Re: [ccp4bb]: preventing oxidation of Selenium

[Frank von Delft <frank@cryst.bioc.cam.ac.uk>]

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>       I apologize that this is not a ccp4 question, but I know many of
> you have experience with selenium-derivatized proteins and was curious to 
> know if there was a consensus on what strategy is best to use to prevent
> oxidation of seleno-methionine.	
> 
>       I will be obtaining a 40mer peptide which oligomerizes and has 1
> selenium/chain. I was wondering if anyone could offer any tips on
> preventing oxidation:  is it enough just to add 10mM DTT or considering
> this is a peptide is it better to use a glove bag purged with nitrogen? 
> Also, how long can a SeMet crystal be manipulated while exposed to air?

In one case, I added DTT to the crystallisation conditions and 10 days
later cryo-stored the xtals.  From the fluorescence spectrum it looked
like there was significant oxidation. 

In the next case, I could not add DTT if I wanted decent crystals, so I
just left it away completely, and the xtals were exposed to oxygen for
some 3 days. But during cryoprotection, about 1 hour before freezing, I
added 3mM DTT to the buffer.  And these crystal seemed nearly completely
non-oxidised, with a proper sharp peak in the fluorescence spectrum.
 
It seems that oxidation is reversible, so you may find this second
treatment adequate, probably in fact more reliable, because DTT goes off
quite quickly, but oxygen diffuses through the Linbro plate plastic (I
have been told).  

Cheers 
Phraenquex