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@ARTICLE{Kroeger:836076,
      author       = {Kroeger, Tobias and Frieg, Benedikt and Zhang, Tao and
                      Hansen, Finn K. and Marmann, Andreas and Proksch, Peter and
                      Nagel-Steger, Luitgard and Groth, Georg and Smits, Sander H.
                      J. and Gohlke, Holger},
      title        = {{EDTA} aggregates induce {SYPRO} orange-based fluorescence
                      in thermal shift assay},
      journal      = {PLoS one},
      volume       = {12},
      issn         = {1932-6203},
      address      = {Lawrence, Kan.},
      publisher    = {PLoS},
      reportid     = {FZJ-2017-05200},
      pages        = {e0177024},
      year         = {2017},
      abstract     = {Ethylenediaminetetraacetic acid (EDTA) is widely used in
                      the life sciences as chelating ligand of metal ions.
                      However, formation of supramolecular EDTA aggregates at pH >
                      8 has been reported, which may lead to artifactual assay
                      results. When applied as a buffer component at pH ≈ 10 in
                      differential scanning fluorimetry (TSA) using SYPRO Orange
                      as fluorescent dye, we observed a sharp change in
                      fluorescence intensity about 20°C lower than expected for
                      the investigated protein. We hypothesized that this change
                      results from SYPRO Orange/EDTA interactions. TSA experiments
                      in the presence of SYPRO Orange using solutions that contain
                      EDTA-Na+ but no protein were performed. The TSA experiments
                      provide evidence that suggests that at pH > 9, EDTA4-
                      interacts with SYPRO Orange in a temperature-dependent
                      manner, leading to a fluorescence signal yielding a
                      “denaturation temperature” of ~68°C. Titrating Ca2+ to
                      SYPRO Orange and EDTA solutions quenched fluorescence.
                      Ethylene glycol tetraacetic acid (EGTA) behaved similarly to
                      EDTA. Analytical ultracentrifugation corroborated the
                      formation of EDTA aggregates. Molecular dynamics simulations
                      of free diffusion of EDTA-Na+ and SYPRO Orange of in total
                      27 μs suggested the first structural model of EDTA
                      aggregates in which U-shaped EDTA4- arrange in an inverse
                      bilayer-like manner, exposing ethylene moieties to the
                      solvent, with which SYPRO Orange interacts. We conclude that
                      EDTA aggregates induce a SYPRO Orange-based fluorescence in
                      TSA. These results make it relevant to ascertain that future
                      TSA results are not influenced by interference between EDTA,
                      or EDTA-related molecules, and the fluorescent dye.},
      cin          = {ICS-6},
      ddc          = {500},
      cid          = {I:(DE-Juel1)ICS-6-20110106},
      pnm          = {553 - Physical Basis of Diseases (POF3-553)},
      pid          = {G:(DE-HGF)POF3-553},
      typ          = {PUB:(DE-HGF)16},
      UT           = {WOS:000400648500123},
      doi          = {10.1371/journal.pone.0177024},
      url          = {https://juser.fz-juelich.de/record/836076},
}