juicymixx At mailinator.com wrote:
>> Hello,
>> This is a pretty simple question; however I can't seem to find an
> answer to it. I have a rather small protein (60 amino acids) in which
> I've mutated in a single cysteine for labeling purposes. There are no
> other cysteines in the protein. Additionally the protein is normally
> unstructured in solution (that is in 30uM phosphate buffer at 25C,
> there is no significant structure). Recently we've noted that after
> purifying the protein (but before labeling it) we appear to have some
> dimer formation which isn't seen in the wild type protein.
>> I was just wondering what the equilibrium constant (reaction rate,
> etc.) for intermolecular disulfide formation from 'free' cysteine would
> be (or better yet, cysteine in a polymer/peptide/protein). Someone
> has probably already done this experiment, however I'm not sure of
> where to look for this number...
Work under inert gas, add some modest reducing agent. Cysteine to
cystine is entirely reversible under mild conditions if not rpevented
beforehand,
http://en.wikipedia.org/wiki/Dithioerythritol.
At high concentrations it induces cleavage of disulfide bonds. At low
concentrations it prevents oxidation of SH groups during
polyacrylamide gel electrophoresis.
--
Uncle Al
http://www.mazepath.com/uncleal/
(Toxic URL! Unsafe for children and most mammals)
http://www.mazepath.com/uncleal/lajos.htm#a2
