Stefan Seyferth1, Geoffrey Lee. 1. Department of Pharmaceutics, Friedrich-Alexander-University, Cauerstr. 4, 91058 Erlangen, Germany.
Abstract
PURPOSE: The purpose of this work was to determine the structure of an insoluble precipitate formed when mixing approximately equimolar amounts of ethylenediamine tetraacetic acid (EDTA) and salmon calcitonin (sCT). METHODS: The interaction between EDTA and sCT was examined by measuring solution turbidity kinetics as a function of pH, ionic strength, and addition of ferric ions. Fourier-transformation infrared spectroscopy (FT-IR) identified changes in peptide secondary structure in presence of EDTA. Scanning and transmission electron spectroscopy revealed the macromolecular structure of the sCT/EDTA precipitate. RESULTS: Aggregation of sCT in a time frame up to 1200 min cannot be induced by either pH (range 3.0-7.0) or ionic strength (up to 200 mM) alone, but is a noncovalent interaction between sCT and EDTA. In the pH range 5.0-7.0, a molar binding stoichiometry of sCT/EDTA in the precipitate of 1-3 was determined. We suggest coulombic binding of the free acidic groups of the EDTA to the side chains of the basic amino acids present in the sCT primary sequence. This results in bridging aggregation of the sCT molecules and their precipitation in aqueous solution. The aggregation reaction was blocked by the addition of ferric ions, which bind preferentially to the acidic groups of the EDTA. The sCT/EDTA precipitate redissolves in water in a pH-dependent manner. FT-IR measurements showed a progressive loss of the random coil structure of sCT in water in the presence of EDTA and a simultaneous strong increase in beta-structure. Scanning electron microscopy revealed a fine, sponge-like morphology of the isolated, solid sCT/EDTA precipitate. Transmission electron microscopy delivered final proof of the existence of extensive fibrillation in the sCT/EDTA mixture. CONCLUSIONS: EDTA induces rapid fibrillation of sCT in water and the partially reversible formation of a noncovalent, insoluble sCT/EDTA precipitate.
PURPOSE: The purpose of this work was to determine the structure of an insoluble precipitate formed when mixing approximately equimolar amounts of ethylenediamine tetraacetic acid (EDTA) and salmon calcitonin (sCT). METHODS: The interaction between EDTA and sCT was examined by measuring solution turbidity kinetics as a function of pH, ionic strength, and addition of ferric ions. Fourier-transformation infrared spectroscopy (FT-IR) identified changes in peptide secondary structure in presence of EDTA. Scanning and transmission electron spectroscopy revealed the macromolecular structure of the sCT/EDTA precipitate. RESULTS: Aggregation of sCT in a time frame up to 1200 min cannot be induced by either pH (range 3.0-7.0) or ionic strength (up to 200 mM) alone, but is a noncovalent interaction between sCT and EDTA. In the pH range 5.0-7.0, a molar binding stoichiometry of sCT/EDTA in the precipitate of 1-3 was determined. We suggest coulombic binding of the free acidic groups of the EDTA to the side chains of the basic amino acids present in the sCT primary sequence. This results in bridging aggregation of the sCT molecules and their precipitation in aqueous solution. The aggregation reaction was blocked by the addition of ferric ions, which bind preferentially to the acidic groups of the EDTA. The sCT/EDTA precipitate redissolves in water in a pH-dependent manner. FT-IR measurements showed a progressive loss of the random coil structure of sCT in water in the presence of EDTA and a simultaneous strong increase in beta-structure. Scanning electron microscopy revealed a fine, sponge-like morphology of the isolated, solid sCT/EDTA precipitate. Transmission electron microscopy delivered final proof of the existence of extensive fibrillation in the sCT/EDTA mixture. CONCLUSIONS:EDTA induces rapid fibrillation of sCT in water and the partially reversible formation of a noncovalent, insoluble sCT/EDTA precipitate.