[RASMB] more on dithionite and Hb

[Leech, AP]

Hello Jo, all,

I'd be interested to know what sort of centre pieces you were using, as
in my (limited) experience of anaerobic experiments, we did largely as
you described and nevertheless found slow oxidation over the course of
the run. We were using charcoal filled epon, which others have also
observed to be problematic. Of course it will also depend on the oxygen
affinity of the sample. Has anybody had the patience to determine how
long charcoal filled epon needs to be deoxygenated before use?

Andrew

Jo Butler wrote:
> Hi Mitra,
> 
> Following Jack's comment, I can point out that there is really no need 
> to have dithionite (or any other reducing agent) present to work 
> anaerobically.
> Some years ago I ran some u/c experiments on a cytochrome in the reduced 
> state and my collaborator was totally opposed to having any reducing 
> agent present during the runs.  Instead we pretreated the assembled 
> cells in vacuum, with a series of nitrogen flushes (i.e. high vac/ N2 
> admission/high vac etc.) and then very rapidly loaded the samples, and 
> sealed the cells, with samples which had also been brought to an 
> anaerobic state.
> Despite the lack of reducing agent, the spectrum of the cytochrome 
> showed that, even at the end of either an equilibrium or velocity run, 
> it was still fully reduced.  Doubtless the vacuum of the chamber helps 
> to prevent any leakage into the cells during the run, but this does show 
> that adequate vacuum/flushing with O2-free N2, can eliminate the 
> residual O2 from cells.
> 
> Jo
> 
> Jack Kornblatt wrote:
>> Hello Mitra
>> Chance, I think, once described dithionite as man's worst enemy. the 
>> concentration that you are using is far in excess of what is need to 
>> keep your solutions anaerobic. The reaction products of dithionite are 
>> too  numerous to list even if I could remember them. If you degas your 
>> solutions just before loading and then add dithionite to 1 mM this 
>> should give you the desired "low" oxygen.
>> Is it really necessary to keep dithionite as low as possible? I have 
>> little experience with Hb but if we use dithionite and cytochrome c 
>> oxidase at 11 mM there are sufficient biproducts generated that 
>> interpreting data is very difficult
>>
>> best
>> jack kornblatt
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> 

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