After mixing Thrombin (2.5mM) with TCEP (30mM) in PBS buffer and waiting for an hour at room temperature I observed a significant turbidity and a slight gelling of the solution. Thrombin was purchased from Sigma Aldrich (T4648) and TCEP was purchased from Thermo Scientific (20490). According to David C. et al, Phys. Chem. Chem. Phys., 2009, 11, 2532–2542 there is no such effect when mixing TCEP and lysozyme in such concentrations. Could anyone please tell me what the reason for such changes in thrombin solution could be?
Thank you for the answer.
If I understood right, in the article is showed the TCEP oxidation in phospate buffer by measuring the absorbance of the product of the reaction with DTNB. So, this is not the oxidized TCEP, but NTB, that leads to turbidity in their case. I still can't understand, what is the reason in my case...
I do not have much experience in biochemistry, so, please, excuse me if i missed something in your answer.
So, as I understood, TCEP is oxidized in PBS buffer, and adding this mixture to the 100 mM DTNB solution leads to DNTB -> NTB liberation, what leads to the absorbance at 412 nm. I still can't get the point, why there is a turbidity when mixing TCEP and thrombin in PBS buffer.
About an hour ago I made a 40mM TCEP solution in PBS and still it is absolutly clear.
Or you mean that Thrombin must be like DTNB in this case and lead to absorbance (what looks kind of strange for me)?
Thank you for paying me so much attention.
Thank you very much.
Could you please clarify, how could the PBS pH (around 7.4) could be changed after adding TCEP or after TCEP interaction with protein? Looks like there should not be any changes in [H+] and [OH-] concentrations.
P.S. Also just observed this effect in albumine (1.5mM) PBS solution after adding TCEP (14 mM)
Thank you so much for your help! I missed the point, that buffer may not cope with COOH dissociation. The real pH of TCEP solution was around 2.3 (!).
the turbidity casued by the aggregation of both BSA & Thrombin as they contain S-S bridges (to stabilized and maintain the active 3D structure) which is cleaved by the reducing agent! ; NTB etc. is quite water soluble
Int J Biol Macromol. 2015 Sep;80:130-8. doi: 10.1016/j.ijbiomac.2015.06.040. Epub 2015 Jun 23.
Kinetic regime of dithiothreitol-induced aggregation of bovine serum albumin.
Borzova VA1, Markossian KA1, Kara DA1, Kurganov B2.
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