CZE of Sulfur-Containing Amino Acids and Peptides and Its Application to the Quantitative Study of Heavy Metal-Caused Thiol Oxidations

Abstract

The redox-active, sulfur-containing amino acids cysteine and methionine, the tripeptide glutathione and their oxidized counterparts cystine, methionine sulfoxide, and glutathione disulfide were separated as anions by capillary zone electrophoresis (CZE) in a 72 cm long fused silica capillary filled with 100 mM phosphate buffer, pH 8.0, at a voltage of ?30 kV in 20 min. The optimized CZE method was suited for the implementation of quantitative metal interaction studies of the biomolecules in a biologically relevant concentration range (lM-mM). Decreasing peak areas of the reduced forms of cysteine and glutathione and simultaneously increasing peak areas of the oxidized forms after incubation of the reduced biomolecules with divalent heavy metal cations indicated redox reactions which could be responsible for toxic metal actions in biological systems. CZE measurements revealed that a 50 % oxidation grade of cysteine was achieved at a molar metal:cysteine ratio of 0.85 in case of Zn(II) addition and of 0.11 in case of Cu(II) addition, respectively. Cu(II) oxidized 50 % of the initial glutathione at a molar Cu:peptide ratio of 0.036, whereas the 50 % oxidation grade was not reached after incubation with Co(II) up to a molar ratio of Co:peptide of 0.25.

Capillary electrophoresis in the analysis of biologically important thiols

“…Schmidt et al. used 100 mM phosphate buffer at pH 6.5 for direct separation of Cys, GSH, and methionine and their oxidized forms. Both thiol forms were monitored upon exposure to different concentrations of metal ions (Cu 2+ , Zn 2+ , and Co 2+ ) and the metal‐induced thiol oxidation could be studied with apparent oxidative power in the following order: Cu > Zn > Co.…”

Recent developments in capillary and microchip electroseparations of peptides (2011–2013)

“…Enhanced sensitivity of peptide detection at ultralow UV region below 200 nm has been utilized in CZE determination of oxidized and reduced forms of GSH, cysteine, and methionine, which were detected at 192 nm , and for detection of dendrigraft poly‐ l ‐lysine at 193 nm .…”

Simultaneous determination of cystine and other free aminothiols in blood plasma using capillary electrophoresis with pH‐mediated stacking

“…Another method involves direct determination of aminothiols in a model mixture using a weakly alkaline BGE (Na phosphate pH 8.0) [15]; however, this method can hardly be applied to the analysis of blood plasma due to low sensitivity even when using a bubble-cell capillary. It is also worth noting here that the use of the acetonitrile-salts-stacking technique, based on the transient pseudo-isotachophoresis mechanism, made it possible, without derivatization, to determine Gln and GlnS at the (sub)micromolar level in plasma when separating analytes in borate or SDS-phosphate-borate buffer (pH 8.0) [16,17].…”