phlogiston
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removing O2 from enzymatic reaction
I need to test if a specific enzymatic oxidation reaction utilizes oxygen as the electron acceptor. To do this, one of the experiments I would like to
do is to simply set up the reaction in the absence of oxygen.
I was hoping some you may have some advice on how to remove oxygen in a way that would leave most enzymes intact? The enzyme I need to test critically
depends on a cysteine residue for its activity, so anything that reacts with sulfhydryl groups is not good.
I do not have an oxygen electrode to measure remaining oyxgen.
For now, what I could come up with is:
1. Purge with nitrogen before starting the reaction (done by addition of the substrate solution which I would also purge with N2). I have no idea how
long to do this for, however, and how effective it would be.
2. Consume any oxygen by means of another enzymatic reaction first, before starting the reaction I need to test. However, all of the reactions I can
think of generate hydrogen peroxide (e.g. glucose oxidase, D-amino acid oxidase, fatty acyl-CoA oxidase, etc.), which reacts with the product of my
enzyme so I need to get rid of the H2O2 efficiently too. I could try adding catalase to do that but I am worried it may not be effective enough, and
it partly regenerates the oxygen I was trying to get rid of in the first place (2 H2O2 --> 2 H2O + O2).
How much oxygen is dissolved in water exposed to atmospheric pressure air anyway?
I would be most grateful for any pointers you guys may have.
[Edited on 30-3-2011 by phlogiston]
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"If a rocket goes up, who cares where it comes down, that's not my concern said Wernher von Braun" - Tom Lehrer |
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ScienceSquirrel
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Have you thought about using helium as a purge gas?
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Ozone
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IIRC, it's about 8 mg/L at rtp. Use helium as a sparging gas (an hour or so seems to work fine) and/or pull a vacuum on it whilst under sonication
(e.g. utrasonic jewelry cleaner). Always run a blank and a spike.
There are also a multitude of chemical O2 scavengers, for example see: http://www.arkema-inc.com/literature/pdf/346.pdf. Of course, care would be needed to pick something that won't interfere with your assay.
Or, you could go the BOD route with a little glucose and some "seed" from the local activated sludge treatment plant  (then autoclave it to kill the bugs and residual enzymes).
Cheers,
O3
[Edited on 31-3-2011 by Ozone]
-Anyone who never made a mistake never tried anything new.
--Albert Einstein
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