How would the dissociation constant vary if I am (1) Titrating 2uM of protein with increasing concentrations of ligand or (2) Titrating 5uM of protein with increasing concentrations of ligand. Isn't the dissociation constant intrinsic property of ligand/binding molecule or it varies with concentration of protein/substrate in the cuvette.
In a simple system the dissociation constant is an intrinsic property of the ligand-protein interaction, and it should not depend on the protein concentration employed in a titration.
However, if the protein exhibits a monomer-oligomer equilibrium, the apparent dissociation constant for the protein-ligand interaction would depend on the protein concentration.
Adrian Velazquez-Campoy Thank you for your reply. I am little uncertain here about Kd calculations. Most of the tutorials simply focus on increasing concentrations of ligand and then calculate Kd from it; where does the fixed concentration of protein/substrate fit in here.
I do not understand what you mean about fixed concentration of protein/substrate.
In a titration you prepare samples with different protein/ligand ratios. You measure a signal that varies according to the ligand saturation fraction of the protein (advance of the reaction) and fitting the data considering the appropriate equilibrium model you can estimate the dissociation constant, Kd.
Depending on the way you do the data analysis you can estimate EC50 (which is concentration dependent) or Kd (which, in the absence of oligomer-monomer equilibrium, is concentration independent).
Adrian Velazquez-Campoy Thank you for your answer. I deduce then that the Kd would be calculated based on Protein:Ligand molar ratios, as observed in ITC titrations.
The molar ratio is informing about the stoichiometry, not the affinity.
To get the affinity from concentrations you need to know the free concentrations, which are unknown. From the total concentrations you get EC50, which is protein concentration dependent and can be quite different from the Kd.
Adrian Velazquez-Campoy I understood the point. Seems we have to consider free ligand concentration in order to calculate Ki(app). The total concentrations would yield EC50, which would be enzyme concentration dependent and most of times are E0/2. Thanks again for your time and explanation.
You can analyze the data in terms of total concentrations, but you must solve chemical equilibrium equations (e.g., a quadratic equation for a 1:1 interaction) for estimating the Kdapp
Adrian Velazquez-Campoy Can you please share link/paper for correct estimation of Kd(app).
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