**2.3. Lectin affinity chromatography**

Lectins which are non-immun proteins are produced by plants, vertebrates and invertebrates. Especially various plant seeds synthesize high levels of lectins [24]. Certain types of carbohy‐ drate residues may be seperated via this method due all lectins have the ability to recognize and bind these types of compounds. Mostly used lectins for affinity columns are concanavalin A, soybean lectin and wheat germ agglutinin [1, 24]. Concanavalin A is specific for α-Dmannose and α-D-glucose residues while wheat germ agglutinin binds to D-*N*-acetylglucosamine. Lectins which are commonly used for the isolation of compunds containing carbohydrates such as polysaccharides, glycoproteins and glycolipids in affinity chromatog‐ raphy are given in Table 4. [1].


Among these amino acids histidine is the most commonly used one. Attachment of histidine tags to the recombinant proteins polypeptides is the most known development in the field of IMAC. Histidine and other metal affinity tags are widely used for protein purification [26]. Adsorbents may be prepared by binding chelators onto the surface and metals to the chelators. Free coordination sites of the metal ions are needed for the analyte to bind to metal ions [25].

Zn2+, Ni2+ and Cu2+ are the most commonly used metal ions. Basic groups on protein surfaces especially the side chain of hisitidine residues, are attracted to the metal ions to form a weak

used in IMAC applications, especially Ni2+ which has six coordination sites and electrochemical stability. The affinity of the metals may be predicted according to the principles of soft acids and bases which is the theory explaining one of the two atoms attached acts as a Lewis acid and the other as a Lewis base. Ligands with oxygen (e.g. carboxylate), aliphatic nitrogen (e.g. asparagin, glutamine) and phosphor (phosphorylated amino acids) are hard Lewis bases, as the ones with sulfur (e.g. cysteine) are soft bases and those with aromatic nitrogen (e.g. histidine, tryptophane) are borderline bases. In case Cu(II), Ni(II), Co(II) or Zn(II) ions are the ions used in the IMAC, the target amino acids on the protein surface are imidazolyl, thiol and indolyl groups; as carboxyl and phosphate groups are of that in case of the use of Fe(III) and Mg(II). Histidine, tryptophane and cysteine are accepted to be the most important amino acids for IMAC due to their strong affinity to metal ions and the retention times. It is reported that histidine residues attached to the protein surface significantly change the retention time of the

, Cu+ and transition metals classified as borderline acids (Co2+, Cu2+, Ni2+) may be

, Ca2+, Mg2+, Fe3+; soft Lewis acids

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The metal ions in the class of hard Lewis acids such as K+

**Chelating Compound Coordination Metal Ions** Aminohydroxamic acid bidentate Fe(III) Salicylaldehyde bidentate Cu(III)

Dipicolylamine (DPA) tridentate Zn(II), Ni(II)

Carboxymethylated aspartic acid (CM-Asp) tetradentate Ca(II), Co(II) *N,N,N'-*tri(carboxymethyl)ethylenediamine (TED) pentadentate Cu(II), Zn(II)

*N-*(2-pyridylmethyl)aminoacetate tridentate Cu(II) 2,6-Diaminomethylpyridine tridentate Cu(II) Nitrilotriacetic acid (NTA) tetradentate Ni(II)

**Table 5.** Some examples of chelating compouns used in IMAC [28]

8-Hydroxy-quinoline (8-HQ) bidentate Al(III), Fe(III), Yb(III) Iminodiacetic acid (IDA) tridentate Cu(II), Zn(II), Ni(II), Co(II)

*ortho-*phosphoserine (OPS) tridentate Fe(III), Al(III), Ca(II), Yb(III)

coordinate bonds [24].

protein of interest [26].

such as Ag+

**Table 4.** Some lectins which are commonly used in affinity chromatography

Enzymes, inhibitors, cofactors, nucleic acids, hormones or cell chromatography can also be utilized as ligands in bioaffinity chromatography types. Examples of these methods include Receptor Affinity Chromatography and DNA Affinity Chromatography [21].

