Chromatography is one of the methods for separating substances. It is used for subsequent qualitative and quantitative analysis of the physical and chemical properties of microparticles. A variation of this technology is affinity chromatography. The idea of differentiating protein compounds using the properties of molecular affinity has been known in science for several decades. However, it received its development only in recent years, after highly porous hydrophilic materials used as a matrix were introduced. This method allows you to solve both analytical problems (separation of substances and their identification), and preparative (purification, concentration).
Essence
Affinity chromatography (from the Latin word affinis - “adjacent”, “related”) is based on affinity interactions, which are the formation of highly specific bonds between a separator molecule (ligand or affinity) and the target molecule. These mechanisms are widespread in nature (the connection of mediators or hormones and receptors, antibodies and antigens, hybridization of polynucleotides and other types of processes). In medicine, affinity chromatography began to be used for practical purposes since 1951.
The separation of the components is as follows:
- a working solution containing the substance to be isolated is passed through a sorbent;
- a ligand deposited on a sorbent matrix retains this substance;
- its concentration occurs (accumulation);
- extraction of the selected substance from the sorbent by washing with a solvent.
This method allows you to select whole cells. The difference from traditional sorption chromatography is that there is a strong biospecific binding of the secreted component to the sorbent, characterized by high selectivity.
Adsorbents
The following substances are used as adsorbents:
- Gel formulations based on agarose - a polysaccharide derived from agar. The most commonly used 3 varieties: 4B Sepharose, CL (crosslinked agarose) and affi gel. The latter composition is a modified gel of agarose and polyacrylamide. It has a greater biological inertness, high chemical and thermal resistance.
- Silica (silica gel).
- Glass.
- Organic polymers.
To eliminate mechanical obstacles in contact with the ligand, additional substances are used that separate it from the carrier (peptides, diamines, polyamines, oligosaccharides).
Equipment
Equipment for affinity chromatography includes the following main components:
- storage tanks for the mobile phase (eluent);
- high pressure pumps for medium supply (most often piston);
- filter for cleaning eluents from dust;
- metering device;
- chromatographic column for separating the mixture;
- a detector for detecting separated components leaving the column;
- chromatogram recorders and microprocessor unit (computer).
In order to reduce the amount of dissolved air, helium is preliminarily passed through the mobile phase. To change the concentration of the eluent, several pumps are installed, controlled by the programmer. Chromatographic columns are made of stainless steel (with increased requirements for corrosion resistance), glass (universal option) or acrylic. For preparative purposes, their diameter can vary from 2 to 70 cm. In analytical chromatography, microcolumns Ø10-150 microns are used.
To increase the sensitivity of the detectors, reagents are introduced into the mixture that promote the formation of substances that absorb more rays in the ultraviolet or visible spectral range.
Methodology
There are 2 main types of liquid affinity chromatography:
- Columnar, in which the column is filled with a stationary phase and a mixture with an eluent flow is passed through it. Separation can occur under pressure or under the action of gravity.
- Thin layer. The eluent moves along a flat layer of adsorbent under the influence of capillary forces. The adsorbent is applied to a glass plate, ceramic or quartz wand, metal foil.
The main stages of work include:
- adsorbent preparation; ligand fixation on a carrier;
- feeding the mixture for separation into a chromatographic column;
- loading of the mobile phase, binding of the component by a ligand;
- phase change to release the bound substance.
Appointment
Affinity chromatography is used to isolate the following types of substances (the type of ligand used in this case is indicated in brackets):
- analogues of enzymatic inhibitors, substrates and cofactors (enzymes);
- bioorganic substances with signs of genetic foreignness, viruses and cells (antibodies);
- high molecular weight carbohydrates, monosaccharide polymers, glycoproteins (lectins);
- nuclear proteins, nucleotidyl transferases (nucleic acids);
- receptors, transport proteins (vitamins, hormones);
- proteins interacting with cell membranes (cells).
This technology is also used to obtain immobilized enzymes, and their binding to cellulose allows the production of immunosorbents.
Chromatography of DNA binding proteins
Isolation of DNA-binding proteins is performed using heparin. This glycosaminoglycan is able to bind a wide range of molecules. Affinity chromatography of proteins of this group is used to isolate substances such as:
- translation initiation and elongation factors (synthesis of nucleic acid molecules and proteins);
- restriction enzymes (enzymes that recognize specific sequences in double-stranded DNA);
- DNA ligases and polymerases (enzymes that catalyze the combination of two molecules to form a new chemical bond and participate in DNA replication);
- serine protease inhibitors, which play an important role in immune and inflammatory processes;
- growth factors: fibroblasts, Schwann, endothelial;
- intercellular matrix proteins;
- hormone receptors;
- lipoproteins.
Advantages
This method is one of the most specific for the isolation of reactive compounds (enzymes and larger aggregates - viruses). However, it is used not only to isolate biologically active substances.
The detection of antibodies in small quantities, the quantitative assessment of polyadenyl acid, the rapid determination of the molecular weights of dehydrogenases, the removal of certain pollutants, the study of the kinetics of activation of the inactive form of trypsin, the molecular structure of human interferons - this is not the whole list of studies that use affinity chromatography. The use in the clinic is due to its advantages such as:
- The ability to effectively purify proteins, polysaccharides, nucleic acids. They slightly differ in their physicochemical properties and lose activity during hydrolysis, denaturation and other types of exposure used in other methods.
- The speed of separation of substances, the dynamic nature of the process.
- The absence of the need for special purification of enzymes and homogenization of isoenzymes to determine dissociation constants.
- The ability to separate a wide range of substances.
- Low consumption of ligands.
- The possibility of separation of substances in large volumes.
- Reversible process of binding of biological macromolecules.
This technique can be combined with others, impose an additional field (gravitational, electromagnetic). This allows you to expand the technical capabilities of chromatography.
Enzyme engineering
Thanks to this method, the active development of a new branch of biotechnology, enzyme engineering, has begun.
Affinity chromatography in relation to the isolation of enzymes has the following advantages:
- the production of enzymes in large quantities as a result of less time spent, as a result - their cost reduction;
- immobilization of enzymes can significantly expand their scope in medicine and industry;
- the connection of enzymes with an insoluble solid substrate makes it possible to study the influence of the microenvironment and the direction of reactions, which play an important role in natural and physiological processes.