[RASMB] more on dithionite and Hb

[Steve Shire]

Hi Jack and Mitra,
    I didn't see the previous E-mail until now so sorry for the  
confusing suggestion. Obviously what I suggested would not address the  
concern of keeping your sample in the AUC deoxygenated. However, if  
one wants to convert met Hb or Met Mb samples to oxy Hb or Mb that  
procedure is a great way to do that.
             Steve
On Feb 5, 2009, at 10:05 AM, Steve Shire wrote:

> 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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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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