Hello!
I am titrating proteins into polystyrene nanoparticles, using ITC. Initially I get good heats evolved which gradually decrease upon nanoparticle saturation. However, just before saturation I see an endothermic 'hump'. However this is not seen for any heats of dilution. My buffer is 20 mM NaPi.
Thank you in advance!
It looks like a cooperative effect on the affinity with the reactant.
See e.g. https://www.cell.com/biophysj/pdf/S0006-3495(06)71899-9.pdf
There are several possible causes for that observation:
- There are different spots in the surface of the nanoparticle with different binding affinities and enthalpies.
- All surface spots for binding have the same affinity and enthalpy, but as the coverage of the surface increases, a cooperative effect (negative cooperativity, because it occurs close to saturation) arises.
- As the surface gets saturated, aggregation of nanoparticles might occur.
The first possibility is not likely because the slope at the equivalence point of the second phase is larger than that of the first phase. The third possibility can be ruled out by measuring the apparent nanoparticle size by light scattering.
Hi Radha,
I think Adrian V-C's 2nd idea of a possible cause is a good one. If the in the interaction can take place involving different extents of the protein surface (involving coverage of different extents of nanoparticles surface area), it may well be the case that increasing protein concentration enough could favor interactions involving lees occluded surface per protein, enabling higher binding density of proteins on nanoparticle surfaces. Something that would not necessarily be endothermic, but in principle it could be (maybe compensated by an increase in entropy associated to higher mobility of parts of the protein).
Another possible explanation I can think of, which could be tested by light scattering measurements, is the following. *If the protein is able to bind multiple nanoparticles*, and if the affinity is high enough to see this at the concentrations you are using, I would expect to form at the beginning of the titration complexes with multiple nanoparticles per protein (2 np : 2 proteins?) and then, as protein concentration increases, lower number of nanoparticles per protein should be favored. To explain your data this idea would require the formation of crosslinked nanoparticles being more exothermic than binding of protein to single nanoparticles.
Good luck.
Best,
Jere
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