Fluorescence result of a HSA-drug system proved that hydrophobic result is the main force .In docking both hydrogen and hydrophobic force are there.but ITC result for the same system give exothermic with negative enthalpy and entropy value.how can i corelate the ITC result with fluorescence and docking result?
Was the fluorescence result you mentioned a blue shift in the fluorescence of the compound, consistent with its being in a hydrophobic environment, compared with water? That is not necessarily the same as the binding being driven primarily by hydrophobic interactions. Binding in a hydrophobic environment can involve multiple forces and does not have to be endothermic.
Thanks all of you for such beautiful explanation..i will consider the inner filter matter
What buffer did you use for ITC? There are no bad buffers in ITC, but, given that proton exchange between complex and solution can be coupled to protein-ligand interaction, the observed enthalpy may contain a contribution from the de/protonation of the buffer. Therefore, the intrinsic interaction enthalpy (buffer-independent) can be very different from the observed enthalpy (magnitude and sign):
dHobs = dHint + nH x dHbuffer
where nH is the net number of proton exchanged upon complex formation. The nH parameter can be zero, positive, or negative.
The buffer ionization can diminish or amplify the overall observed enthalpy. As a consequence, an ITC experiment performed in phosphate, acetate, or cacodylate provide an enthalpy value close to the intrinsic enthalpy because of their small ionization enthalpy. However, an ITC experiment performed in Tris, imidazole, or Mops can provide you an enthalpy value even of opposite sign regarding the intrinsic enthalpy (depending on nH and dHbuffer).
Then, you did an ITC experiment and the observed enthalpy was negative. What buffer did you use?
Dear Adrian
i have used phosphate buffer of pH=7.4.but if i change my experimental temperature then the exothermic rxn changed to endothermic...i think its the temp which is responsible for this .
With phosphate you do not have to worry about protonation effects.
Of course temperature will change the enthalpy because of a non-zero interaction heat capacity.
You should read more about binding thermodynamics, and also about calorimetry and other binding techniques.
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