Agilent 7500cs icp ms

Microwave digestion of the C. violifolia leaf and root samples from China and C. pratensis biomass was carried out in a CEM Mars-5 digestion system (CEM; Matthews, NC, USA). 50 mg of the samples were mixed with 5.0 ml HNO₃ in PTFE digestion tubes and after 24 h 3.0 ml H₂O₂ was added prior to the microwave digestion process. The pressure was raised to 250 psi over 20 min and held for 15 min. Total Se concentration was determined with an Agilent 7500cs ICP-MS (Agilent Technologies, Santa Clara, CA, USA) on the ⁷⁷Se and ⁸²Se isotopes by the method of standard addition using rhodium (¹⁰³Rh) as an internal standard. The limit of quantification for Se was ~0.05 mg/kg DW in plant samples.

Protein concentrations were determined by the Bradford assay (Bio-Rad) or via the calculated extinction coefficient at 280 nm (10,470 L/mole-cm). Metal ions were removed by dialysis against a buffer containing 50 mM EDTA, 50 mM HEPES pH 8.0, 100 mM NaCl, 1 mM DTT and reconstituted using the same buffer minus the EDTA and including 10 mM Zn. The presence of transition metal ions was determined by ICP-MS using an Agilent 7500cs ICP-MS with argon as the carrier gas and commercially available multi-element standards (Fluka or High Purity Standards). Metal binding was also evaluated via fluorescence quenching experiments using tyrosine fluorescence excitation at 275 nm and emission at 305 nm on a SPEX fluorimeter. Metal binding stoichiometry was determined by plots of relative fluorescence intensity versus metal/Fur ratio. Binding constants were determined by fits of relative fluorescence intensity versus logarithm of metal concentration data to a three parameter sigmoidal dose–response curve as implemented in Sigma Plot 12.5.