[RASMB] more on dithionite and Hb

[Steve Shire]

Hi Jack and MItra,
    One of the ways to minimize damage from excess dithionite is to  
use a procedure described by Dixon and McIntosh, Nature (London) 213,  
399 (1967). Essentially one uses a G25 Sephadex (or equivalent )column  
to load a band of dithionite and then follow with your heme protein.  
The protein obviously migrates faster through the column and the time  
in contact with dithionite is greatly reduced. I tried this once long  
time ago with myoglobin and showed identical absorption spectra and  
oxygen equilibrium measurement with oxy myoglobin prepared directly  
from whale tissue (those were the good old days in Frank Gurd's lab at  
Indiana). At any rate check out the reference and give that a try.
             Steve
On Feb 5, 2009, at 8:03 AM, 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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Steven J. Shire, Ph.D.
Staff Scientist and Group Leader
Late Stage Pharmaceutical and Device Development Dept.
MS #96A
Genentech, Inc.
1 DNA Way
S. San Francisco, CA 94080
650-225-2077 (VOICE)
650-467-2388 (FAX)
shire.steve at gene.com

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