### **2.4. Dye-ligand affinity chromatography**

Development of the dye-ligand affinity chromatography could be attributed to observation of some proteins irregular elution characteristics during fractionation on gel filtration column in presence of blue dextran. Blue dextran consists of a triazine dye (cibacron blue F3G-A) covalently linked to high molecular mass dextran. Some proteins bind triazine dye and this allows to its use as an affinity adsorbent by immobilization [24]. This method is especially popular tool for enzyme and protein purification [21]. Dye-ligand adsorbents are of interest due to inexpensiveness, ease of availablity and immobilization process. These adsorbents may be used in analytical, preparative analysis and large scale studies. Although dye-ligand affinity technique for pharmaceuticals may be preferred owing to these advantages, concerns about leakage and toxicity has stopped its use. Therefore proteins purified using this technique is convenient for analytical or technical uses. Procion Red HE3b, Red A, Cibacron Blue F3G-A are some examples of dye-ligands which are used for purification [9].

### **2.5. Metal-chelate affinity chromatography (Immobilized-metal (Ion) affinity chromatography)**

In 1970s, first application of metal-chelate affinity chromatography which is later named as "immobilized-metal (ion) affinity chromatography (IMAC) was perfomed. Metal-chelate chromatography technique exploits selective interactions and affinity between transition metal immobilized on a solid support (resin) via a metal chelator and amino acid residues which act as electron donors in the protein of interest [25-26]. As well as aromatic and heterocyclic compounds, proteins such as histidine, tyrosine, tyriptophane and phenylalanine posses affinity to transition metals which form complexes with compounds rich in electrons [25,27]. Among these amino acids histidine is the most commonly used one. Attachment of histidine tags to the recombinant proteins polypeptides is the most known development in the field of IMAC. Histidine and other metal affinity tags are widely used for protein purification [26]. Adsorbents may be prepared by binding chelators onto the surface and metals to the chelators. Free coordination sites of the metal ions are needed for the analyte to bind to metal ions [25].

**Lectin Source Sugar specificity Eluting sugar**

ELB Elderberry bark Sialic acid or *N*-acetyl-β- D -glucosamine Lactose

AAA Freshwater eel α – L-fucose <sup>L</sup> -fucose

are some examples of dye-ligands which are used for purification [9].

**2.5. Metal-chelate affinity chromatography (Immobilized-metal (Ion) affinity**

In 1970s, first application of metal-chelate affinity chromatography which is later named as "immobilized-metal (ion) affinity chromatography (IMAC) was perfomed. Metal-chelate chromatography technique exploits selective interactions and affinity between transition metal immobilized on a solid support (resin) via a metal chelator and amino acid residues which act as electron donors in the protein of interest [25-26]. As well as aromatic and heterocyclic compounds, proteins such as histidine, tyrosine, tyriptophane and phenylalanine posses affinity to transition metals which form complexes with compounds rich in electrons [25,27].

**Table 4.** Some lectins which are commonly used in affinity chromatography

**2.4. Dye-ligand affinity chromatography**

**chromatography)**

90 Column Chromatography

GNL Snowdrop bulbs α -1→ 3 mannose α -methyl mannose

Receptor Affinity Chromatography and DNA Affinity Chromatography [21].

Enzymes, inhibitors, cofactors, nucleic acids, hormones or cell chromatography can also be utilized as ligands in bioaffinity chromatography types. Examples of these methods include

Development of the dye-ligand affinity chromatography could be attributed to observation of some proteins irregular elution characteristics during fractionation on gel filtration column in presence of blue dextran. Blue dextran consists of a triazine dye (cibacron blue F3G-A) covalently linked to high molecular mass dextran. Some proteins bind triazine dye and this allows to its use as an affinity adsorbent by immobilization [24]. This method is especially popular tool for enzyme and protein purification [21]. Dye-ligand adsorbents are of interest due to inexpensiveness, ease of availablity and immobilization process. These adsorbents may be used in analytical, preparative analysis and large scale studies. Although dye-ligand affinity technique for pharmaceuticals may be preferred owing to these advantages, concerns about leakage and toxicity has stopped its use. Therefore proteins purified using this technique is convenient for analytical or technical uses. Procion Red HE3b, Red A, Cibacron Blue F3G-A

Con A Jack bean seeds α- D-mannose, α- D -glucose α - D -methyl mannose WG A Wheat germ *N*-acetyl-β- D -glucosamine *N*-acetyl-β- D -glucosamine PSA Peas α- D -mannose α- D -methyl mannose LEL Tomato *N*-acetyl-β- D -glucosamine *N*-acetyl-β- D -glucosamine STL Potato tubers *N*-acetyl-β- D -glucosamine *N*-acetyl-β- D -glucosamine PHA Red kidney bean *N*-acetyl-β- D -glucosamine *N*-acetyl-β- D -glucosamine

Zn2+, Ni2+ and Cu2+ are the most commonly used metal ions. Basic groups on protein surfaces especially the side chain of hisitidine residues, are attracted to the metal ions to form a weak coordinate bonds [24].

