Good day fellow researchers,
I am investigating the kinetics of enolase in E. coli w1110. The assay is performed by measuring the disappearance of phosphoenolpuruvic acid (PEP). Using the UV spec I first did a spectrum measurement, from 190nm to 600nm. This showed me that PEP absorbs between 240 and 250 nm, agreeing with literature values. I then completed the assay measuring absorbance at 240nm as the literature protocol stated.
The problem is that for the same crude cell free extract I can see definite activity for enolase using NMR spectroscopy in the first five to ten minutes of the assay, but when the assay is reapeated on the UV spec, under the exact same conditions there is no reaction.
I have even tested the reverse assay options, linking PGM with enolase made up from purified freezer stock, starting the assay with 3pg and looking for PEP accumulation. This works perfectly...I see very nice curves for PEP appearance. This led me to believe that it is a problem of the Keq not favouring the reverse reaction, but this cannot be, as time and time again the NMR assays from PEP to 2pg and then 3pg works perfectly.
Asay mixtures are the same for both UV and NMR experiments and contain ell free extract, substrate and MgCl2 as cofactor.
Hi Christiaan,
Does the UV spectrum of the crude extract without PEP have a UV absorbance around 240 nm? If there are other components of the assay mixture that absorb at ~ 240 nM, they could obscure the ability to detect any PEP that accumulates.
There is an enolase assay available from Sigma-Aldrich* that includes a peroxidase substrate that reacts with an intermediate during the 3pg --> PEP conversion, yielding a product that absorbs at 570 nM. This may be a viable alternative to the assay you are using.
Keith
* http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/2/mak178bul.pdf
Thank you very much Keith,
I will definitely have a look and test both of your suggestions.
Have a nice day.
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