Affinity chromatography procedure is a type of liquid chromatography used to separate, purify or analyze samples of components. Instrumentation of affinity chromatography is it, uses the reversible biological or molecular recognition of affinity that refers to the forced attraction of atoms in different degrees between them, causing them to be combined.
Affinity Chromatography Definition
Since the introduction of affinity chromatography procedure 50 years ago, traditional purification methods based on the strength of ionic ions, pH or temperature, have been It is replaced with this new and sophisticated method. It is reported that 60% of purification processes are techniques rely on affinity methods.
The broad application of this procedure is based on the reality that every biomolecule one wants to purify generally is composed of an inherent recognition spot can be connected to either a natural or artificial molecule. So, we can conclude the affinity chromatography procedure is primarily built on the molecular recognition of targeted molecules by a molecule attached to the column.
Principle of Affinity Chromatography
Affinity chromatography procedure works on the principle of mutual recognition forces between a ligand and receptor. The major determinants, responsible to provide specificity are shape complementarity, electrostatic, hydrogen bonding, vander waal interaction between the groups present on the ligand-receptor pair.
Dissociation constant is specific to the receptor-ligand pair and number of interaction between them. When a crude mixture is passed through an affinity column, the receptor present on the matrix reacts with the ligand present on different molecules.
Figure 1: shows the principle of Affinity Chromatography
The mutual collision between receptor on matrix and ligands from different molecule test the affinity between them and consequently the best choice bind to the receptor whereas all other molecules do not bind and appear in flow through. A wash step removes remaining weakly bound molecules on matrix. Subsequently, a counter ligand is used to elute the bound molecule through a competition between the matrix bound molecule and counter ligand.
Types of Affinity Chromatography
Affinity chromatography is further divided into the different types based on the nature of receptor present on matrix to binds tag present on the analyte molecule. Different types of affinity chromatography procedure are
i) Bio-affinity chromatography
Biomolecules are used as receptor present on matrix and it exploit the biological affinity phenomenon such as antibody-antigen. In addition, enzyme-substrate or enzymeinhibitor is also belong to this class. Ex. GST-Glutathione.
ii) Pseudo-affinity chromatography
A non-biological molecule is used as receptor on matrix to exploit the separation and purification of biomolecules. There are two specific example to this class.
iii) Dye-affinity chromatography
Matrix is coupled to the reactive dye and the matrix bound dye has specificity towards a particular enzyme. For ex. Cibacron Blue F3G-A dye coupled to the dextran matrix has strong affinity towards dehydrogenases.
iv) Metal-affinity chromatography
Transition metals such as Fe2+, Ni2+ or Zn2+ is coupled to the matrix and the matrix bound metal form multidentate complex with protein containing poly-his tag (6x His).
Instrumentation of Affinity Chromatography
i) Choice of matrix
The choice of matrix solely depends on the affinity tag present on the recombinant protein produced after genetic engineering.
ii) Receptor Generation
The receptor molecule present on the matrix can be produced either by genetic engineering, isolation from the crude extract or in the case of antibody, it is produced in the mouse/rabbit model and purify. The generation of receptor molecule is beyond the discussion in the current lecture and interested student are advised to follow it from other relevant course.
iii) Receptor Coupling
Once the receptor molecule is available, it can be couple to the matrix by following steps. (1) Matrix activation (2) covalent coupling utilizing reactive group on ligand. (3) deactivation of the remaining active group on matrix.
Affinity Chromatography Procedure
Affinity column material packed in a column and equilibrate with a buffer containing high salt (0.5M NaCl) to reduce the non-specific interaction of protein with the analyte.
ii) Sample Preparation
The sample is prepared in the mobile phase and it should be free of suspended particle to avoid clogging of the column. The most recommended method to apply the sample is to inject the sample with a syringe.
Figure 2: Shows the steps in Affinity Chromatography Procedure
There are many ways to elute a analyte from the affinity column. (1) increasing concentration of counter ligand, (2) changing the pH polarity of the mobile phase, (3) By a detergent or chaotrophic salt to partially denature the receptor to reduce the affinity for bound ligand.
iv) Column Regeneration
After the elution of analyte, affinity column requires a regeneration step to use next time. column is washed with 6M urea or guanidine hydrochloride to remove all non-specifically bound protein. The column is then equiliberated with mobile phase to regenerate the column. The column can be store at 40⁰C in the presence of 20% alchol containing 0.05% sodium azide.
Applications of Affinity Chromatography
- Purification of biomolecules: GST Based Purification- Glutathione S-transferase (GST) utilizes glutathione as a substrate to catalyze conjugation reactions for xenobiotic detoxification purposes. The recombinant fusion protein contains GST as a tag is purified with glutathione coupled matrix.
- Protein-Protein interaction: Protein-protein interaction can be studied through multiple techniques or approaches. Affinity column also can be used as a tool to study or isolate interacting partner of a particular protein.
- Enzymatic Assay: Affinity chromatography procedure can be used to perform enzymatic assay such as protease assay.
- Clinical diagnosis: Receptor present on the matrix provides a unique tool to isolate, detect and characterize biomolecules from the crude mixture. For example, matrix containing boronic acid is used to separate and quantify glycosylated hemoglobin from diabetic patient’s blood. Ribonucleoside in patient urine can be identifying by an affinity matrix containing boronic acid followed by the reverse phase chromatography.
- Immuno-purification: The avidin-biotin system is used to capture and isolate cytokines from immune cells. Biotinylation of antibodies allows immobilization of antibodies in the correct orientation on the streptomycin coated glass beads. Lymphocyte lysate is passed to the column packed with the glass beads containing antibodies binds cytokines. The cytokines are eluted by flowing buffer of decreasing pH or by chaotrophic ions. The antibodies remain bound to the column due to strong affinity between avidin-biotin which is resistant to this chemical treatment.
Advantages of Affinity Chromatography
- Specificity: Affinity chromatography procedure is specific to the analyte in comparison to other purification technique which are utilizing molecular size, charge, hydrophobic patches or isoelectric point etc.
- Purification Yield: Compared to other purification method, affinity purification gives very high level of purification fold with high yield. In typical affinity purification more than 90% recovery is possible.
- Reproducible: Affinity purification is reproducible and gives consistent results from one purification to other as long as it is independent to the presence of contaminating species.
- Easy to perform: Affinity purification is very robust and it depends on force governing ligand-receptor complex formation. Compared to other techniques, no column packing, no special purification system and sample preparation required for affinity purification.
Limitations of Affinity Chromatography
- It takes a lot of skill to handle it. It interferes with the structure. Transfer and the leakage of metal ion lead to protein loss.
- Sometimes ligands leakage is observed. The volume of the sample is limited. The carrier gas used must be pure such as pure nitrogen.
- The ligands used in affinity chromatography procedure are costly. Relatively low productivity. It has a non-specific adsorption.
- Degradation of the solid support. Metal-ion transfer and metal ion leakage lead to loss of protein.