Date: Thu, 16 Jan 1997 12:09:30 -0600
Message-Id: <v01530504af051cf9f7cb@[128.252.197.88]>
To: abrfhyp@cco.caltech.edu
From: rskubish@pharmdec.wustl.edu (Richard Skubish)
Subject: PepSyn-disulfide exchange
>Date: Wed, 10 Jul 96 17:15:52 EST
>From: "Thomas Andersen" <tandersen@ccgateway.amc.edu>
>Old-To: abrf@aecom.yu.edu, jack.moore@ualberta.ca (Jack Moore)
>Subject: Re: PepSyn-disulfide exchange
>To: Recipients of ABRF List <abrf@aecom.yu.edu>
>Sender: Association of Biomolecular Resource Facilities
><abrf-request@aecom.yu.edu>
>Precedence: bulk
>
> I always like to know the end use of the peptide so that I won't make
> stupid suggestions, but in the absence of that knowledge, here goes:
>
> If the protein in which those Cys existed were one containing disulfides,
> your user may be talked into using the peptide in an alkylated form
>(I like
> the carboxamidomethyl). Many users do not realize that generating a
> sulfhydryl where the protein had the disulfides may not be advisable.
>[Of
> course, if the user needs those for coupling purposes, then this was a
> stupid suggestion.]
>
> It may be that it would not interfere with the end use to introduce a
> reducing agent: we have been finding glutathione advantageous for
>some of
> our work recently, though it would be inappropriate for many uses.
>If you
> do AAA after introducing glutathione, don't forget to correct for it.
> Mercaptoethanol may be useful but will probably form the mixed disulfide;
> dithiothreitol probably won't form the mixed disulfide. Again, end
>use is
> the driving consideration.
>
> Of course, it seems as though you have already tried the low pH range
>that
> would usually be recommended to avoid disulfide interchange; have you
> tried a combination of intermediate pH and gassing it with nitrogen or
> argon to avoid oxygen?
>
> Finally, if the peptide is simply forming disulfides, can they not be
> reduced back immediately before, or during, the end use?
>
> I hope SOME of these were not stupid suggestions.
>
>
>----------------------------------------------------------------
>Thomas T. Andersen e-mail: tandersen@ccgateway.amc.edu
>Dept Biochem Molec Biol voice: 518 262-5368 (alt: 6474)
>Albany Medical College fax: 518 262-6475 (alt: 5689)
>Albany, NY 12208
>----------------------------------------------------------------
>
>
>
>
>______________________________ Reply Separator
>_________________________________
>Subject: PepSyn-disulfide exchange
>Author: jack.moore@ualberta.ca (Jack Moore) at Internet-Mail
>Date: 7/10/96 4:39 PM
>
>
>I am working on a synthetic peptide that seems to be exchanging disulfides and
>would appreciate any suggestions on how to minimize this problem. The
>sequence
>is CYIQNCPLGGKR. Both terminals are free and the two cys residues form a
>disulfide. After purification, oxidation and another purification I have
>my desired product at a purity level of greater than 95%. The problem is
>that I can't stop the peptide from forming new oxidation products. This
>occurs after a couple of days as a lyophilized powder. It happens on both
>the benchtop and the freeze drier. I'm sure that the desired peptide has
>been made as I've characterized it to death so it's not just a case of the
>reduced peptide forming random oxidation products. The exchange happens as
>a TFA salt, an HCl salt and also seems to be happening as an acetate salt.
>It is most stable as a dilute solution (~0.1 mg/mL) but unfortunately the
>end user can't have it like this.
> Does anybody have any ideas?
>
>Thanks - Jack Moore
>
>P.S. Two other peptides, one minus the arg and the other minus the lys and
>the arg are also doing the same thing.
>
>
>
Richard Skubish
rskubish@pharmdec.wustl.edu
314-362-0283
Washington University Medical School
Box 8103 - PNACL
660 S. Euclid
St. Louis, MO 63110