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 property of molecular affinity has been known in science for several decades. However, it has received its development only in recent years, after the introduction of highly porous hydrophilic materials used as a matrix. This method allows solving both analytical problems (separation of substances and their identification) and preparative problems (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 spacer molecule (ligand or affinant) and a target molecule. These mechanisms are widespread in nature (connection of mediators or hormones and receptors, antibodies andantigens, hybridization of polynucleotides and other types of processes). In medicine, affinity chromatography has been used for practical purposes since 1951
The components are separated as follows:
- working solution containing the substance to be isolated is passed through the sorbent;
- ligand deposited on the sorbent matrix retains this substance;
- it is concentrated (accumulation);
- extraction of the isolated substance from the sorbent by washing with a solvent.
This method allows you to isolate whole cells. The difference from traditional sorption chromatography is that there is a strong biospecific binding of the isolated component to the sorbent, which is characterized by high selectivity.
Adsorbents
The following substances are used as adsorbents:
- Gel compounds based on agarose, a polysaccharide obtained from agar. The most commonly used are 3 varieties: sepharose 4B, CL (cross-linked agarose) and affi-gel. The latter composition is a modified gel of agarose and polyacrylamide. It has greater biological inertness, high chemical and thermal resistance.
- Silica (silica gel).
- Glass.
- Organic polymers.
To eliminate mechanical obstacles during ligand contact, additional substances are used to separate it from the carrier (peptides, diamines, polyamines, oligosaccharides).
Equipment
Affinity chromatography equipment includes the following main units:
- storage tanks for the mobile phase (eluent);
- high pressure pumps for medium supply (most often reciprocating);
- filter for cleaning eluents from dust;
- dosing device;
- chromatographic column for mixture separation;
- 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 first passed through the mobile phase. To change the concentration of the eluent, several pumps controlled by the programmer are installed. Chromatographic columns are made of stainless steel (for 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 µm are used.
To increase the sensitivity of the detectors, reagents are introduced into the mixture, which contribute to the formation of substances that absorb more rays in the ultraviolet or visible region of the spectrum.
Methodology
There are 2 main types of liquid affinity chromatography:
- Column, in which the column is filled with a stationary phase and a mixture is passed through it with a floweluent. Separation can occur under pressure or under gravity.
- Thin layer. The eluent moves along the flat adsorbent layer under the influence of capillary forces. The adsorbent is applied to a glass plate, ceramic or quartz rod, metal foil.
Main stages of work include:
- preparation of the adsorbent, fixation of the ligand on the carrier;
- feeding the separation mixture to the chromatographic column;
- mobile phase loading, component binding by ligand;
- phase replacement to isolate the bound substance.
Destination
Affinity chromatography is used to isolate the following types of substances (the type of ligand used is indicated in brackets):
- analogues of enzymatic inhibitors, substrates and cofactors (enzymes);
- bioorganic substances with signs of genetic alienness, viruses and cells (antibodies);
- high molecular weight carbohydrates, monosaccharide polymers, glycoproteins (lectins);
- nuclear proteins, nucleotidyltransferases (nucleic acids);
- receptors, transport proteins (vitamins, hormones);
- proteins interacting with cell membranes (cells).
This technology is also used to obtain immobilized enzymes, and binding them to cellulose allows the production of immunosorbents.
Chromatography of DNA-binding proteins
Isolation of DNA-binding proteins is performed usingheparin. This glycosaminoglycan is capable of binding a wide range of molecules. Affinity chromatography of proteins of this group is used to isolate substances such as:
- factors of initiation and elongation of translation (synthesis of nucleic acid molecules and proteins);
- restrictases (enzymes that recognize certain sequences in double-stranded DNA);
- DNA ligases and polymerases (enzymes that catalyze the joining of two molecules to form a new chemical bond and are involved in DNA replication);
- serine protease inhibitors that play an important role in immune and inflammatory processes;
- growth factors: fibroblast, Schwann, endothelial;
- proteins of extracellular matrix;
- hormone receptors;
- lipoproteins.
Dignity
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.
Detection of antibodies in small quantities, quantitative assessment of polyadenylic acid, rapid determination of the molecular masses of dehydrogenases, removal of certain pollutants, 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 in which affinity is used. chromatography. The use in the clinic is due to its advantages such as:
- Effective cleaning capabilityproteins, polysaccharides, nucleic acids. They differ slightly in their physical and chemical properties and lose activity during hydrolysis, denaturation and other types of treatment used in other methods.
- The speed of separation of substances, the dynamic nature of the process.
- No need for special enzyme purification and isoenzyme homogenization to determine dissociation constants.
- Able to separate a wide range of substances.
- Low consumption of ligands.
- Possibility of separation of substances in large volumes.
- Reversible process of binding biological macromolecules.
This technique can be combined with others, to impose an additional field (gravitational, electromagnetic). This allows you to expand the technical capabilities of chromatography.
Enzymatic engineering
Thanks to this method, the active development of a new branch of biotechnology - enzyme engineering began.
Affinity chromatography for enzyme isolation has the following advantages:
- obtaining enzymes in large quantities as a result of less time, as a result - their reduction in price;
- immobilization of enzymes can significantly expand the scope of their application in medicine and industry;
- The association of enzymes with an insoluble solid support 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.
