Affinity Chromatography - Department of Molecular and Cellular ...

Binding equilibria: Selective elution or competitive elution
The examples shown have related to the changes in K D caused by non-selective elution
techniques for affinity chromatography. However, competitive elution can also be interpreted
in terms of changes in the binding equilibrium, as in the illustration below showing
elution by adding a competing free ligand. A similar situation applies when adding a
competing binding substance.
L +
T
LT
Binding equilibrium for competing ligand.
C + T
CT
At equilibrium
K DComp = [C][T]
[CT]
K DComp is the equilibrium dissociation constant
[C] is the concentration of free competing ligand
[T] is the concentration of free target
[CT] is the concentration of the competing ligand/target complex
Graves and Wu in Methods in Enzymology 34, 140–163 (1974) have shown that:
Eluted target r
~
Total bound target r + 1
rC 0
rC 0 + K DComp L 0
K D
r is the ratio between the volume of competitor added and the pore
volume in the gel, assumed to be in the range 1–10
K D is the dissociation constant, coupled ligand
K DComp is the dissociation constant, free competing ligand
C 0 is the concentration of competing ligand, usually 10 -2 - 10 -1 M
L 0 is the concentration of coupled ligand, usually 10 -4 - 10 -2 M
Again the derivation of the equation relies on some assumptions and simplifications and
can only be expected to give a qualitative picture of what happens during binding.
If K DComp and K D are similar then the concentrations of competing and coupled ligand
should be similar to achieve effective elution.
If K DComp is 10 x K D (i.e. the free competing ligand binds more weakly) then the concentration
of competing ligand will need to be 10 x higher to achieve effective elution.
If the competing ligand is not very effective in capturing the target protein at low concentrations
so that the target is eluted from the column as a very broad peak, then a higher
concentration of the competing ligand will be required to achieve elution. Since competing
ligands are often expensive this is not a desirable situation.
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