[RASMB] more on dithionite and Hb

[Tom Laue]

Hi Mitran-
All of the steps need to be done in a glove box that is flushed with N2 
or Ar. All of the solutions need to be degassed in a weak vacuum or 
sparged with N2 or Ar to make sure they are O2 free.
Best wishes,
Tom

mitrana at mail.utexas.edu wrote:
> Concerning soaking the centerpieces in dithionite - is this all done 
> under anaerobic conditions? Somehow the first thing that came in my 
> mind is - how do you clean the centerpieces after the dithionite soak? 
> -Mitra
>
>
> Quoting Tom Laue <Tom.Laue at unh.edu>:
>
>> Hi-
>> The epon centerpieces do leach O2, and soaking them, then storing them
>> in an N2 or Ar atmosphere is necessary. I recall, too, that he was
>> concerned about the Al-filled epon, though properly passivated
>> Al-filled centerpieces should be no more reactive than charcoal-filled
>> centerpieces. Though this probably does not apply to your work, KelF
>> centerpieces are even worse when it comes to O2 leaching.
>> Best wishes,
>> Tom
>>
>> John Philo wrote:
>>> Regarding handling oxygen-sensitive materials in the centrifuge, 
>>> first I
>>> agree fully with Jack Kornblatt's statements about dithionite and 
>>> had said
>>> something similar to Mitra privately yesterday, and recommended he 
>>> try an
>>> enzymatic oxygen scavenging recipe I used for Hb kinetics studies in 
>>> the
>>> past. Second, one additional point is that Todd Schuster told me  
>>> years ago that
>>> the Epon centerpieces are a significant source of oxygen. I believe the
>>> old-timers would soak the centerpieces in dithionite solutions for 
>>> several
>>> days to deplete that oxygen and then the need for oxygen scavenging 
>>> capacity
>>> is much reduced.
>>>
>>> John -----Original Message-----
>>> From: rasmb-bounces at rasmb.bbri.org 
>>> [mailto:rasmb-bounces at rasmb.bbri.org] On
>>> Behalf Of Jack Kornblatt
>>> Sent: Thursday, February 05, 2009 8:04 AM
>>> To: RASMB
>>> Subject: [RASMB] more on dithionite and Hb
>>>
>>> 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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>>>
>>
>> -- 
>> Department of Biochemistry and Molecular Biology
>> University of New Hampshire
>> Durham, NH 03824-3544
>> Phone: 603-862-2459
>> FAX:   603-862-0031
>> E-mail: Tom.Laue at unh.edu
>> www.bitc.unh.edu
>> www.camis.unh.edu
>>
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>
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-- 
Department of Biochemistry and Molecular Biology
University of New Hampshire
Durham, NH 03824-3544
Phone: 603-862-2459
FAX:   603-862-0031
E-mail: Tom.Laue at unh.edu
www.bitc.unh.edu
www.camis.unh.edu