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Active enzyme goes on ion exchange column -- dead enzyme comes off. What am I do


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#1 Luria Bertani

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Posted 06 June 2012 - 01:56 AM

Hello,

I have a metalloenzyme which I test the crude extract and there is heaps of it and it is very active.

I load it into a Q Sepharose column - Buffer A is 50 mM TRIS buffer pH 7.8, and the said enzyme is eluted with Buffer B (Same as buffer A but with 1M NaCl).

The purification looks all nice on the chromatogram, but when I go to assay the activity - all the activity is gone. What have I done wrong?

I'm thinking maybe I should add zinc to the buffer (50 uM or 100 uM???) or perhaps the pH is far too high
(is pH 7.8 too high, the predicted theoretical pI of my protein is 6.5).


This is very frustrating and is happening with other metalloenzymes that I put through my Q Sepharose FF column.
The column itself is brand new and cleaned.

Any help or suggestions would be muchly appreciated!!

#2 mdfenko

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Posted 06 June 2012 - 12:12 PM

are you performing a step elution or a gradient?

the salt concentration may to high for the enzyme to exhibit activity.

as for pH, you can perform a pH curve assay to see what pH is and isn't suitable for activity.

you may be purifying it away from a necessary cofactor.

if the metalloenzyme uses zinc then try adding zinc to the assay (1mM should be more than enough to see if it is required)
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#3 Luria Bertani

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Posted 06 June 2012 - 02:00 PM

Hello mdfenko,

Thank you for your quick reply! I am doing an elution by salt gradient - the protein comes off at about 30% NaCl. The gradient itself goes from 0 M NaCl linearly up to 1M NaCl.

This is very frustrating for me, I will try dialysis of the dead protein against lower pH buffer and zinc present.

#4 Luria Bertani

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Posted 10 June 2012 - 01:05 AM

I am trying more zinc in the buffer - increasing this to 500 uM...
We will see if this does the trick.




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