How to deplete Oxygen from enzyme assay?

John Philo jphilo at amgen.com
Fri Sep 19 15:42:24 EST 1997


William P. Tschantz wrote:
> 
> Hi
> 
> 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.
> 
> Any ideas appreciated.
> 
> Thanks in advance.
> 
> Bill

Based on my 'former life' as a hemoglobin researcher, you might consider
the following things:

1) the strongest oxygen destroyer is sodium dithionite, but this is also
a powerful reductant and may reduce metals in the active site of your
enzyme (if it really uses oxygen I am assuming you are dealing with a
metalloenzyme).  Dithionite can also have some nasty contaminants and
byproducts of its activity.  In the hemoglobin field there is much lore
regarding brands of dithionite, and the common lab chemical suppliers
don't have high grade dithionite, so you may want to secure some from a
hemoglobin lab.

2) I had a lot of success with an mixed enzyme system: glucose oxidase,
catalase, and glucose.  The drawback of this system is that the glucose
oxidase converts dioxygen to peroxide, and that may destroy your enzyme
before the catalase gets to it.  I believe this system is still commonly
used for hemoglobin work by Gary Ackers lab (Washington U. Med School).

3) A second possible enzyme system is protocatechuate dioxygenase +
protocatechuic acid.  This one has the advantage that it destroys
dioxygen in one step.

With any of these systems, the key is to use them to remove traces of
oxygen remaining AFTER you have already done a good job of degassing
with nitrogen or argon.  You don't want to make them try to remove all
the oxygen in the solution.

'Hope this helps,

John Philo, Protein Chemistry, Amgen Inc.



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