Na polyphosphate

A solution mimicking cytosol was prepared in 20 mM ammonium acetate (AA) (Sigma-Aldrich) pH 6.5 containing (final concentrations) 2 mM Na(citrate) (Fisher Chemical)^{13 (link)}, 5 mM GSH (Sigma-Aldrich)¹⁴ , 500 μM GSSG (Sigma-Aldrich)^{15 (link)}, 5 mM Na₂ATP (Sigma-Aldrich)^{16 (link)}, 500 μM NaADP (Sigma-Aldrich)^{16 (link)}, 200 μM Na₂AMP (Sigma-Aldrich)^{16 (link)}, 100 μM cysteine (Sigma-Aldrich)^{17 (link)}, 50 mM Na(glutamate) (Sigma-Aldrich)^{13 (link)}, 5 mM aspartate (MP Biomedicals)^{13 (link)}, 70 μM histidine^{13 (link)}, 5 mM Na₂PO₄ (Sigma-Aldrich)¹⁸ , and 3 kDa-filtered Na(polyphosphate) (Sigma-Aldrich)¹⁸ . The composition of the pFTS was designed to mimic the potential Ni-binding ligands in the cytosol and near to cytosolic concentrations. The pFTS had lower salt concentration and lacked species that were considered unlikely ligands to Ni. Individual standard nickel complexes were prepared by mixing NiSO₄ and stock solutions of individual ligands (previously described in ref. 10 (link)) to a final concentration of 2 μM Ni and various desired, final concentrations of the ligand. Standards were prepared fresh on the day of analysis and stored anaerobically at 5 – 10 °C prior to injecting them onto columns.