1. Affinity chromatography

2. What is Affinity Chromatography?
A chromatographic technique for the separation of the compound on the basis of a reversible interaction between a compound and a specific ligand.
Ideal for purification of compound from complex mixtures several thousand folds in concentrated form.
Originally developed for purification of enzymes
Also purifies nucleotides, nucleic acids, immunoglobins, membrane receptors, whole cells and cell fragments.
High selectivity and high resolution

3. Principle
When a complex mixture containing specific compound is passed through a column containing immobilized ligand, the specific compound will bind to the ligand.
Ligand is immobilized to an insoluble solid matrix.
A biological reversible interaction between a compound and a specific ligand occurs as follows:
Compound (M) + Ligand (L)  ML complex
This interaction occurs due to
Electrostatic or hydrophobic interactions
Van der Waals' forces or
Hydrogen bonding.

4. Principle…
Compounds not specifically bound are washed away with the buffer.
Compounds specifically bound are recovered from ligand by reversing the interaction
There are two methods of elution
Specific
Non-specific

5. Types of elution Non-specific elution:
Change of pH or ionic strength or polarity.
pH shift using dilute acetic acid or ammonium hydroxide causes a change in the state of ionization of groups in the ligand and the compound.
A change in ionic strength causes elution due to a disruption of the ligand-compound interaction.
Specific elution:
Use of soluble competitive ligand.

6. Matrix/ Resins Most common matrices
Cross-linked dextrans, agarose, polymethylacrylate, polyacrylamide, polystyrene, cellulose, porous glass and silica.
Matrix particles are uniform, spherical and rigid.
An ideal matrix must have the following characteristics:
Should possess chemical groups to which the ligand may be covalently coupled.
Should be stable during binding of the compound and its subsequent elution
Should interact only weakly with the compound to minimize non-specific adsorption
Should exhibit good flow properties

7. Ligand Ligand must specifically bind to one particular compound.
Ligand that display group selectivity selected as it will bind to closely related groups of compounds that posses similar chemical specificity.
Ligand must possess a chemical group that should not reversibly bind the ligand to the compound but should attach the ligand covalently to the matrix.
Most common chemical groups are
–NH2, -COOH, -SH and –OH.

8. Spacer arm Spacer arm interposed between ligand and matrix.
It prevents ligand-matrix attachment that interfere with the ability to bind the compound.
Optimum length of the spacer arm is 6-10 carbon atoms.
Their chemical nature is critical for separation.
Some spacers hydrophobic - consists methylene (CH2) groups
Others hydrophilic - consists carbonyl (CO) or imido (NH) groups.
Spacers important for small immobilized ligands and not for macromolecular ligands.
Matrix supports available with a variety of spacer arms and ligands attached for immediate use.

9. Spacer arm

10. Different group specific ligands and their affinity.
5’AMP
NAD+ dependent dehydrogenases, some kinases
2’5’-ADP
NAPP+ dependent dehydrogenases
Calmodulin
Calmodulin binding enzymes
Avidin
Biotin containing enzymes
Fatty acids
Fatty acids binding proteins
Heparin
Lipoproteins, lipases, coagulation factors, DNA polymerases, steroid receptor proteins, growth factors, serine protease inhibitors
Proteins A and G
Immunoglobulins
Concanavalin A
Glycoproteins containing -D-mannopyranosyl and -D-glucopyranosyl residues
Soybean lectin
Glycoproteins containing N-acetyl -(or ) -D-galactopyranosyl residues
Phenylboronate
Glycoproteins
Poly (A)
RNA containing poly (U) sequences, some RNA specific proteins
Lysine
rRNA
Cibacron Blue FF3G-A
Nucleotide-requiring enzymes, coagulation factors

11. Method 1. Affinity medium is equilibrated in binding buffer.
2. Sample is applied. Specific binding of the target molecule to the ligand, but reversibly. Unbound material washes through the column
3. Target protein is recovered by changing conditions to favor elution of the bound molecules.
4. Affinity medium is re-equilibrated with binding buffer.
Method

12. Some typical biological interactions
Enzyme- substrate or inhibitor or cofactor
Antibody- antigen or virus or cell
Lectin – polysaccharide or glycoprotein or cell surface receptor, cell
Nucleic acid - complementary base sequence or histones or nucleic acid polymerase or nucleic acid binding protein.
Hormone/vitamin – receptor or carrier protein.
Glutathione - glutathione-S-transferase or GST fusion proteins.
Metal ions - Poly (His) fusion proteins or native proteins with histidine, cysteine, tryptophan residues on their surfaces.

13. Loading affinity column. Figure 2.
Proteins interact with affinity ligand with some binding loosely and others tightly.
Figure 1.
Loading affinity column.
Figure 2.
Proteins sieve through matrix of affinity beads.
Figure 6.
Elute proteins that bind tightly to ligand and collect purified protein of interest.
Figure 4.
Wash off proteins that do not bind.
Figure 5.
Wash off proteins that bind loosely.

14. Applications
Purification of enzymes, immunoglobulins and receptor proteins
Isolation of nucleic acids. eg. mRNA by selective hybridization of poly (U) – Sepharose 4B by exploiting its poly (U) tail.
Isolation of complementary RNA and DNA using immobilized single stranded DNA.
Isolation of proteins involved in nucleic acid metabolism by immobilized nucleotides