[RASMB] more on dithionite and Hb

[Dj Scott]

Hello All,
Andrew brings up a good point, which is that oxygen can dissolve in the
centrepieces and is then slowly released over the course of the run. The
way to get round this is placing the centrepieces for 24-48 hours in an
anaerobic chamber and then assembling them under N2. This strategy was
employed by an old York AUC user, George Kellett when he measured Hb way
back in the early 70's (and relayed to me when I was a Post-doc at
York), and it works very well indeed. You can then check the degree of
oxidation via a wavelength scan before and after the run. The incubation
step under N2 really does make a big difference, those oxygen molecules
get everywhere....

Hope this brings some comfort,

Dave.

 
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
Dr. David J. Scott
Associate Professor in Physical Biochemistry
National Centre for Macromolecular Hydrodynamics
School of Biosciences
Sutton Bonington Campus
University of Nottingham
Leicestershire. LE12 5RD.
United Kingdom.
+44 115 951 6221
+44 115 951 6142
www.nottingham.ac.uk/ncmh
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

-----Original Message-----
From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org]
On Behalf Of Leech, AP
Sent: 06 February 2009 11:03
To: RASMB
Subject: Re: [RASMB] more on dithionite and Hb

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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