Mary,
If you want to do this "off resin", here is a reasonable approach.
Do a thiol-disulfide interchange reaction with cystine (Cys-Cys) at say
5-10 fold molar excess over peptide sulfhydryl concentration at pH 8. You
could use 0.1 M tris or 0.1 M ammonium bicarb etc. I would add a chelator
to minimize metal- catalyzed sulfhydryl oxidation. Your main fear here is
the formation of peptide dimers (linked via a disulfide bond) which will
decrease your final yield. 10 mM EDTA or EGTA should suffice. The exchange
reaction is virtually quantitative. I'm guessing 4h at RT will work but
you might have to play with this. Ok, now you have your target molecule
and need to purify it away from other reaction components. I doubt RP-HPLC
will resolve the unreacted peptide from the target cystine peptide but you
could give it a go. A better approach would be to use thiophilic
chromatography such as TNB-thiol Agarose (Pierce). Here you are trying to
REMOVE unreacted peptide from target and any dimers which may have formed.
Once again a thiol-disulfide interchange reaction is operating. In this
case any unreacted peptide will displace the TNB anion (in organic jargon
it is a "good leaving group". By the way that phrase and steric hinderance
are GREAT for partial credit) and your target peptide and any peptide
dimers will NOT BIND to the resin. Run unbound eluate on standard
C18RP-HPLC to obtain desired product (here I think dimer should be easily
resolved from target!). Check mass via MS and lyophilize. Move on to your
next project.
Best regards,
>
Dan L. Crimmins
Washington University School of Medicine
Dept. Pathology/Division of Laboratory Medicine
660 S. Euclid Ave., Box 8118
St. Louis, MO 63110
Phone: 314-454-8514; Fax: 314-454-5208
e-mail: crimmins@labmed.wustl.edu