On-line cleavage of disulfide bonds by soluble and immobilized tris-(2-carboxyethyl)phosphine using sequential injection analysis

Reduction of disulfide bonds is – in many cases – a critical pretreatment step for the determination of thiols in real samples. This study reports the first systematic investigation of the potentials of the on-line reduction of disulfide bonds under flow conditions in a sequential injection setup. One of the most promising reducing agents, tris-(2-carboxyethyl)phosphine (TCEP) was selected for this purpose while the Ellmanʹs disulfide (DTNB) was used as model compound. The study involved the investigation of several parameters that affected the kinetics and efficiency of the reaction, including stopped-flow experiments. Both soluble and immobilized TCEP on agarose beads were examined. The results confirmed that both forms of TCEP can be used as an advantageous on-line reducing reagent for disulfide bonds under flow conditions.