Free Sulphydryl detection

"Marianne Leverone ", BIO leverone at CHUMA.CAS.USF.EDU
Wed Mar 20 11:53:23 EST 1996

Mike:  I have used DNTB to quantify free sulfhydryl groups after 
acyl-CoAs have been cleaved with hydroxylamine, pH 7.  It should work for 
other sulfhydryl groups:

10 uL 10 mM DTNB
5-50 uL of coumpund being tested

Allow the reaction to react for 30 sec;  add 0.3 ml 0.1 M Potassium 
phosphate, pH 8.  Add water to bring final volume to 1.0 mL.

Record Absorbance at 412 nm.  DTNB Coefficient is 13,700 M-1, cm-1.

Reference: Fong and Schulz.  1981.  Methods in Enzymology.  71:390-398.

                                 Marianne Leverone
                                 Univ. of South Florida
                                 Tampa, FLorida 

On Tue, 19 Mar 1996, Achim Recktenwald wrote:

> Michael Daws 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?
> > Thanks.
> > 
> > Mike Daws
> A gel is easy. Just run a SDS-gel, but run two samples.Treat one sample with 
> reducing SDS-buffer, that means it contains mercaptoethanol or DTT, the other 
> sample with a SDS-buffer without reducing agent.
> If you are correct, the reduced sample should show only one band at 2200 Da, but 
> the non-reduced sample a major band at 4400 Da, and a minor one at 2200 Da.
> You'll probably have to run a gradient gel or a highly crosslinked one. 2200 Da is 
> at the resolution limit for normal gels. If you have access to a Pharmacia 
> Phastsystem use the 10 - 25% gradient gel. This should work, I used it myself in 
> this range.
> Achim

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