.while i think that DTT affect the rate of enzyme reaction. so maybe DTT can not solve our problem
you can use elman's reagent and follow the change of absorbance
thank you so much for your reply but i want to confirm thlol-disulfide exchange between a disulfide bond and a thiol group of a cystein residue in protein to find the reason of protein misfolding
You can react the protein with a fluorescent cysteine-modifying reagent such as fluorescein maleimide, digest the protein completely with trypsin, and separate the peptides with HPLC, using a fluorescence detector to map the elution times of the labeled peptides. Thiol-disulfide exchange would cause a change in the peptide map, since different peptides would be labeled before and after. You could also do peptide mapping by LC-MS, which would even allow you to identify the peptides involved.
Dear Dr Fanaei,
If case the SS of your interest is related to the protein folding, the reply by Dr Shapiro covers ultimately.
Some SS is/are buried in the properly folded protein and the other exposed out of the protein. Most SH reagents such as Elman's only react to the exposed SH and do not reach the buried SH. Taking advantage of this property, you can find probable change in protein folding by the distinctive number of reactive SS.
In case, as you mentioned, DTT affects the rate of the enzyme reaction, DTT might have converted SS to SH which is closely related to the enzyme reaction, this/these SH is/are exposed out of the protein. DTT does not change protein folding and does not react to the buried SS.
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