Sulfhydryl reagents to protect S=S bond

Emir Khatipov ekhatipo at NOSPAMmidway.uchicago.edu
Tue Jun 26 12:04:20 EST 2001


Hi everybody,
I have such a problem to solve. I have a 30-mer peptide that has 2 cysteine
residues (something similar to S-S constrained phage display libraries),
that are bound via S=S bond, thus making a peptide having a loop in its
structure. I would like to figure out if this peptide is binding an
intracellular protein it is supposed to bind. To do that I treat mammalian
cells with that peptide labelled with fluorescent probe, wash the cells
extensively, and analyze retention of the fluorescence by microscopy or flow
cytometry. However, it is known that due to reduced nature of intracellular
environment, disulfide bond is going to be eventually cleaved after the
peptide gets into the cell, possibly pretty fast. To avoid intracellular
reduction of S=S, I am willing to try using reagents that would link those
cisteines covalently, so that the loop structure of the peptide will be
preserved after it penetrates the cell. Ideally I would like to have
something like -Cys-S-R-S-Cys-, where R should be as small as possible.

I sort of figured out that sulfhydryl p-chloromercuribemzioate are possible
candidates. the p-CMB would react with S=S to make S-Hg-S, which would be
fine for me as long as it works. However, p-CMB being used mainly to
inactivate SH enzymes at millimolar concentrations, I am not sure it will be
same reactive with disulfides in the peptide molecule. I am also not aware
if there are other potential "small" bifunctional reagents that I could use.
I checked Pierce, but it does not seem that they have anything applicable
for my case.

I understand that this is not a trivial problem, because most often these
days we are interested in reducing S=S, e.g. when we boil proteins for
SDS-PAGE, with DTE or BME, or maleimides. However, I would greatly
appreciate if someone experienced in protein and peptide chemistry, and
applications of sulfhydryl reagents in particular, would provide any
feedback.

Thank you.
Emir





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