Our equipment of ITC does not reach the molar ratio (N=1) of a titration of EDTA with Ca2+ in MES buffer pH 6. We have cleaned with DCN 90 and HCl pH 2, but the N value is very low, around N=0.89.
Would anyone have any recommendations?
Which solutions have you used?
How did you prepare the solutions?
Which instrument are you using?
What values for enthalpy and affinity are you getting?
Could you show a picture of the experimental data?
Hi, Adrian thank you for your interest.
Below I answer your questions.
Which solutions have you used? I have used a solution of EDTA 0.1011 mM (cell) and CaCl2 1.0011mM (syringe) in MES buffer 10.0mM at pH 6.
How did you prepare the solutions?
MES 10mM solution. An amount of MES powder was dissolved in water, the pH was adjusted with NaOH pellets to pH 6.
EDTA 0.1011 mM. A necessary amount of EDTA powder was dissolved with MES buffer and the volume flask was filled to reach the desired volume.
CaCl2 1.0011mM. A necessary amount of CaCl2 powder was dissolved with MES buffer and the volume flask was filled to reach the desired volume.
Which instrument are you using? We are using a VP-ITC by LG.
What values for enthalpy and affinity are you getting?
the binding affinity of Ka=2.91E6 1/M and enthalpy ∆H=-3.99 kcal/mol (averages values of 4 experiments)
Could you show a picture of the experimental data? I added an image of a set of 4 experiments. Peaks.pdf shows the heat releases of interaction Ca-EDTA and Fit-pdf shows integrated heat data with fitting.
I hope my answers help you understand the problem with our instrument or our methodologies.
Regards.
Ricardo Hipólito.
The binding parameters you are getting are close to the expected ones, considering the experimental error.
If the EDTA or calcium concentrations are not the precise, due to experimental error, the binding parameters would be slightly different from the expected ones. And depending which concentration is incorrect, different binding parameters would be affected. For example, if calcium concentration is OK, but EDTA concentration is lower than the expected one, the enthalpy and the affinity would be OK, but the stoichiometry would be lower. If EDTA is OK, but calcium concentration is higher than the expected one, all paremeters would be affected (but mainly enthalpy and stoichiometry).
I suggest employing commercial solutions from which you prepare working dilutions or employing the standard Ca-EDTA system provided by MicroCal.
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