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.
Highlights
We suggest the first structural model of Ethylenediaminetetraacetic acid (EDTA) aggregates at the atomistic level and how these can interact with SYPRO Orange
These results demonstrate an interference of EDTA and SYPRO Orange that leads to a temperature-dependent change in fluorescence intensity resembling that observed when a protein unfolds
These results clearly identified the EDTA4- species to be involved in the occurrence of fluorescence in the presence of SYPRO Orange
Summary
Ethylenediaminetetraacetic acid (EDTA) and its parent molecule ethylene glycol tetraacetic acid (EGTA) are widely used in biology, biochemistry, pharmaceutical industry, and food. We failed to reproduce the melting temperature of the protein of ~86 ̊C previously measured by CD spectroscopy [10]; instead, we observed a sharp change in fluorescence intensity at 68 ̊C (Fig 1) We hypothesized that this change results from SYPRO Orange/EDTA interactions rather than from interactions of SYPRO Orange with the unfolded protein. We provide evidence that suggests that at a practically relevant pH > 9 [11, 12], EDTA in its quadruple negatively charged state interacts with SYPRO Orange in a temperature-dependent manner, leading to a fluorescence signal similar to that when proteins unfold, with a “denaturation temperature” of ~68 ̊C under the conditions chosen here. Given the widespread use of EDTA in biology, pharmacy, and food technology, and TSA for investigating the thermal stability of proteins under varying conditions [13], our results make it relevant to ascertain that future TSA results are not influenced by interference between the chelator and the fluorescent dye
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