stabilization of disulfide bonds by oxidation: HOW?

Kresten 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
> sepharose
> 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
amine–thiol 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.

HTH
Kresten


>
> Frank
>
>

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Kresten Lindorff Larsen, Dept. Yeast Genetics


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