Saturation and Competitive Binding Expriments
Anton Zavialov
Uppsala
Binding is an essential functional step
of many processes
Protein-ligand interactions
Hydrophobic effect
Van der Waals interactions
Hydrogen bonds
Salt bridges
Hydrophobic effect
Saturation Binding Experiment
Main Parameters:
1. Affinity: dissociation constant (Kd)
DGdo=- RT ln(Kd)
2. Total amount of receptors (RT) or stoichiometry
of the binding (n, m)
Additional information:
Type of the binding (cooperative or noncooperative), site accessibility, site
heterogeneity, etc.
Saturation Curve and Scatchard Plot
[RL]=RT [L] / (Kd+[L])
[RL] / [L] =RT / Kd - [RL] /Kd
Effects
Experimental setup
Technical considerations
1. RT<<LT, which means that [L] is close to LT
-
life is easier , but to create high concentration of the ligand we
have to add non-radioactive ”cold” ligand
2. Which range of concentrations to use?
- around Kd and above: e.g. expected Kd=2-4 mM, use LT=2-30 mM
Protein concentration?
About 0.2 mM in this case
My Last Weekend’s Binding Experiment
Competitive Binding Experiment
Advantage:
Cheep
Ligand is labeled,
whereas inhibitors are
not –
I1, I2, I3, I4 ...
My Last Weekend’s Binding Experiment
[RL]i/[RL]=1/(1+[I]/IC50), Ki=IC50/(1+LT/Kl ),
but only when RT<<LT and RT<<IT