Affinity chromatography

1.HISTORY OF AFFINITY CHROMATOGRAPHY
2.PRINCIPLE
3.TYPES
Lectin Affinity Chromatography
Immuno Affinity chromatography
Metal Chelate Chromatography
Dye Ligand Chromatography
Covalent chromatography
4.APPLICATIONS

1. S.GOKULAKRISHNAN M.Pharm (Pharmaceutics) – I Year, Department of Pharmaceutics, College of Pharmacy, Mother Theresa Post Graduate and Research Institute of Health Sciences, (A Government of Puducherry Institution) Puducherry . AFFINITY CHROMATOGRAPHY GOKULAKRISHNAN CHROMATOGRAPHY 1

2. HISTORY OF AFFINITY CHROMATOGRAPHY     1930s, first developed by A.Wilhelm Tiselius-a swedish biochemist, won the Nobel Prize in 1948.   Used to study enzymes and other proteins.   Relies on the affinity of various biochemical compounds with specific properties. GOKULAKRISHNAN CHROMATOGRAPHY 2

3. AFFINITY CHROMATOGRAPHY: Discovered by Pedro and Meir Wilcheck . Affinity chromatography is a type of chromatography that makes use of a specific affinity between a substance to be isolated and a molecule that it can specifically bind. GOKULAKRISHNAN CHROMATOGRAPHY 3

4. PRINCIPLE Based on specific affinity between substance to be isolated and a molecule that it can specifically bind(a ligand). M + L ML Macromolecule Ligand (attached Complex to matrices) GOKULAKRISHNAN CHROMATOGRAPHY 4

5. EXAMPLES Antigen -Antibody Antibody- Antigen Substrate -Enzyme DNA- Histon Hormone -Binding Protein/Receptor GOKULAKRISHNAN CHROMATOGRAPHY 5

6. SPECIFICITY OF AFFINITY CHROMATOGRAPHY   Specificity is based on three aspect of affinity: Matrix : for ligand attachment. Spacer arm: used to bind ligand to matrix. Ligand: molecule that binds reversibly to a specific target molecule(site of interaction ). GOKULAKRISHNAN CHROMATOGRAPHY 6

7. PROCEDURE   The Sample is injected into the equilibrated affinity chromatography column . Only the substance with affinity for the ligand are retained on the column .   The substance with no affinity to the ligand will elute off . The substances retained in the column can be eluted off by changing the pH of salt or organic solvent concentration of the eluent. GOKULAKRISHNAN CHROMATOGRAPHY 7

8. GOKULAKRISHNAN CHROMATOGRAPHY 8

9. MATRIX  The matrix is an inert support to which a ligand can be directly or indirectly coupled. It has some peculiar qualities like:- Does not itself adsorb molecules to a significant amount. Ligand must be coupled without altering its binding properties. Stability under a wide range of experimental conditions such as high and low pH, detergent and dissociating conditions. The most useful matrix materials are agarose and polyacrylamide GOKULAKRISHNAN CHROMATOGRAPHY 9

10. SUPPORT MATERIALS USED IN AFFINITY CHROMATOGRAPHY GOKULAKRISHNAN CHROMATOGRAPHY 10 SUPPORT MATERIALS TRADE NAME         Agarose Sepharose 2B,4B,6B     Cross-linked dextran Sephadex     Polyacrylamide Bio-gel-P     Cross linked cellulose Matrex Cellufine

11. LIGAND: The ligand is the molecule that binds reversibly to a specific molecule or group of molecules ,enabling purification by affinity chromatography . The selection of the ligand for affinity chromatography is influenced by two factors : Ligand must exhibit specific and reversible binding affinity for the target substance(s ) It must have chemically modifiable groups that allow it to be attached to the matrix without destroying binding activity. GOKULAKRISHNAN CHROMATOGRAPHY 11

12. COMMENLY USED LIGANDS GOKULAKRISHNAN CHROMATOGRAPHY 12 LIGAND AFFINITY         Concanavalin A Glycoproteins and polysaccharides     Calmodulin Calmodulin binding enzymes     Avidin Biotin containing enzyme     Heparin Lipoproteins, lipases , coagulation factors, DNA     polymerases , steroid receptor proteins     serine protease inhibiter     Proteins A and G Immunoglobins

13. TYPES Lectin Affinity Chromatography Immuno Affinity chromatography Metal Chelate Chromatography Dye Ligand Chromatography Covalent chromatography GOKULAKRISHNAN CHROMATOGRAPHY 13

14. LECTIN AFFINITY CHROMATOGRAPHY Used for purification of glycoproteins particularly membrane receptor proteins. Lectins are a group of proteins produced by plants & animals, which have the ability to bind carbohydrates and glycoproteins.   Used to separate mixtures of cells by taking advantage of the saccharide components of their outer membrane.   Commonly used lectins are: ConcanavalinA , Soyabean lectin,etc . GOKULAKRISHNAN CHROMATOGRAPHY 14

15. IMMUNO AFFINITY CHROMATOGRAPHY Exploited in the isolation & purification of a range of proteins including antigens, membrane proteins of viral origin. Used for purification of antibodies. Ligands used is Protein A and protein B. METAL CHELATE CHROMATOGRAPHY Special form of chromatography in which an immobilised metal ions such as Cu 2 + ,Zn2 +,Mn2 +,Ni2 + etc. are used . Used for purification of proteins containing imidazole groups or indole groups . Commonly metal ions are immobilised by attachment to an imino-diacetate or tris ( carboxymethyl ) ethylenediamine substituted. GOKULAKRISHNAN CHROMATOGRAPHY 15

16.    DYE LIGAND CHROMATOGRAPHY Uses a number of triazine dyes as ligands. Most widely used dye is Cibracron Blue. F3G-A . Used for purification of lipoproteins, interferons , coagulation factors etc . COVALENT CHROMATOGRAPHY Developed specifically to separate thiol containing proteins . Most commonly used ligand is a disulphide 2’-pyridyl group . Used for purification of a number of proteins but its use is limited by its cost and rather difficult regeneration stage. GOKULAKRISHNAN CHROMATOGRAPHY 16

17. AFFINITY CHROMATOGRAPHY Can be used,   Purify and concentrate a substance from a mixture into a buffering solution. Reduce the amount of a substance in a mixture .   Discern what biological compounds bind to a particular substance, such as drugs .   Purify and concentrate an enzyme solution. GOKULAKRISHNAN CHROMATOGRAPHY 17

18. APPLICATIONS Used in Genetic Engineering nucleic acid purification Production of Vaccines antibody purification from blood serum   Basic Metabolic Research protein or enzyme purification from cell free extracts. GOKULAKRISHNAN CHROMATOGRAPHY 18

19. ADVANTAGES OF AFFINITY CHROMATOGRAPHY Extremely high specificity. High degrees of purity can be obtained .   The process is very reproducible.   The binding sites of biological molecules can be simply investigated. GOKULAKRISHNAN CHROMATOGRAPHY 19

20. DISADVANTAGES OF AFFINITY CHROMATOGRAPHY Expensive ligands Leakage of ligand   Degradation of the solid support   Limited lifetime   Non-specific adsorption   Relatively low productivity GOKULAKRISHNAN CHROMATOGRAPHY 20

21. REFERENCE Hand book of Affinity chromatography, principles and Method from GE Healthcare Practical Biochemistry, Principles and techniques by Keith Wilson and John Walker, Cambridge University Press   Affinity Chromatography: A Review ,Journal of Pharmacy Research;May2011, Vol. 4 Issue 5, p1567 GOKULAKRISHNAN CHROMATOGRAPHY 21