Biosynthesis of Cd-bound phytochelatins by Phaeodactylum tricornutum and their speciation by size-exclusion chromatography and ion-pair chromatography coupled to ICP–MS

Abstract

Cd-bound phytochelatins (Cd-PCs) have been synthesised by incubation of Phaeodactylum tricornutum cell cultures with Cd and purified by size-exclusion chromatography-UV-Vis. These complexes, which were identified in previous work, have now been used as model substances to develop and optimise ion-pair chromatography (IPC) coupled to inductively coupled plasma-mass spectrometry (ICP-MS) for analysis of Cd-PCs. Subsequent analysis of samples taken from Silene vulgaris plants cultivated under heavy metal stress conditions revealed Cd signals but no Cd-PC signals. By use of isotopically enriched (116)Cd-PCs the sample preparation steps were verified to determine the stability of the analytes. We observed species transformation between Cd-PCs and other unidentified Cd complexes. Consequently, the kinetic and thermodynamic lability of Cd-PCs are decisive factors in their detection.

Study of phytochelatins and other related thiols as complexing biomolecules of As and Cd in wild type and genetically modified Brassica juncea plants

“…10,20 Apparently, Cd-PC complexes are believed to be labilized to a great extent in the vacuoles 8 since they are very unstable under those acidic conditions (contrary to the As complexes) and this fact could explain why only minor traces of this compound can be detected. 15…”

“…In this regard, the use of mass spectrometric techniques can be extremely useful for detecting and characterizing the chemical structures of these complexes in plant tissues. In fact, inductively coupled plasma mass spectrometry (ICP-MS) has proved to be an extraordinary tool for selective detection of As 12,13 and Cd 14,15 containing species, previously separated in a chromatographic system, in plant tissues. Complementary to element selective detection by ICP-MS, electrospray ionization mass spectrometry (ESI-MS) has become the technique of choice for elucidating the structure of several of these organometallic/metalloid complexes.…”

Binding of Cd2+ and Zn2+ with the Phytochelatin (γ‐Glu‐Cys)4‐Gly: A Voltammetric Study Assisted by Multivariate Curve Resolution and Electrospray Ionization Mass Spectrometry

“…Knowledge of PC n complexation becomes very important because of the extended use of plants and other organisms for bioremediation of heavy-metal contaminated soils and waters [1]. A variety of techniques have been used for the analysis and identification of the different metal-PC n complexes: UV-vis spectroscopy [2], size-exclusion chromatography [3], Electrospray Ionization Mass Spectrometry (ESI-MS) [4 -7] and electroanalytical techniques [8 -10].…”

An HPLC-ICP-MS technique for determination of cadmium–phytochelatins in genetically modified Arabidopsis thaliana

“…The cadmium-containing fractions are therefore collected and the phytochelatins are further separated by a reversed-phase highperformance liquid chromatography (RP-HPLC) with pre-column or post-column derivatization using UV-vis or fluorescence detection. Loreti et al recently showed this second separation coupled to ICP-MS [25]. The separation conditions used in the RP-HPLC, as well as the derivatization protocol used in these techniques, require an acidification step wherein the cadmium-phytochelatin complexes are dissociated and are rather used to characterize the apo-phytochelatins.…”