Affinity chromatography

Its one of the important techniques in chromatography.

1. AFFINITY CHROMATOGRAPHY MD NADIR HASSAN

2. CONTENTS 1.INTRODUCTION 2.HISTORY 3.PRINCIPLE 4.COMPONENTS 5.TYPES 6.EXPERIMENTAL PROCEDURE 7.APPLICATIONS ,ADVANTAGES AND LIMITATIONS 8.CONCLUSION

3. AFFINITY CHROMATOGRAPHY Biomolecules are purified using techniques that separate according to differences in specific properties. PROPERTY TECHNIQUE Charge Ion exchange chromatography Size Gel filtration chromatography Biorecognition Affinity chromatography It is a technique that enables the purification of biomolecule on the basis of its biological function. Interaction between a biomolecule and a specific ligand hold the basis of separtion. SAMPLE LIGAND Enzyme Co factor, inhibitor Antibody Antigen, virus Lectin Polysaccharide,glycoprotein Nucleic acid Histones, bases Hormones Receptors,carrier proteins

4. HISTORY In1910, German scientist Emil Starkenstein published an article describing the concept of resolving macromolecules via their interaction with an immobilised substrate. The credit for the invention and term of affinity chromatography goes to Pedro Cuatecasas and Meir Wilchek. Their article(1968) briefly describe the technique of enzyme purification via immobilised substrate and inhibitors. In 1987,Pedro and Meir won Wolf Prize in medicine for their invention.

5. PRINCIPLE Separation of desired biomolecule s relies on the reversible interaction between the sample to be purified and affinity ligand. Most of the biomolecule (say protein ) have an inherent recognition site that can be used to select the appropriate affinity ligand. The binding between the protein of interest and the chosen ligand must be both specific and reversible . There is a covalent affixing of molecules to the ligand.

6. COMPONENTS Using affinity chromatography requires the following components: 1. MATRIX Characteristics features of a matrix are: a) Low non specific adsorption. b) Good flow characteristics c) Chemical and mechanical stability over a broad range of pH , ionic strengths, and denaturant concentrations. d) Availability, in large numbers,of chemical groups capable of being activated. e) High effective porosity .

7. TYPES OF MATRIX AGAROSE Often used and ferry all the desirable features. Only disadvantage is its susceptibility to contraction when denaturant solutions are used. POLYACRYLAMIDE Lack porosity and undeseribility increase with substitution. CONTROLLED POROSITY GLASS BEADS Mechanical rigidity, chemical inertness, and good flow rates are its feature but it has high non specific adsorption.

8. LIGAND The selection of ligand should have two most important requirements: a) Ligand interaction should be less with desired macromolecule. b) The ligand should possess functional groups that can be modified to form covalent linkage with the supporting matrix. COUPLING OF LIGAND TO MATRIX INVOLVES: Activation of matrix functional groups Coupling of the ligand to these activated groups

9. 3. SPACER ARMS To avoid the encounter steric repulsion between ligand and activated groups of matrix . The ligand projects out the macromolecule to prevent repulsion. E.g.: 1)Hexamethylene, 3,3’-diamino propylamine 2) 1,6-diamino hexane 3) 6-amino-hexanoic acid 4) 1,4-bis-(2,3-epoxypropoxy) butane Has two functional sites :one to react with matrix and other to ligand.

10. LIGAND IMMOBILIZATION The most common procedure is to link a coupling agent to the matrix and then add the liagand. Most of the common couplers are : 1. CYANOGEN-BROMIDE-ACTIVATED AGAROSE Cyanogen bromide(CNBr) reacts strongly with the amino group. Useful in coupling enzymes, coenzymes, inhibitors, antigens, nucelic acid. 2. AMINOETHYL- AND HYDRAZIDE-ACTIVATED POLYACRYLAMIDE More ligands can be coupled in this. Capacity of polyacrylamide support column is greater than agarose. The most common linkers are -CONH-CH2-CH2-NH2 CONH-NH2 3. THIOPROPYL-AGAROSE Has 2-thiopyridyl groups linked to agarose by a spacer of a 2-hydroxypropyl residues. 2-thiopyridyl group react with terminal thiol groups.

11. TYPES On the basis of types of ligand ,affinity has few categorisation: 1.IMMUNO AFFINITY CHROMATOGRAPHY Antibodies is used as immobilised ligand. Purify range of proteins Monoclonal antibodies may be linked with agarose matrix. 2.METAL CHELATE CHROMATOGRAPHY Immobilised metal ion such as Cuprous,Zinc or transition metal such as Nickel is used. Reacts with imidazole group of histidine,thiol group of cysteine,indole group of tryptophan residues of proteins. Formation of co ordinate bond is there.

12. 3.COVALENT AFFINITY CHROMATOGRAPHY Ligand contain a disulphide group. Specifically developed to separate thio containing proteins. The most commonly used ligand is disulphide 2’-pyridyl group. The ligand is attached to an agarose matrix i.e Sepharose 4B.

13. EXPERIMENTAL PROCEDURE Select gel and ligand Swell gel in buffer and couple the ligand. Prepare gel for column. Pack gel in glass column and set up column equipment. Equiliberate column with buffer. Apply sample Wash column to elute unbound molecules. Elute bound molecules. Collect and analyse eluent.

14. ELUTION Generally two methods of elution are there: First is by washing the column with a solution of a compound which competes with bound ligand and has higher affinity. Require large volumes of eluent . Increasing the competitor concentration does not alter time require to remove desire molecule. The second approach is to drastically change the environment of the complex. The methods are Change ionic strength Change of pH This does not totally unfold the protein.

15. APPLICATIONS Purification of substances from biological mixtures. Separation of native from denatured form of protein. Purify and concentrate an enzyme in a solution. Purification of immunoglobin. Purification of small amount of biological material from high level of contaminating substances.

16. ADVANTAGES Single step purification. The matrix can be reused rapidly. The matrix is solid ,can easily be washed and dried. Give purified product with high yield. Also removes specific contaminants like proteases. The technique can be used for substances of low concentration

17. LIMITATIONS Non specific adsorption can totally be not eliminated,can only be minimised. Limited availability High cost of immobilised ligand If required pH is not adjusted ,protein get denatured. Creates problem when two macromolecules has same affinity for a particular ligand .

18. CONCLUSION Affinity chromatography is one of the most reliable chromatographic technique. This technique offers high selectivity,hence high resolution and usually high amount of protein of interest. It is theoretically capable of giving absolute purifications even from complex mixtures in a single process.

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