[RASMB] more on dithionite and Hb
Jo Butler
pjgb at mrc-lmb.cam.ac.uk
Fri Feb 6 06:49:49 PST 2009
Dear Andrew,
We were routinely using aluminium filled epon centrepieces. We did leave
them degassing overnight at a high vacuum, before going on to the N2
flushes. I do not remember the O2 affinity of the cytochrome, but it was
notoriously easily oxidised – hence the measurement of the spectrum at
the end of the runs.
Jo
Leech, AP wrote:
> 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
>>> _______________________________________________
>>> RASMB mailing list
>>> RASMB at rasmb.bbri.org
>>> http://rasmb.bbri.org/mailman/listinfo/rasmb
>>
>
--
Dr P.J.G. Butler,
MRC Laboratory of Molecular Biology,
Hills Road, Cambridge, CB2 0QH, UK.
Tel. +44 (0)1223 402296
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