Speciation of heavy metal binding non-protein thiols in Agropyron elongatum by size-exclusion HPLC–ICP-MS

Abstract

An analytical approach based on the coupling of size-exclusion HPLC with ICP-MS was used for studying the speciation of heavy metal binding non-protein thiols (NPTs) in extracts of Agropyron elongatum. A non-oxidizing (N 2) preparation environment and a −85 °C preservation condition were employed to avoid the oxidation of sulfhydryl groups and corresponding decomposition of the heavy metal binding NPTs. The accumulated free heavy metal ions on the column were cleaned by a 10 mmol/l 2-mercaptoethanol eluate so as to avoid these free heavy metal ions in exchanging with the metals from NPT complexes. When these precautions were taken, the on-line size-exclusion HPLC–ICP-MS method allowed very low levels of procedural blanks for Cd, Cu and Zn, and the limits of detection were 0.4, 2.1 and 2.3 μg/l for Cd, Cu and Zn, respectively. Then, the method was applied to the study of heavy metal binding NPTs in roots of Agropyron elongatum. Results showed that the nutrient heavy metals (Cu and Zn) were bound to GSH and Cysteine both under Cd stress and in the control. Under Cd stress, phytochelatins (PCs) were induced, but, these PCs induced by Cd were not only bound to Cd, but also to Cu. Interestingly, neither GSH–Cd nor Cysteine–Cd, which was presumed to be the precursors of the PC–Cd, was detected in the extracts. This leads to the question about GSH–Cd or Cysteine–Cd as precursors of PCs, which warrants further investigation.