Evaluation of the arsenic binding capacity of plant proteins under conditions of protein extraction for gel electrophoretic analysis.
As prerequisite for the investigation of arsenic-binding proteins in plants, the general influence of different extraction parameters on the binding behaviour of arsenic to the plant protein pool was investigated. The concentration of the extraction buffer affected the extraction yield both for proteins and for arsenic revealing an optimal buffer concentration of 5mM Tris/HCl, pH 8. The addition of 1 or 2% (w/v) SDS to the extraction buffer produced a two- to threefold enhancement of the total protein extraction yield but strongly suppressed the simultaneous extraction of arsenic from 80+/-8% extraction yield obtained without SDS to 48+/-2% in presence of 2% (w/v) SDS. The arsenic binding capacity of the protein fraction obtained after extraction with Tris buffer and protein precipitation by trichloroacetic acid in acetone was estimated to be 1.4+/-0.6% independently on the original spiking concentration of arsenic provided in the form of monomethylarsonate to the extracts. Due to the low total protein concentrations of the plant extracts that varied in the range from 75 to 412 microgmL(-1) depending on the extraction parameters, high arsenic concentrations of 263-1001 mg (kgproteinmass)(-1) resulted for spiking concentrations of 10 mgAsL(-1). The optimized protein isolation procedure was applied to plants grown under arsenic exposure and revealed a similar arsenic binding capacity as for the spiked protein extracts.
Schmidt
Anne-Christine
AC
TU Bergakademie Freiberg, Faculty of Chemistry and Physics, Institute of Analytical Chemistry, Leipziger Str. 29, D-09596 Freiberg, Germany.
Steier
Sandra
S
Otto
Matthias
M
D001151:Arsenic / Q000032:analysis* / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D010940:Plant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding
D010940:Plant Proteins; D001151:Arsenic