Procedures for EDTA removal from protein samples and detection of EDTA.
(A) The protein samples containing 1 mM EDTA were subjected to the indicated purification procedures. Left panel: Samples of BSA or vimentin were subjected to spin column gel filtration as detailed in methods. Right panel: protein samples were applied to Millipore Amicon Ultra filter units (10 K pore size) and subjected to two rounds of ultrafiltration, as described in the text. (B) Colorimetric determination of EDTA present in protein samples after diverse purification procedures using the PAR competition assay. Results shown are mean ± SD of 4 (dialysis plus gel filtration), 2 to 7 (ultrafiltration), or 3 to 7 (ultrafiltration plus dialysis) assays. (C) NMR analysis. Upper panel: 600MHz 1D-proton NMR spectrum of an ultrafiltrated sample of vimentin (1.8 μM final concentration). Signals of buffer and additives used in the purification (glycerol, from the ultrafiltration filters, and DTT) are observed. Protein signals appear at baseline noise level and are not recognizable. Middle panel: The same sample analyzed in the upper panel monitored after addition of 150 μM of ZnCl2. A quadruplet that appears at 3.92 ppm corresponds to trifluoroethanol added for referencing. Lower panel: monitorization of the sample after addition of 20 μM EDTA. The signal pattern of the AB system at 3.32 and 3.25 ppm and the singlet at 2.79 ppm corresponding to the Zn2+-EDTA chelate are clearly visible.