Investigation of deferiprone binding to different essential metal ions using microscale thermophoresis and electrospray ionization mass spectrometry

In this study, the microscale thermophoresis (MST) method was applied to investigate the interaction of deferiprone with Fe3 +, Cu2 +, Zn2 +, Co2 +, Ni2 +, Mn2 +, Mg2 +, and Ca2 +. Experiments were performed on a MST (Monolith NT.115 LabelFree®) system. Pretest scanning indicated good fluorescence intensity of deferiprone allowing the use of label-free MST experiments. Different concentrations of the intended metal ions in the range of 0.048 to 100.0 μM were titrated against 100 μM fixed concentration of deferiprone dissolved in 0.1 M Tris buffer pH 7.4. MST measurements were performed in standard capillaries at 50% excitation power and 20% MST power. The results indicated significant interactions of deferiprone with Fe3 +, Cu2 +, Zn2 +, Co2 +, and Ni2 +. The data fitted to the Hill model with Hill coefficients of 1.8, 1.5, 3.2, 1.6, and 1.5 for Fe3 +, Cu2 +, Zn2 +, Ni2 +, Co2 +, respectively, thus indicating more than 1:1 stoichiometry. EC50 values for the binding of deferiprone to Fe3 +, Cu2 +, Zn2 +, Co2 +, and Ni2 + were calculated to be 20.6 ± 3.34, 38.1 ± 3.39, 39.5 ± 4.90, 51.1 ± 6.86, and 101.1 ± 22.70 μM, respectively. No binding was observed for deferiprone with Mn2 +, Mg2 +, and Ca2 +. Electrospray ionization mass spectrometry was used as a complementary technique under similar conditions; however, Tris buffer was replaced by ammonium acetate buffer to be compatible with the mass system. Electrospray ionization mass spectrometry confirms the results obtained by MST indicating stable molecular ion peaks for the complexes of deferiprone with either Fe3 +, Zn2 +, Cu2 +, Co2 +, Ni2 +, and Mn2 +, but no binding was observed for deferiprone with either Ca2 + and Mg2 + under a gas phase state. MST shows a fast and simple approach to study the binding of deferiprone to different essential metal ions. Moreover, the complexes were stable for investigation by mass spectrometry under a soft ionization technique like used electrospray ionization, which aided to confirm binding stoichiometry.