Affinity chromatography separates proteins on the basis of a reversible interaction between a protein (or group of proteins) and a specific ligand coupled to a chromatographic matrix. The technique is ideal for a capture or intermediate step in a purification protocol and can be used whenever a suitable ligand is available for the protein(s) of interest. With high selectivity, hence high resolution, and high capacity for the protein(s) of interest, purification levels in the order of several thousand-fold with high recovery of active material are achievable.
Target protein(s) is collected in a purified, concentrated form. Biological interactions between ligand and target molecule can be a result of electrostatic or hydrophobic interactions, van der Waals’ forces and/or hydrogen bonding. To elute the target molecule from the affinity medium the interaction can be reversed, either specifically using a competitive ligand, or non-specifically, by changing the pH, ionic strength or polarity. In a single step, affinity purification can offer immense time-saving over less selective multistep procedures. The concentrating effect enables large volumes to be processed.
Target molecules can be purified from complex biological mixtures, native forms can be separated from denatured forms of the same substance and small amounts of biological material can be purified from high levels of contaminating substances. Affinity chromatography owes its name to the exploitation of these various biological affinities for adsorption to a solid phase. One of the members of the pair in the interaction, the ligand, is immobilized on the solid phase, whereas the other, the counterligand (most often a protein), is adsorbed from the extract that is passing through the column. Examples of such affinity systems are listed in Table 1.1.
Affinity sorption requires that the compound to be isolated is capable of reversibly binding (i.e., sorption-elution) to a sorbent which consists of a complementary substance (i.e., the so-called ligand) immobilized on a suitable insoluble support, i.e., the so-called carrier.
Schematic represantation of bioaffinity is shown in Figure 1.1. Affinity chromatography demonstrated in this figure is based on the simple principle that every biomolecule usually recognize another natural or artificial molecule. A wide variety of ligands may be covalently attached to an inert support matrix, and subsequently packed into a chromatographic column.
The term affinity chromatography has been given quite different connotations by different authors. Sometimes it is very broad, including all kinds of adsorption chromatographies based on nontraditional ligands, in the extreme all chromatographies except ion exchange. Often it is meant to include immobilized metal ion affinity chromatography (IMAC), covalent chromatography, hydrophobic interaction chromatography, and so on. In other cases it refers only to ligands based on biologically functional pairs, such as enzyme-inhibitor complexes.
The term not only to include functional pairs but also the so-called biomimetic ligands, particularly dyes whose binding apparently often occurs to active sites of functional enzymes although the dye molecules themselves of course do not exist in the functional context of the cell. Thus chromatography based on the formation of specific complexes such as enzyme-substrate, enzyme-inhibitor, etc., i.e on biological recognition, is termed bioaffinity or biospecific chromatography and the respective interaction-biospecific adsorption or bioaffinity.
The original term “affinity chromatography” acquired a broader meaning also including hydrophobic chromatography, covalent chromatography, metal-chelate chromatography, chromatography on synthetic ligands, etc., i.e chromatography procedures based on different, less specific types of interaction. The broad scope of the various applications of affinity has generated the development of subspecialty techniques, many of which are now recognized by their own nomenclature. Table 1.2 summarizes some of these techniques. As can be seen from Table 1.2, some of these subcategories have become accepted useful techniques.
Very often the use of affinity chromatography requires that the investigator synthesizes the adsorbent. The methods for doing this, which are described later, are well worked out and are also easily adopted for those not skilled in synthetic organic chemistry. To further simplify the task, activated gel matrices ready for the reaction with a ligand are commerically available. The immobilization of a ligand can, in the best cases, be a very simple affair. In addition, immobilizations are just as easy for proteins as for small molecules.
A property that needs special consideration is the association strength between ligand and counterligand. If it is too weak there will be no adsorption, whereas if it is too strong it will be difficult to elute the protein adsorbed. It is always important to find conditions, such as pH, salt concentration, or inclusion of, for example, detergent or other substances, that promote the dissociation of the complex without destroying the active protein at the same time. It is often here that the major difficulties with affinity methods are encountered.
Ligands can be extremely selective, but they may also be only group specific. The latter type includes glycoprotein-lectin interactions, several dye-enzyme interactions, and interactions with immobilized cofactors. However, these interactions have also proved to be extremely helpful in solving many separation problems. Good examples are ligands that are group selective against immunoglobulins (e.g., staphylococcal protein A or streptococcal protein G).
Dye-Ligand Afinity Chromatography
In affinity chromatography a molecule having specific recognition capability (“ligand” or “binder”) is immobilized on a suitable insoluble support (“matrix” or “carrier”), which is usually a polymeric material in bead or membrane form. The molecule to be isolated (“analyte” or “target”) is selectively captured (“adsorbed”) by the complementary ligand immobilized on the matrix by simply passing the solution containing the target through the chromatographic column under favorable conditions.
The target molecules are then eluted (“desorbed”) by using proper eluants under conditions favoring desorption, by adjusting the pH, ionic strength or temperature, using specific solvents or competitive free ligands, so that the interaction between the ligand and target is broken and the target molecules are obtained in a purified form. Since its first introduction, thousands of different molecules (enzymes, antibodies, hormones, vitamins, receptors, many variety of other proteins and glycoproteins, RNA, DNA, etc.), even bacteria, viruses, and cells have been separated or purified by affinity chromatography.
A wide variety of functional molecules, including enzymes, coenzymes, cofactors, antibodies, amino acids, oligopeptides, proteins, nucleic acids, and oligonucleotides may be used as ligands in the design of novel sorbents. These ligands are extremely specific in most cases. However, they are expensive, due to high cost of production and/or extensive purification steps. In the process of the preparation of specific sorbents, it is difficult to immobilize certain ligands on the supporting matrix with retention of their original biological activity.
Precautions are also required in their use (at sorption and elution steps) and storage. Dye-ligands have been considered as one of the important alternatives to natural counterparts for specific affinity chromatography to circumvent many of their drawbacks, mentioned above.
Dye-ligands are able to bind most types of proteins, especially enzymes, in some cases in a remarkably specific manner. They are commercially available, inexpensive, and can easily be immobilized, especially on matrices bearing hydroxyl groups, stable against biological and chemical attack, storage adsorbent without loss of activity, reusable: cleaning and sterilization, high capacity. Although dyes are all synthetic in nature, they are still classified as affinity ligands because they interact with the active sites of many proteins by mimicking the structure of the substrates, cofactors, or binding agents for those proteins.
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