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

Jenn via proteins%40net.bio.net (by juicymixx At mailinator.com)
Wed Jan 10 12:19:54 EST 2007


DK wrote:
> In article <1168370284.242721.50740 At o58g2000hsb.googlegroups.com>, 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...
>
> There is no single number to answer your question. "It depends".
> Lots of factors will influence propensity of your cysteins dimerizing:
> their precise pKa (depends on envirnoment; put a Lys next to it and
> it will be A LOT more reactive), degree of solvent exposure and
> steric accessibility, amound of O2 and pH of your buffer,
> the amount of traces of metals, etc, ect. Just work as fat as you can,
> perform labeling and, if the experiment demands it, purify monomer
> away from dimer on gel-filtration or by affinity chromatography
> against the probe.
>
> DK


Thanks for the reply.   I understand that the local environment around
the CYS makes a difference (as well as the bulk environment).   Is
there any literature (or a journal article) that expands on this?
Someone must have tried to measure the reaction rate under some certain
condition...?

Thanks again.



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