Determination of Thiols and Free Thiol Content in a Protein with Liquid Chromatography Coupled to Amperometric Detection at a Silver Based Carbon Paste Electrode

Abstract

Liquid chromatography (LC) coupled to an electrochemical detector was applied for the determination of small thiols such as cysteine (Cys), glutathione (GSH) and dithiothreitol (DDT) and the free thiol content in a protein. The thio-compounds were first derivatized by 2,2-dipyridyldisulfide (DPDS) at pH4.5 to yield the corresponding disulfide and 2-thiopyridone (2-TP). The reaction mixture was separated isocratically on a reversed-phase C-18 column with amperometric detection (AD) at a carbon paste electrode modified with silver microparticles and polarized at 0.080V versus Ag/AgCl. The LC-AD response corresponding to the generated 2-TP allowed for the indirect determination of the studied thiol derivatives in the 1x10(-5)-1x10(-4)M range for Cys and GSH and 5x10(-6)-5x10(-5)M range for DTT. The LOD and LOQ values were in the range of 9x10(-7)-4x10(-6)M and 3x10(-6--)5.5x10(-6)M, respectively for the three studied compounds. Application to the determination of the free thiol content of human and bovine serum albumin as model proteins was realized. The LC-AD results were validated with respect to a developed spectrophotometric method performed under identical experimental derivatization conditions.