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We have used ACM-cys like you propose with success in the past. For us,
the next step after oxidation is HPLC purification, so we avoid DMSO as
the peptide will not stick to the column when dissolved in it. Some
general things we do when we form disulfide bridges:
dissolve the peptide in acidic buffer (1-20% acetic acid;
acetonitrile/water with 0.1% TFA; etc, etc,). If you dissolve in a
basic buffer, you run the risk of polymerization (concentration of
peptide is very high as it initially dissolves). It doesn't happen very
often, but I've seen it happen. Concentration depends on if the
disulfide bridge is intra-molecular (more dilute-- 1mg/ml or less) or
inter-molecular (1 mg/ml or more). Add ammionium bicarb to raise the pH
to 8. Monitor the HPLC of the peptide to determine when reaction is
complete-- the retention time will shift. For disulfide bridges that
don't want to form, add a little ferricyanide to push it (just until the
solution is yellow -- the color will go away as the reaction proceeds).
If you only have a few mgs, you might want to dissolve it and add
ferricyanide to ensure you have a reaction. I hope this helps. Good
luck!
> ----------
> From: alessandro tossi[SMTP:tossi@univ.trieste.it]
> Sent: Monday, January 26, 1998 1:50 AM
> To: Recipients of ABRF List
> Subject: Pepsyn
>
>
> Dear ABRFers,
>
> A student of ours is working on a peptide with two cysteines, with
> which
> we would like to make parallel and antiparallel homodimers. At the
> moment we have made two versions of the peptide: peptide1 with a free
> Cys towards the N-terminal and an Acm protected Cys towards the
> C-terminal, and peptide2 which is vice-versa.
>
> For the parallel dimer we thought of air oxidation of peptide one, to
> form a first intermoloecular S-S bridge via the free cysteines,
> followed
> by removal of Acm and second bridge formation. I am told oxidation in
> the presence of DMSO is most efficient, but I suppose it must then be
> removed someway before the Acm can be removed from the remaining two
> cysteines. We thought of using iodine.
>
> For the antiparallel peptide we need to initially form an S-S bridge
> between Peptide1 and Peptide2 via the free cysteines. I suppose that
> to
> do this we need to preactivate the free cys on one peptide before we
> add
> it to the other peptide. I have read that Ellman's reagent can be used
> for this but have not been able to find a good protocol. We could then
> form the second S-S bridge by removing Acm.
>
> One problem is that we have only a few mg of each peptide. Any
> suggestions would be very welcome. If you feel that alternative
> protection schemes requiring resynthesis would be better we would also
> welcome these suggetsions.
>
>
> Thanks , Alex Tossi
>
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Dr. Alessandro Tossi
Antiinfective peptides group
Dept. of Biochemistry, Biophysics and Macromolecular Chemistry
University of Trieste
Phone -- 39 40 6763673; Fax -- 39 40 6763691
email: tossi@univ.trieste.it
tossi@icgeb.trieste.it
http://www.univ.trieste.it:80/~nirdbbcm/antimic.html
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