AFFINITIY CHROMATOGRAPHY

Affinity Chromatography

The attracting force exerted in different degrees between atom which causes them to enter into it and remain in combination is the type of adsorption chromatography in which there is high degree of specificity in the interaction between adsorbent and the compound to be separated. The technique requires that material to be separated is capable of binding reversibility to a specific ligand that is attached to an insoluble matrix.

M + L ® ML

(Macromolecules) (Ligand) (Complex)

Affinity chromatography was originally devloped for purification of enzymes but extended to nucleotides, nucleic acid and immunoglobulins. In case of the purification of ligand attached to adsorbent is usually a powerful inhibitor which will bind only one enzyme in a complex mixture.

Figure 1: Binding of ligand and macromoleuclar complex (Matrix)

An ideal matrix for affinity chromatography must possess following characteristics:

1. It must be stable during binding of macromolecules and in subsequent elution.

2. It must be at the most interactive with a specific compound and only weakly with other macromolecules to minimize non specific adsorption.

3. It must contain suitable and sufficient chemical group to which ligand may attched and it must be stable under condition of attachment.

4. It should exhibit good flow properties i.e. the matrix has to be macroporous to allow large molecules assess to the binding site and need to have good flow properties.

5. It has to be devoid of non-specific adsorption sites but must contain functional group to which ligand can be attached.

The most common matrixes that are used in Affinity chromatography:

· Dextran

· Porous glass

· Polyacrylamide

· Agarose

· Cellulose

The ligand is covalently attached to the supporting matrix so that the chromatography materials can be designed for a specific purification task. The chemical nature of ligand is determined by biological specificity of the compound to reseparate it. For e.g. in case of enzyme purification, the ligand attached to matrix is usually a powerful inhibiton which will bind only one enzyme in a complex mixture.

While selecting ligand one should consider that the selective ligand will bind one particular compound (compund to be separated). Ligand should be specific.

It is essential that the ligand should possess the suitable chemical group that will be involed in the binding of ligand to the macromolecules to be separated. Ligand should possess functional group that will bind with matrix. The most common such groups are amine groups, carboxyllic groups, phenolic groups and alcoholic groups.

Ligand	Affinity
1. Nucleotides 5¢ AMP 2; 5¢ AMP	NAD⁺ dependent dehydrogenase, some kinase NADP⁺ dependent dehydrogenase
2. Fatty acids	Fatty acid-binding proteins
3. Proteins A and G	Immunoglobulins
4. Soyabean lectin	Glycoprotein containing N-acetyl -µ or b D galactopyrabosyl residues
5. Phenylboronate	Glycoproteins
6. Poly (A)	RNA containing poly (U) sequences
7. Lysine	RNA

Spacer arms ® 1,6 diaminohexane

® 6 amino hexanoic acid

® 1,4 bis (2,3 epoxypropoxy) butane

Spacer arm is nearly inserted between matrix and ligand so that large molecule can get assess the binding sites. The spacer arm positions the ligand at the certain distance from the matrix. In practice spacer arms of 2-10 C atoms has to be found optimum.

The principle of affinity chromatography.

Figure 2: Binding of specific molecules in the affinity resin

When the complex mixture contains specific compound to be purified is added to the ligand contained in chromatographic column only specific compound bound to ligand. All the washed and bound compound can be away and subsequently purified can be recovered.

Purification of acetylcholine esterase by Affinity chromatography

Acetiylcholineesterase is membrane bound enzyme which are extracted fron mice brain to rat brain.

Steps:

1. Preparation of an affinity column / material

The most common method of attachment of ligand to the matrix involves treatment of matrix with cyanogen bromide activated sepharose 48. Cyanogen bromide is toxic and causes explossion which is impure.

2. Addition of Spacer arm.

The spacer arm is introduced next by reacting the activated sepharose with diaminobutanne then lengthen by addition of succinic anhydride so that the ligand 3 amino N methyl pyrimide ion can be linked to the activated spacer arm.

The affinity material prepared is N-methyl 3 amino pyridine agarose.

Figure 3: Coupling of Ligand

3. Packing of column

Column of 10 cm x 1 cm size is used. After liquid attachment , the affinity material ions packed into the column. Method of packing of column is similar to that of column chromatography.

4. Sample Application

It is done with micropipette at the top of affinity column.

5. Binding

The ligand attached to the sepharose i.e. 3 amino N methyl pyridine ion is strong inhibitor of acetylcholine esterase so when crude (impure) extract is added to the column, the acetylcholine esterase molecule specifically bind to the ligand.

6. Washing

The other proteins present in the extract donot bind to affinity material and are removed from the column by washing. The washing is achieved by running the solution contaning the protein through the column followed by elution buffer until protein is no longer detected in the eluent. NaCl 1 mol/L in the triton is used as elution buffer.

7. Elution

The bound enzyme i.e. acetylcholine esterase is eluted from the column by incorporating an inhibitor becamethomium bromide in the elution buffer which has an even higher affinity for acetylcholine esterase than that shown by ligand.

8. Reactivation of Acetylcholine esterase

Reactivation means the removal of bound acetylcholine esterase with demethomium bromide. This is carried out by adding the cation exchange resin, amberlite G-120 which strongly binds the decathomium bromide. Thus pure form of acetylcholine bromide can be obtained. After this ion exchange chromatography is run.

Application

1. It has been used in purify a wide range of enzyme and proteins including Ig. For e.g.: DNA related enzyme can be purified by employing affinity chromatography . Hepain agrose is used to purify the hepain binding domain of platelet i.e thrombrspondin can be purified from blood platelets using affinity chromatrography an heprose agqrose.

2. Messenger RNA are routively isolated by using poly(u)sepharose 4B

3. Clinical application: It includes immuno assay. Affinity chromatrography are widely used in theapentic drug monitoring and allergy testing.

4. It has also been applied in concentration of dillute protein solution and storage of unstable protein in the immobilized form.