stabilization of disulfide bonds by oxidation: HOW?
kresten at my-deja.com
Fri Aug 20 08:20:34 EST 1999
In article <37BD45E1.3072FD43 at uni-konstanz.de>,
Frank.Fackelmayer at uni-konstanz.de wrote:
> Hi all,
> We have immobilized a cystein containing protein on thiopropyl
> beads where is bound via a single disulfide bond. The protein can be
> quantitatively recovered from the beads by treatment with DTT. Now, we
> want to make this bond resistant to DTT, and have thought about
> oxidation of the bond to a sulfonate. What would be the best method to
> do that? The reaction should be simple to perform, quantitative, and
> with minimal side reactions with other amino acids
> including methionine.
> Any ideas?
Well, this is not really an answer to your question (sorry).
Perhaps you could cross-link your protein to the column using a
bifunctional cross-linking reagent. One possibility is of course simply
to use a thiol-thiol cross-linker to link your protein to the column
(starting over again with protein-SH and column-SH), but I would guess
you would have to play around with concentrations to avoid crosslinking
column with column or protein with protein.
Another idea is to use another bifunctional cross-linking reagent eg. an
aminethiol crosslinker to cross-link protein lysines to remaining
column thiols after your protein-SS-column disulfide formation. Then
your protein would stick to the column even after reduction. There are
of course still the problems with disturbance of structure etc.
The address kresten at my-dejanews.com is for
spambots only. Please mail me at LysLeuLeu at crc.dk
transforming the pre at -part into my initials.
Kresten Lindorff Larsen, Dept. Yeast Genetics
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