The metal ions in the class of hard Lewis acids such as K+ , Ca2+, Mg2+, Fe3+; soft Lewis acids such as Ag+ , Cu+ and transition metals classified as borderline acids (Co2+, Cu2+, Ni2+) may be used in IMAC applications, especially Ni2+ which has six coordination sites and electrochemical stability. The affinity of the metals may be predicted according to the principles of soft acids and bases which is the theory explaining one of the two atoms attached acts as a Lewis acid and the other as a Lewis base. Ligands with oxygen (e.g. carboxylate), aliphatic nitrogen (e.g. asparagin, glutamine) and phosphor (phosphorylated amino acids) are hard Lewis bases, as the ones with sulfur (e.g. cysteine) are soft bases and those with aromatic nitrogen (e.g. histidine, tryptophane) are borderline bases. In case Cu(II), Ni(II), Co(II) or Zn(II) ions are the ions used in the IMAC, the target amino acids on the protein surface are imidazolyl, thiol and indolyl groups; as carboxyl and phosphate groups are of that in case of the use of Fe(III) and Mg(II). Histidine, tryptophane and cysteine are accepted to be the most important amino acids for IMAC due to their strong affinity to metal ions and the retention times. It is reported that histidine residues attached to the protein surface significantly change the retention time of the protein of interest [26].


**Table 5.** Some examples of chelating compouns used in IMAC [28]

Multidentate chelating compounds are widely used in order to strengthen the complex which is comprised of chelator, metal ion and protein. Different length of spacers is used to bind the chelator onto the surface of the support. Type of the chelator influences the strength of the chelation and retention power, for instance metal binds to the nitrogen atom and two carbox‐ ylate oxygens and reveals three free sites in case of tridentate iminodiacetic acid (IDA); tetradentate nitrilotriacetic acid (NTA) binds the metal by an additional carboxylate oxygen and this provides stronger chelation, but a weaker retention power. IDA is the chelator which is commonly used in the applications of IMAC. Although most of the chelators are carboxy‐ methylated amines, there also some other compounds which are commonly used such as dyeresistant yellow 2KT, OPS and 8-HQ [26]. Some examples of chelating compounds are given in Table 5.

stationary phases for chiral seperations. Protein-based and carbohydrate-based ligands may be used as the stationary phases in the analysis of chiral compounds via HPLC [1]. Orosomu‐

whites) are some examples of protein-based stationary phases, while cyclodextrins (especially

There are a number of areas related to affinity chromatography that have also been of great interest in pharmaceutical and biomedical analysis. One such area is the use of affinity chromatography in drug discovery [12]. In drug discovery explaining the mechanism of action of bioactive compounds, which are used as pharmaceutical drugs and biologically active natural products, in the cells and the living body is important. For this pupose isolation and identification of target protein(s) for the bioactive compound are essential in understanding its function fully. Affinity chromatography is a useful method capable of isolating and identifying target molecules for a specific ligand, utilizing affinity between biomolecules such as antigen–antibody reactions, DNA hybridization, and enzyme–substrate interactions. Since the development of affinity chromatography in the early 1950s, various types of target proteins for bioactive compounds have been isolated and identified. Selected samples of the target proteins isolated by affinity chromatography are listed in Table 6. Since then, affinity chro‐ matography has been gaining renewed attention as a widely applicable technique for the

**Bioactive compound Molecular structure Target protein**

NH O

N

Cl

H

N

H

OO

O

OH

O

O

OH

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Cathepsin B, H, L

Glyoxalase 1

β-cyclodextrin) are of carbohydrate-based stationary phases [1,29].

**3. Affinity chromatography and drug discovery**

discovery the target proteins for bioactive compounds [30].

H

CH3O


coid (α<sup>1</sup>

E-64

(cystein protease

Indomethacin (antitumor drug)

inhibitor) <sup>N</sup>

H2N
