[Protein-analysis] Re: equilibrium const for disulfide bond formation

[Uncle Al]

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
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 (Toxic URL! Unsafe for children and most mammals)
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