My calcium EDTA binding experiments at pH 4.5 and 5.5 using iTC200 in 20 mM sodium acetate are endothermic. The Ca-EDTA kit provided by Microcal gives exothermic peaks in 20 mM MES buffer, pH 5.7. My HODs (CaCl2 into buffer) are insignificant. Any idea why I am getting endothermic peaks?
in general concept "bond breaking gives endothermic peak where as bond making gives exothermic". As the case here, when calcium binds with EDTA, it must be an exothermic reaction but when you carried out with iTC200 in 20 mM sodium acetate, there must be some extra reaction happening which breaks down some of the existing bond. This can lead to endothermic peak. You must check the literature about the reaction of Sodium acetate with EDTA first. Hope you can find the exact answer there.
Sudhanshu
As it occurs in many binding reactions, when EDTA binds calcium (in general, any cation), protons are exchanged between EDTA and buffer. The extent of this poton exchange depends on the pKa of the carboxylic groups in EDTA, the change in those pKa values upon cation binding, and the pH of the solution. At acidic pH EDTA should release some protons when binding calcium. The protons exchanged between the complex and the solution are captured by the buffer, thus maintaining a constant pH. Therefore, the observed enthalpy you determine by ITC contains a contribution from the ionization enthalpy of the buffer. In that way, just changing the buffer you may get a positive or negative enthalpy. You are using acetate, whereas the kit from MicroCal has been prepared in MES. These two buffers have different ionization enthalpies.
Take a look at this article where we performed calcium and magnesium tittrations with EDTA under different conditions, and you will also find an explanation about the influence of the buffer on the experimentally determined enthalpy:
http://www.ncbi.nlm.nih.gov/pubmed/19899171
But what happens to the entropy ? Do you have the reliable data on it ?
There are three coupled processes: 1) calcium binding to EDTA; 2) deprotonation of carboxylic groups in EDTA; and 3) protonation of bufffer. At a given pH each process has certain enthalpy, entropy and Gibbs energy changes associated. However, when selecting the buffer you are modifying only the thermodynamic parameters of process 3. If the number of protons exchanged is significant and the buffers exhibit significantly different ionization enthalpies (and entropies), then the observed enthalpy and entropy of binding (that is, the overall enthalpy and entropy) can be markedly different in the experiments performed in the two buffers. That is why a single experiment cannot provide you the "intrinsic enthalpy of binding", unless there is no net proton exchange upon complex formation, or you are performing the titration using a bufffer with negligible ionization enthalpy (e.g. acetate at pH 5). An interesting point is that the binding affinity determined does not depend on the buffer employed as long as the pKa of the buffer is close enough to the experimental pH (and this is something we take care of).
The data available in the NIST database for complexation of Ca2+ by EDTA at 0M, 0.1M, 0.5 and 1.0M ionic strength are -5.3, -5.7, -6.3 and -6.4 kcal/mol respectively. You need to take into account protonation reaction occuring in your buffered media as well as interaction of the metal ion with acetate. When working in calorimetry you need to make sure your solution systems are comparable, otherwise you are comparing apples with oranges! Break your system down into manageable components and carry out the individual experiments. You can then use the NIST database to calculate and check your data.
Thanks for all the input re Endothermic vs Exothermic plots. I am getting clean plots and expected Kd values at pH 4.5 (1.3 mM). However I noticed that no matter how many times I did Ca-EDTA binding experiments, the N values is never close to 1 but is around 0.35 -0.42. I suppose it should be close to 1??
I am using 200 mM CaCl2, pH 4.5 + 20 mM Sodium acetate, pH 4.5 buffer in Syringe and 20 mM EDTA, pH 4.5 + 20 mM sodium acetate, pH 4.5 in cell. Each inj is 1 ul. Any Ideas?
When you perform ITC experiments on protein-ligand interactions, it is usual to get fractional N values (e.g. N = 0.76), even if the the concentrations are precisely determined by spectrophotometry. That would mean that, after the purification procedure, there is a significant fraction of the protein that is not properly folded and is not binding-competent, or there are some contaminants contributing to the absorbance measurement. As I mentioned, another possibility is that of incorrectly determined concentrations. However, in the case of the calcium-EDTA interaction, there no possibility for contaminants (or could there be?), or non-binding-competent fraction of EDTA. You may be facing a problem with incorrect concentrations.
If you have a calcium-EDTA from kit MicroCal you may try the following: 1) perform a titration using the EDTA from your preparation and calcium from MicroCal; 2) perform a titration using calcium from your preparation and EDTA from MicroCal. In this case, having EDTA and calcium in different buffers (and pH) in a given experiment should not be a problem, because you will be only interested in the N value for each titration. Hopefully, the buffer mismatch will only affect the observed enthalpy and affinity, and probably you will get non-zero "heat of dilution". If you consider that MicroCal concentrations are correct, then, you will be using both MicroCal solutions for calibrating your solutions. For example, if the first titration gives you an N value of 0.45, then, your EDTA concentration must be corrected by that factor (true concentration is only 45% of the initially thought concentration).
It is highly unlikely that I made a mistake in weighing EDTA and calcium chloride. I made these solutions multiple time and each time I am getting the same result. I made similar solutions at ph 5.5 which gave me N = 1. only at pH 4.5, I get N< 0.5. However your idea of checking using Microcal kit is brilliant. I will try that. Thanks for help.
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