[Miscman85]

I apologise for what is probably a simple question, but I don't have a strong chem background. I am working on a protein with 4 disulfide bonds, which we restore by dialysing the denatured protein solution against a redox buffer consisting of 10 mM sodium acetate, 10 mM reduced glutathione and 1 mM oxidised glutathione pH 5.0.

How does this work, considering that the reaction looks to be reducing rather than oxidising?

[rjb]

Miscman,

That's actually a pretty tough question to answer! You're right, the GSH:GSSH containing buffer is slightly reducing, but that's actually a very positive thing for the refolding process. It would be entirely possible to use reagents that restored the S-S bond rapidly, but we don't want to do that, chances are the bonds formed are going to be mispairs (especially with larger proteins) and you'll end up with a big mess rather than the structure you actually want. By using refolding buffer, there is constant reversible exchange of S-S bonds ('Shuffling') which allows the protein in time to find its own native stable structure. It is this stability which is in competition with the reducing conditions, the former winning in the end...

Hope that sort of helps

R   

[Miscman85]

That does help, thanks!

[John009]

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