Affinity Chromatography

Principle of Chromatography:

1. Equilibration
   
2. Sample application
   
3. Binding and washing
   
4. Desorption and elution
   

Generally, during the process of equilibration, the specific ligand would be attached to the matrix of the column. After that, the sample would be inserted into the column. Only target protein would binds to the ligand, which is bound onto the column matrix, while the non-target protein would be washed out. Finally, during the process of desorption, the target protein, which is attached to the ligand initially, would get eluted out simply by changing the eluent.

 

Choosing the Correct Ligand

These are the properties of the ligands that are highly favourable:

1. Form reversible complex with protein sample.
   
2. High complex constant for stable complex
   
3. Easy to dissociate the complex
   
4. Easy mobilization
   

The ligand chosen must be able to form reversible complex with the protein sample as you would want to obtain the purified protein back during the process of desorption at the end of the purification. However, the complex formed must be stable enough to give sufficient retardation (retention) to the column. In addition to the requirement that the complex formed between the ligand and the protein sample must be reversible, it should be easy to dissociate the complex by simply changing the medium or buffer of the column, without creating any change or denaturing the protein or ligand.

The types of ligand:

1. Monospecific low molecular weight ligand
   
2. Monospecific macromolecular weight ligand
   
3. Group specific low molecular weight ligand
   
4. Group specific macromolecular weight ligand
   

Monospecific low molecular weight ligand

Monospecific means that ligand binds to single or very small amount of protein in cell extract or body fluid.

Group specific low molecular weight ligand

The ligand used here are most likely enzymes and its analogues such as biomimetic dye, boronic acid derivatives, vitamins, amino acids, etc.

Monospecific macromolecular ligand

This kind of ligand is about groups of specific protein-protein interactions. Due to the high specificity of antibodies, antigen and antibodies is normally good candidate for Monospecific macromolecular ligand. The mobilized proteins are also known as immunoadsorbent. These immunoadsorbent can be used to purify cells, soluble proteins, peptides, solubilised membrane protein and also viruses. It is good to use antigen as ligand due to its uniform binding to the target protein. Besides that, the use of monoclonal antibodies allow a constant supply of a highly uniform antibody, which give rise to high reproducibility from batch to batch of immunoadsorbent. This is extremely advantageous as such uniform antigen binding would produce sharp desorption peak. However, the disadvantages of such ligand are mainly due to its high cost of production, as well as high risk of fouling. This is because the antibody will only recognize and bind to corresponding antigenic determinant of actual protein. Thus, the antibody would fail to observe if the protein has been modified or degraded. Furthermore, if the sample is crude extract, the sample might be denatured chemically via proteolysis.

 

Choosing the correct matrix

The following properties are important when choosing a matrix:

1. Macroporous
   
2. Hydrophilic and neutral
   
3. Presence of functional group for derivation
   
4. Chemically stable
   
5. Physically stable
   
6. Readily available
   

The best candidate would be spontaneously gel-forming galactan agarose, as it possesses most of the characteristic mentioned above for an ideal affinity chromatographic matrix. Although agarose gel might be less chemically and physically stable, it still can be used in affinity chromatography due to its chemical cross-linking of the physically cross-linked junction zones in the agarose gel structure.

Spacer arm

Spacer arm is a linker between the matrix and the ligand. If the length of the arm is too short, it might be ineffective. However, if the length of the arm is too long, it might result in non-specific binding due to hydrophobic interaction. The spacer arm can be added via the following two methods:

1. Immobilize a spacer arm with a terminal primary amine; increase the length by reaction with succinic anhydride.
   
2. Immobilize a spacer arm with a terminal carboxyl group, increase the length by reaction with 1,7-diamino-4-azaheptane with the aid of condensation reaction.
   

Ligand Immobilization

Action of the matrix can be done by introducing an electrophilic group into matrix. The ligand is either coupled directly to the activated matrix or otherwise. The deactivation of the matrix can be done by a large excess of suitable low molecular weight substance. When affinity adsorbents are prepared, a stable bond should be formed between matrix and the ligand to prevent leakage of ligand.

Evaluation of the Affinity Adsorbent

The ligand density can be used to evaluate the affinity adsorbent by a few methods. One of the methods is indirect method, which estimates the uncoupled ligand. The amount of immobilized ligand can be calculated by the difference between the amount of ligand originally added and the amount of ligand recovered in the liquid phase and pooled washing after finished coupling.

 

Desorption Process

The process of desorption is about changing the binding equilibrium for the absorbed substance from the stationary to the mobile phase. Affinity chromatography is all about the interaction between the sample proteins and the ligand, which is a combination of electrostatic, hydrophobic and hydrogen bonds interaction. Hence, any agent that can weaken such an interaction would be effective non-specific chemical used for desorption. However, during the process of desorption, it is always a compromise between the harshness and risk of denaturing the protein.