The research papers have variations regarding use of buffer system for the same protein in kinetics studies (Tris-HCl, HEPES or Phosphate).
Is the selection based on purification buffer used for enzyme or It's an independent choice?
Thanks.
Hi there,
You can't tell which is the best until you have actually tested. Basically the best buffer for protein stability during purification might not be the best buffer for activity measurement.
Hi, you may need to do some testing, but here are a few hints:
Tris/HCl is extremely universal and works with many enzymes while being inexpensive.
HEPES is often chosen for sensitive enzymes, but is more expensive.
Phosphate buffer can be problematic if phosphate is e.g. one of the products of the enzymatic reaction, as this influences the equilibrium.
Furthermore, the ionic strength your enyzme needs is a very significant factor (salt concentration), as well as the requirement for reducing agents such as DTT (weak agent) or mercaptoethanol (very strong) to reduce Cystein-Cystein S-S double-bonds to -SH.
So, in summary, it is a complex picture. The more you know about your protein, the less you need to test.
Hi,
Tris-HCl buffer sometimes can play role as activator, you have to keep mind. And purification buffer is not necessarily used for kinetic studies.
Phosphate buffer. The pKa is 7.2. Thus, it is useful between pH 6.2 and 8.2.
There are many buffers which work well in the pH range 7 to 8. However, you have to first establish the nature of the enzyme assay and the enzyme itself. If your enzyme is a metallo-enzyme, phosphate buffers are not recommended, if you are assaying a phosphatase enzyme, phosphate buffers are inhibitory and therefore not recommended. Tris-HCl buffers can have an effect on phosphatase activity by acting as an acceptor molecule in competition with water and show increased rates.
The best thing to do is to measure the reaction rates of your enzyme in the presence of increasing molarity of your buffer, keeping the ionic strength constant, and see which buffer has the minimum effect on the reaction rate itself.
So, phosphate buffers, Tris-HCl, HEPES are probably your best bet.
One of the best, is the MOPS buffer, this "Good buffer" is most stable than TRIS or another, its pKa is 7.2. Tris have a pKa value of 8.1, therefore, it's not the best choice for the kinetic enzymatic assays.
My enzymatic assays (PEPCK, MDH, PK) was done using MOPS as buffer, another reliable choice is the BES salt, it have a pKa value of 7.1.
Thank you all for the inputs. My enzyme is a metallo enzyme and I am using tris buffer. I have found that the enzyme works well at 10mM buffer at pH 7.5. Yet, I have observed many people have used different buffers for same enzyme for same sort of analysis. This has amazed me.
Jose is right in one thing. MOPS is a very stable buffer and also resistant to pH shift if you change the temperature. This is a trick not everybody takes into account when changing temperature of the assay or measuring activation energy. Tris has a very strong drift and should be prepared every time in a temperature it is supposed to be used while MOPS has almost no repose to the range of T we are interested in (usually 20-70o C).
The issue of Tris being sometimes an activator is also important. We have tested our metalloenzyme in HEPES and Tris/HCl, pH 7.5 and got different activation energy of the reaction. So this is also one thing to keep in mind.
Thanks Maciej for your clarifications. I know about the pH shift due to temperature fluctuations in case of Tris buffer. I am avoiding extreme range of temperature variation for the assay to avoid any dramatic change in kinetics. So far I have got consistent results with triplicate repeats.
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