Indirect Detection of Disulfides

Disulfides can be reduced chemically, electrochemically or enzymatically. Chemical reduction is often used prior to derivatisation of thiols to fluorophores, but it can also be used with HPLC-ED. Common reducing agents are mercapto-ethanol, DTT, dithioerythritol, cyanide and sodium borohydride, but their efficiency varies towards different disulfides. Cyanide or borohydride rapidly reduce cystine at room temperature, but require over 24 h to reduce cysteine-D-penicillamine disulfide completely, whilst the sterically hindered D-penicillamine disulfide is not reduced at all.  

A yield of 89% for D-penicillamine from its homodisulfide using a 10-fold excess of DTT at 60 °C (4 h) has been claimed. Comparative studies with cysteine-D-penicillamine disulfide or protein-bound D-penicillamine were not reported. Kelly et al. reduced cysteamine disulfides and protein-cysteamine with DTT prior to HPLC-ED. However, excess DTT can produce analytical problems since both DTT and its oxidised form are readily separated by RPLC and can be detected electrochemically. 

---