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Determination of Thiols and Free Thiol Content in a Protein with Liquid Chromatography Coupled to Amperometric Detection at a Silver Based Carbon Paste Electrode
Author(s) -
Aydogmus Zeynep,
Sarakbi Ahmad,
Kauffmann JeanMichel
Publication year - 2016
Publication title -
electroanalysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.574
H-Index - 128
eISSN - 1521-4109
pISSN - 1040-0397
DOI - 10.1002/elan.201600192
Subject(s) - amperometry , chemistry , thiol , derivatization , chromatography , dithiothreitol , glutathione , cysteine , bovine serum albumin , detection limit , high performance liquid chromatography , electrode , electrochemistry , organic chemistry , enzyme
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 pH 4.5 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 0.080 V 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 1×10 −5 –1×10 −4 M range for Cys and GSH and 5×10 −6 –5×10 −5 M range for DTT. The LOD and LOQ values were in the range of 9×10 −7 –4×10 −6 M and 3×10 −6−‐ 5.5×10 −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.