Hi all,
I have an unhappy protein. I managed to take it down to 2M urea and previous studies have shown that it is refolded in 4M urea. So I am happy with the protein staying in 2M urea. The problem is doing ITC experiments with that 2M urea buffer. I read somewhere that it is alright as long as i have the same buffer for both cells. Anyone has a comment on this?
Also, I have been looking through all the buffer guidelines recommended for ITC. All suggest to use low ionisation enthalpy. I have trouble finding the ionisation enthalpy of urea. Anybody has any clue?
Thanks.
Urea is not an ion. It cannot be used as a buffer itself. However, urea gradually decomposes into ionic species.
For ITC experiment it is critical to use same buffer for the protein and the ligand/binding partner. If there is a buffer mismatch you might get false positive result. Therefore, first make sure that your ligand/binding partner is happy with the buffer that you have used for your protein.
Just for your information, urea has a pKa value of 0.1; therefore, it is not a suitable molecule to be employed as a buffer.
You can try to do titrations with urea 2 M, using an apropriate buffer. But you must be sure the reactant molecules are OK in urea 2 M, and be careful enough to guarantee no composition mismatch between titrand and titrant solutions.
@ Dear Wong
Urea is a denaturant. it denatures protein. its better to avoid urea as buffer. Rather you can use phosphate buffer as best as it has low ionization energy.
I agree with the other comments that urea by itself is not a buffer. If you do want to use urea as an additive in your ITC samples to keep the protein soluble, the concentration needs to be matched exactly in your protein and binding partner solutions, and that may be very difficult to achieve with urea.
You also need to make sure the urea does not crystallize. 2 M urea at 25 deg C should not crystallize, but if you go higher in urea and/or lower in temperature you can get urea crystals which can clog up the washing system, injector, automation (if you have an automated ITC), and other ITC components. Also if any urea remains in the ITC cell or syringe, that can crystallize and cause corrosion of some wetted surfaces of the ITC. Be sure you thoroughly rinse out the entire system well when the experiment is completed.
I agree with all recommendations, but I would like to say something about the preferential use of low ionization enthalpy buffers in ITC. It is true that using a low ionization enthalpy buffer (e.g. phosphate) the contribution of buffer de/ionization to the observed enthalpy will be negligible, no matter how many protons are exchanged upon complex formation. But this is fine if the intrinsic interaction enthalpy is not close to zero (under the experimental conditions tested) and it is measurable. However, what happens if it is close to zero? We can change the conditions or, if protons are exchanged upon complex formation, we can use a buffer with large ionization enthalpy (e.g., Tris). That way the interaction is monitored indirectly through de de/ionization of the buffer. Of course, the affinity should be hardly affected, but the observed enthalpy will not coincide with the intrinsic interaction enthalpy. In that case, experiments with different buffers with different ionization enthalpies will help in estimating the buffer-independent enthalpy and the number of protons exchanged upon complex formation.
I have studied protein-ligand interactions by ITC that could not be detected when using phosphate, but were evident when using Tris or Pipes, just because the intrinsic interaction enthalpy was quite small. The important point is to know how different processes may influence the outcome in ITC and apply the appropriate procedures for data analysis.
Please, let's avoid demonizing certain useful buffers.
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