I study protein-small molecule interaction. And it is a constant observation that I find. However referring to a literature I hardly come across such significant differences. What should be taken care of while assessing such interactions on different platforms so that quantitative assays agree with each other? Or is it not necessary?
There are many experimental techniques that can be employed to measure binding. Although all of them can be used to determine the value of Kd (that is, the binding equations, derived from equilibrium and mass conservation euqations, are the same, irrespective of the experimental technique), the experimental set-up, the experimental procedure and the signal measured is different. You must consider the advantages, limitations and subtleties associated (physical origin of the signal, potential interferences, time course of the measurement and signal recording) with each experimental techniques, because they will condition the ability to determine the "true" or the "model" Kd.
There are mainly two types of techniques regarding the output: those that allow determining equilibrium concentrations of reactants (e.g., ultracentrifugation, size exclusion chromatography, dialysis), and those that report a signal proportional to the progression of the binding or the saturation of the protein (e.g., isothermal titration calorimetry, most types of fluorescence -based techniques, circular dichroism).
And there two main types of techniques regarding time course of the output: those that report a steady-state signal after long equilibration (e.g., fluorescence-based reading after long equilibration time after mixing protein and ligand), or those that report the transient effect (e.g., fluorescence-based reading after rapid mixing of protein and ligand).
ITC belongs to this second category. ITC provides the transient heat effect resulting after mixing protein and ligand solutions. Any slow process would be overlooked. Therefore, if the interaction proceeds through two steps with markedly different kinetic association rates, the fast step would dominate the output (first encounter between protein and ligand), the information about the second step (reorganization of the compex towards a higher affinity complex) would hardly be accessible, and only an apparent Kd larger than the "true" Kd would be determined. With a steady-state state technique it would be possible to measure an apparent Kd similar to the "true" Kd, since the output signal is recorded after long equilibration.
The discrepancy between the Kd obtained from different techniques will be larger for slow-binding ligands.
All this long explanation is to say that I think the Kd values obtained using different experimental techniques can be significantly different.
Kou Hayakawa
Interesting comments. Can you elaborate on the part that FP and others are not quantitative methods please.
Kou Hayakawa
Since the question is not necessarily about an enzyme-substrate interaction, Km is not appropriate, but Kd, which is the equilibrium dissociation constant for protein-ligand interaction. Rigorously, Km is the ratio between kinetic rate constants (kon, koff, and kcat).As Vikrant Kumar is asking, which are the reasons for considering those techniques (FP, ITC, MST, NMR...) are not quantitative? What is the connection of Lamber-Beer law with ITC or NMR, for example?
Adrian Velazquez-Campoy Can you please provide literature support for such ITC experiments where the fast step dominates the average KD value and thus increases the overall binding constant value?
I did not say anything about a fast step dominating the interaction and modulating the interaction affinity.
I said that the dissociation constant (i.e., binding affinity) for a given interaction may somewhat vary depending on the employed experimental technique. Although the same interaction is taking place, we would be monitoring different signals and following the process with different approaches depending on the technique.
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