? Intramolecular Disulfide Bond Determination ?

Newswatcher Newswatcher at sickkids.on.ca
Thu Jan 28 17:31:11 EST 1999


> Dear All:
> We are going to determined the present of intramolecular disulfide bond
> in our protein of interest.  We have run the SDS-PAGE of  the reduced 
> and non
> reduced samples.  The protein band of the non- reduced sample
> was shown to be of lower than the band of the reduced one.
> According to the amino acid sequence deduced from the known DNA
> sequence, there are only two cys  in the molecule.
> Is this evidence is enough to say that there is a disulfide bond in the
> molecule formed by those two cys?  If not, what should I do to confirm
> the present of this disulfide bond?
> Thanks you very much for any suggestions and comments.

   The way that you can confirm this really depends on the amount of
protein that you have available.   If you have a fair amount and can
quanitate it, you can use one of the classic cysteine specific reagent
like Ellman's reagent to react with the free cysteines produced after
reduction.   You can react your protein with the reagent before reduction
and after reduction.   From the data you can quantitate the number of free
cysteines present in the molecule before and after.    If you see 2 free
after and 0 free before (+/- experimental error) that would certainly be
suggested of those cysteines being involved in a disulphide bond.    If
you find Ellman's original paper (1960's), the method is fairly well
established there.   It is also a fairly simple colorimetic assay followed
on a spectrophotometer.   There are slight updates to the method, but the
old method works fairly well.   

   You could also try doing a literature search on Medline or something
like that and see if anybody has used the method more recently and see
what they have done. ;-)

Best of Luck,


Scott Bukovac
bukovac at sickkids.on.ca

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