Derivatization of phytochelatins from Silene vulgaris, induced upon exposure to arsenate and cadmium: Comparison of derivatization with Ellman's reagent and monobromobimane
Fec. Sneller et al., Derivatization of phytochelatins from Silene vulgaris, induced upon exposure to arsenate and cadmium: Comparison of derivatization with Ellman's reagent and monobromobimane, J AGR FOOD, 48(9), 2000, pp. 4014-4019
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.