Free Sulfhydryl detection

Jeffrey Weidner jeffrey_weidner.immunology at merck.com
Mon Mar 18 15:20:14 EST 1996

Michael Daws <Michael.Daws at ucmp.umu.se> wrote:
>I have a 19-mer peptide (2200 MW) with a C-terminal cysteine which I 
>purchased and received as a lyophilised powder.  The company told me 
>that the peptide would be greater than 95% monomer, however the results 
>I have been getting would suggest that the majority of the peptide is 
>disulphide bonded.  Is there any nice easy colorimetric reagent I can 
>use to detect free sulphydryl groups so that I can determine just how 
>much of my peptide is disulphide bonded?  Alternatively does anybody 
>know a recipe for a gel that I could use to detect such a difference? 
>Mike Daws
You need to use bis-Dithionitrobenzoic acid (DTNB) also called Ellman's 
reagent.  It is readily available from Sigma or Aldrich.  This turns 
bright yellow in the presence of free thiols.  You can do the assay in 
pH 7.5 buffer and read A412.  The Assay is quite sensitive and will 
detect nmoles of free SH.

As a peptide chemist, I would like to point out that the lyophilized 
peptide may all be monomer which is oxidizing to the dimer after it has 
been solubilized.  Slightly acidic solutions will stabilize the dimer 
while basic solutions will promote spontaneous oxidation.

Good Luck,

Jeffrey R. Weidner, Ph.D.
Merck Research Laboratories

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