[RASMB] more on dithionite and Hb

mitrana at mail.utexas.edu mitrana at mail.utexas.edu
Thu Feb 5 12:53:53 PST 2009


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
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