How to deplete Oxygen from enzyme assay?

David Moss david.moss at ifia.fzk.de
Fri Sep 26 14:40:44 EST 1997



William P. Tschantz <tschantz at galactose.mc.duke.edu> wrote
> I am looking for an easy way to deplete a system of oxygen.  I suspect
> that the enzyme i am working on uses molecular oxygen in its mechanism
and
> would like to test it.  I have tried degassing my samples and reaction
> vial with nitrogen, but have gotten messy results.  Enzyme linked assays
> would be the easiest i think.

I did some experiments on ways to deplete reaxtion mixtures of oxygen
during my doctorate. I found degassing with nitrogen leaves large, variable
residual concentrations. A few grains of dithionite is the most efficient
method,
but the strong reducing conditions may damage your enzyme. More mild
but nearly as good is glucose oxidase + glucose, as long as you don't mind
having a second enzyme around. You could also try anthraquinone sulfonate:
you can photoreduce it with light from a slide projector in the presence of
HEPES or similar buffers, then it autooxidizes, consuming all the oxygen
around.
The reaction is autocatalytic (quinone-mediated), so you get down to very
low oxygen tensions very fast. As soon as you switch off the lamp, you
no longer have a damagingly strong reducing agent. I ended up using
anthraquinone sulfonate and glucose oxidase together, with a trace of
catalase
to dispose of any hydrogen peroxide formed.

Regards,
David Moss
 



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