I do not really understand the question, because your are mixing two different issues.

Should the thermodynamic binding parameters obtained by fluorescence be the same as those determined by ITC (assuming that the same experimental conditions are employed)? The answer is: Not always. Although you intend to determine the same binding parameters with two different techniques, each technique employs a different procedure: some of them are "transient effect" techniques (e.g., ITC) and some of them are "steady state" techniques (e.g. fluorescence). Therefore, slow binding ligands may exhibit a different interaction strength depending on the technique employed.

If different experimental conditions are employed, very likely the binding parameters will be different. In particular, if a different temperature is employed, maybe the binding affinity (or the Gibbs energy) will be similar, but the binding enthalpy and entropy will be quite different (because of a non-zero binding heat capacity). Binding affinity and Gibbs energy are not as sensitive to temperature as the enthalpy or the entropy.

Remember also that another important source of discrepancy between the estimated binding parameters is that in ITC you measure directly the binding enthalpy and in fluorescence you estimate the binding enthalpy through the van 't Hoff relationship. In addition, uncertainties in reagent concentrations and other issues will result in uncertainties in the binding parameters.

Dear Adrian

thank u so much for your explanation.i got my answer

Hi, Samima

I could recomend this paper. It explain in a really friendly way the thermodynamic analysis of biomolecular interactions. I personally like the comments the author put on some references. Here is the link to the PDF file.  http://europacat.co.uk/staff/alanc/CurrOp1999.pdf

I also included the pdf file