Fig 1.
Proton NMR spectra of EDTA and its Ca2+-complexed forms at different pH.
EDTA (3 mM) and CaCl2 (1.5 mM) were mixed in deuterated Tris buffer to yield equimolecular amounts of free and Ca2+-complexed EDTA. Proton NMR spectra of the mixture were acquired at different pH: (A) pH 8.2, (B) pH 7.0, (C) pH 5.9. The signals corresponding to protons of the free and Ca2+-complexed forms of EDTA are indicated. The NMR spectrum of EDTA alone is identical to that of free EDTA presented. The moieties responsible for each of the components of the signals are depicted in bold. The signal corresponding to Tris buffer is labeled with *.
Fig 2.
Detection and quantitation of EDTA in protein samples.
(A) NMR analysis. Upper panel: 500MHz 1D-proton NMR spectrum of a commercial sample of vimentin (0.7 μM) with added 150 μM ZnCl2. Signals for the protons of sucrose, a stabilizer present in the sample as indicated in specifications, and those of PIPES buffer are easily identified. An additional singlet at 2.79 ppm and two coupled doublets at 3.25 and 3.32 ppm (2JHH = 17.3 Hz) are also observed. Middle panel: reference proton spectrum of EDTA in presence of Zn2+, the two coupled doublets corresponding to the AB spin system of the methylene protons of the four acetyl groups, non-equivalent due to the structure of the metal chelate, and the singlet corresponding to the four equivalent protons of the ethylenediamine moiety are apparent. Lower panel: reference proton spectrum of EDTA at pH 7.2, only the two characteristic singlets of uncomplexed EDTA appear. (B) Detection of EDTA by colorimetric analysis. Upper panel: Calibration curve showing the dependence on EDTA concentration of the absorbance at 492 nm of mixtures containing 100 μM PAR and 10 μM ZnCl2. Lower panel: Amount of EDTA remaining in samples from vimentin and BSA subjected to extensive dialysis as determined from the absorbance at 492 nm after incubation with PAR and ZnCl2, using the calibration curve. Initial EDTA concentration in the samples was 1 mM. Data shown are mean ± SD of 4 assays.
Fig 3.
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.