For a metallo-enzyme activity, the suggested pH for assay is 9.5, which buffer systems to consider? Tris-HCl or glycine buffers are discussed in the literature. In addition, literature also mention that enzyme needs to be activated in the presence of metal salt solution such as Ni, Mn, or Co and therefore, should I activate enzyme solution containing metal ions alone or buffer in addition as well? With my initial trial, activation of enzyme for 10 min in buffer containing both metal salt (Mn) and glycine did not produce the reaction product. Thanks in advance.
Hi there,
Tris buffer is not appropriate as pH 9.5 is outside pKa +/- 1 (pKa of Tris being 8.1). The amine function of glycine might be more appropriate as a buffer as its pKa is around 9. Bicarbonate/carbonate system might also be an alternative but the inconvenient is that it is actually volatile. You may also have look at the following for more ideas:
https://www.sigmaaldrich.com/technical-documents/protocols/biology/ion-exchange-chromatography/non-volatile-and-volatile-buffer-systems.html
If you know which metal ion activates your enzyme, and the optimal concentration of it, then include that in the experiment buffer used to prepare all the solutions. If those are parameters you want to test, then keep the metals out of the buffer, and prepare separate solutions containing each metal at 3X the final desired concentration. Then you can mix equal volumes of 3X enzyme, 3X substrate(s), and 3X metal solution to make the final 1X reaction solution. All three solution can be prepared in 1X buffer.
To measure the baseline activity in the absence of metals, include 1 mM EDTA to make sure there is no trace metal contamination.
Try https://www.google.ru/url?sa=t&source=web&rct=j&url=https://www.goldbio.com/documents/3581/User%2BGuide%2Bfor%2BGoldBio%2BBuffers%2B1.3.pdf&ved=2ahUKEwipq8jxmvXbAhXid5oKHUZmBpgQFjACegQIBhAB&usg=AOvVaw3-X9p7SOCKOhpml2oQpFVO
Tris-HCl buffers is usually at best between pH 8-9... I would rather suggest you use Glycine buffer for pH between 9 and 11.
Depending on What you are studying on the Enzyme, the addition of the chelating agent, EDTA (1 mM) to the buffer could be a welcome idea.
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