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

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 (μM–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.