
3. Final result: exclusively the protein that is specific to this particular ligand is adsorbed on the polymer column. All other (ballast) proteins pass straight through unhindered, without being retained.

Class and exam questions

What else to study on this topic

• The three-dimensional structure and properties of dextran polysaccharide chains.
• The chemical nature of insoluble ion-exchange resins.
• Mechanisms of covalent attachment of a ligand to an inert polymer.
• The nature of complementary protein-ligand binding.

Other topics in the section "Structure, Properties, and Functions of Proteins"

Prepared by WhiteCoat Pro faculty in accordance with the medical school curriculum. For educational purposes only.