Derivatization of phytochelatins from Silene vulgaris, induced upon exposure to arsenate and cadmium: Comparison of derivatization with Ellman's reagent and monobromobimane

Phytochelatins (PCs) are a family of thiol-rich peptides, with the general structure (gamma-Glu-Cys)(n)-Gly, with n = 2-11, induced in plants upon exp osure to excessive amounts of heavy metals and some metalloids, such as ars enic. Two types of PC analyses are currently used, i.e., acid extraction an d separation on HPLC with either precolumn derivatization (pH 8.2) with nro nobromobimane (mBBr) or postcolumn derivatization (pH 7.8) with Ellman's re agent [5,5'-dithiobis(2-nitrobenzoic acid), DTNB]. Although both methods we re satisfactory for analysis of Cd-induced PCs, formation of (RS)(3)-As com plexes during extraction of As-induced PCs rendered the DTNB method useless . This paper shows that precolumn derivatization with mBBr, during which th e (RS)(3)-As complexes are disrupted, provides a qualitative and quantitati ve analysis of both Cd- and As-induced PCs. In addition, derivatization eff iciencies of both methods for the oligomers with n = 2-11 (PC2-4) are compa red. Derivatization efficiency decreased from 71.8% and 81.4% for mBBr and DTNB derivatization, respectively, for PC2 to 27.4% and 50.2% for PC4. This decrease is most likely due to steric hindrance. Correction of measured th iol concentration is therefore advised for better quantification of PC conc entrations in plant material.