Derivatization of Phytochelatins from Silene vulgaris, Induced upon Exposure to Arsenate and Cadmium: Comparison of Derivatization with Ellman's Reagent and Monobromobimane

Abstract

Phytochelatins (PCs) are a family of thiol-rich peptides, with the general structure (γ-Glu-Cys)_n-Gly, with n = 2−11, induced in plants upon exposure to excessive amounts of heavy metals and some metalloids, such as arsenic. Two types of PC analyses are currently used, i.e., acid extraction and separation on HPLC with either precolumn derivatization (pH 8.2) with monobromobimane (mBBr) or postcolumn derivatization (pH 7.8) with Ellman's reagent [5,5‘-dithiobis(2-nitrobenzoic acid), DTNB]. Although both methods were satisfactory for analysis of Cd-induced PCs, formation of (RS)₃−As complexes during extraction of As-induced PCs rendered the DTNB method useless. This paper shows that precolumn derivatization with mBBr, during which the (RS)₃−As complexes are disrupted, provides a qualitative and quantitative analysis of both Cd- and As-induced PCs. In addition, derivatization efficiencies of both methods for the oligomers with n = 2−4 (PC_2-4) are compared. 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 thiol concentration is therefore advised for better quantification of PC concentrations in plant material. Keywords: Silene vulgaris; phytochelatins; heavy metal; arsenic; cadmium; Ellman's reagent; monobromobimane