Hi All! I am using ITC to study binding of lectin Concanavalin A to glycopolymer decorated with around 40 saccharide moieties (MW ~ 16 000 Da), which is multivalent-type ligand. Binding of corresponding, free saccharide is 1:1 binding process with Ka = 1.8x10^3 M^-1. My question concerns the concentration, which should be used for data analysis. Should I use the glycopolymer concentration, which is 0.11 mM, or should I regard the concertation to saccharide concentration which is 4.5 mM? In the first case I get Ka = 9.9x10^4 M^-1, while in the second Ka equals 2.5x10^3 M^-1. Thanks for your help!
A polymer concentration of 1.9 μM and a monomer concentration of 4.5 mM do not agree with a chain consisting of 40 monomers.
Anyway, depending on your decision to quantitate the macromolecule concentration (per chain or per monomer), the association constant will be different, as well the ligand binding stoichiometry.
Depending on the structural and the physico-chemical properties of the polymer, you may use a model considering a model considering: 1) a single ligand binding site (taking the concentration of binding subunit and if bound ligands would not make contact) ; 2) N identical and independent ligand binding sites (taking the concentration of polymer chain and if bound ligands would not make contact); or 3) N identical and overlapping ligand binding sites with or without cooperativity (McGhee-von Hippel model).
Adrian Velazquez-Campoy thank you for your reply. You are of course right about the mismatch between concentrations expressed per glycopolymer and per monomer. I made a mistake, which I have already corrected.
I will consider to run and compare curve fitting to the suggested models.
I also wonder if depending on the way of expressing the ligand concentration I can conclude that: Ka derived at concentration calculated per monomer characterize average binding affinity of single saccharide moiety on glycopolymer chain, while Ka obtained once applying glycopolymer concentration characterize the binding affinity of the whole macromolecule?
Let's consider the simplest case: a (homodimeric) macromolecule with two identical and independent ligand binding sites (i.e., they are far apart in such a way they do not interfere reciprocally and the ligand binding to one of the sites does not induce a conformational change affecting the other ligand binding site).
Let's say you perform a titration with 10 μM protein (as dimer) and 200 μM ligand. Then, you will get the association constant (Ka), the binding enthalpy (ΔH) and the binding stoichiometry (n=2, if everything goes well and all concentrations values are correct and reflect the active concentrations of reagents).
Now, if you recalculate the protein concentration to express in a monomer (subunit) basis, the concentration would be 20 μM (as monomer). Then, the molar ratio ([L]T/[P]T) will decrease, and the stoichiometry will be n=1. But the binding enthalpy will remain unchanged (it does not depend on the titrand concentration, but on the titrant concentration), and the association constant will also remain unchanged (in the equilibrium equations the parameter n is always multiplying the protein concentration [P]T, and, therefore, a change in [P]T will result in a change in n, with no changes in Ka or ΔH).
But, if you titrate the protein into the ligand, other considerations will apply, and the analysis may be more complicated (and potential cooperativity can further increase the complexity).
At this point, I wonder if you are injecting ConA into polymer or polymer into ConA. What is your setup?
The simplest and less problematic setup is the titration of ConA into polymer.
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