[RASMB] more on dithionite and Hb

[Jo Butler]

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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-- 
Dr P.J.G. Butler,
MRC Laboratory of Molecular Biology,
Hills Road, Cambridge, CB2 0QH, UK.
Tel. +44 (0)1223 402296