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11: Binding proteins- Antibodies, Myoglobin/Hemoglobin

Reading & Problems:LNC p. 174-178, 157- 163; p. 184 prob. 1

I. Affinity chromatography methods - very powerful, can often purify a protein in a single step.

 

A. Ligand affinity chromatography - beads are derivatized with a molecule that is specifically bound by the protein that you wish to purify. This molecule is referred to as a "ligand". Often this is an inhibitor of an enzyme to be purified. You must know of a such a molecule to use this method. A mixture of proteins in loaded onto the column. The protein of interest binds and other proteins flow through during washing. Elute your column with free ligand or other agent that will release the target protein from the column.

 

B. Immunoaffinity chromatography - also called "antibody affinity chromatography".

 

1. Antibodies (immunoglobulins) are proteins produced by the vertebrate immune system that have binding sites that have the correct shape and chemistry to bind a specific ligand (a small molecule that is bound), or epitope (a specific surface region of a larger molecule).

 

2. external link: Antibodies consist of four protein chains, two identical light chains and two identical heavy chains.

antibody structure.jpg
AntibodyCHODisulfide.jpg
IgG Antibody Structure: light chains are in green and dark blue, heavy chains in light blue and orange, disulfide bonds in yellow spacefill, carbohydrate in red wireframe.

3. For Immunoaffinity chromatography, antibodies specific to your protein are bound to beads and used to purify your protein. Can sometimes be eluted with salt, but may require stronger means (concentrated urea, SDS) to elute protein.

Procedure for antibody production:

  1. Purify a small amount of your protein.
  2. Inject a sample into a mammal (usually a rabbit, mouse, rat or goat) or a chicken.
  3. Wait and do a second injection to increase immune response.
  4. The animal's immune system produces antibodies that will bind tightly to the injected protein.
  5. Harvest blood (mammal) or egg (chicken).
  6. Use blood serum directly or do additional purification from this "antiserum".
  7. Lipids and other contaminants in egg yolk must be separated from antibodies prior to use.

Antibodies are bound to beads for use in immunoaffinity chromatography (antibody affinity chromatography).
Antibodies labeled with radioactive, fluorescent or histochemical (an attached enzyme that will make color from a colorless compound) can be used to detect proteins that have been run on a gel.

II. Myoglobin and hemoglobin.

 

A. Mb and Hb include a heme prosthetic group that can bind oxygen reversibly.

heme.jpg

B. Mb binds and releases O2 and the kinetics and saturation can be analyzed mathematically.

 

C. Hb can bind 4 oxygens and the kinetics of this cooperative binding can also be modeled.

external link: Comparison of Hb and Mb O2-binding curves
external link: Myoglobin structure in JSmol (external link: Myoglobin structure in Jmol), external link: Hb structure in JSmol; external link: Mb and Hb structure tutorial.

D. Other molecules affect the O2 binding by Hb (and their binding is in turn affected by O2 binding).

 

Some take home information:

Ligand affinity chromatography - can purify a protein in a single step, but you have to have identified a ligand specific for the protein in order to use this method.
Immunoaffinity chromatography - an antibody can be generated to almost any protein, but you must have some of your protein (or a region of your protein) purified in order to generate the antibody to make the column.

Oxygen binding equations:
O2Binding.jpg

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