Rapid Screening and Estimation of Binding Constants for Interactions of Fe3+ with Two Metalloproteins, Apotransferrin and Transferrin, Using Affinity Mode of Capillary Electrophoresis

The interaction behavior of Fe3+ with transferrin and apotransferrin (iron-free form) was investigated in this study using affinity capillary electrophoresis. Change in the mass and charge of protein upon binding to the metal ion in the capillary tube led to variation in its migration time and was used to measure the noncovalent binding interactions by fast screening method. Acetanilide was used as the electroosmotic flow (EOF) marker to avoid possible errors due to the change in EOF during the experiment. The binding results were calculated from the mobility ratios of protein (Ri) and EOF marker (Rf) using the formula (Ri − Rf)/Rf or ∆R/Rf. For more comprehensive understanding, the kinetics of the interaction was studied and binding constants were calculated. Results showed that the Fe3+ displayed insignificant interaction with both proteins at lower metal ion concentrations (5–25 μmol/mL). However, transferrin exhibited significant interactions with the metal ion at 50 and 100 μmol/mL (ΔR/Rf = 0.0114 and 0.0201, resp.) concentrations and apotransferrin showed strong binding interactions (ΔR/Rf = −0.0254 and 0.0205, resp.) at relatively higher Fe3+ concentrations of 100 and 250 μmol/mL. The binding constants of 18.968 mmol−1 and −13.603 mmol−1 were recorded for Fe3+ interaction with transferrin and apotransferrin, respectively, showing significant interactions. Different binding patterns of Fe3+ with both proteins might be attributed to the fact that the iron-binding sites in transferrin have already been occupied, which was not the case in apotransferrin. The present study may be used as a reference for the investigation of protein-metal ion, drug-protein, drug-metal ion, and enzyme-metal ion interactions and may be helpful to provide preliminary insight into the new metal-based drug development.

Chelator-based parameterization of the 12-6-4LJ molecular mechanics potential for more realistic metal ion-protein interactions

Metal ions are associated with a variety of proteins and play critical roles in a wide range of biochemical processes. There are multiple ways to study and quantify protein-metal ion interactions, including by molecular dynamics simulations. Recently, the Amber molecular mechanics forcefield was modified to include a 12-6-4LJ potential, which allows better description of non-bonded terms through the additional pairwise Cij coefficients. Here, we demonstrate a method of generating Cij parameters that allows parametrization of specific metal ion-ligating groups in order to tune binding energies computed by thermodynamic integration. The new Cij coefficients were tested on a series of chelators: EDTA, NTA, EGTA and the EF1 loop peptides from the proteins lanmodulin and calmodulin. The new parameters show significant improvements in computed binding energies relative to existing force fields and produce coordination numbers and ion-oxygen distances that are in good agreement with experimental values. This parametrization method should be extensible to a range of other systems and could be readily adapted to tune properties other than binding energies.

Versatile Colorimetric Chemosensor Based on Bis(rhodamine) B Hydrazone for Rapid Detection of Multi-Analytes (Fe3+, Bi3+, Cu2+, and Hg2+) in Aqueous Media

A new bis(rhodamine) B hydrazone L, bearing two rhodamine chromophores, has been designed and synthesised in 70% yield. The chemical structure of the new ligand L was characterised using different spectroscopic techniques. The receptor L was utilised for rapid ‘naked eye’ detection of Fe3+, Bi3+, Cu2+, and Hg2+ in THF–H2O (1:1, v/v) by displaying significant colour changes. The mechanism of detection of metal ions was based on the ring-opening of the spirolactam rings of L upon recognition. UV/Vis spectrometry was employed to investigate L–metal ion interactions in THF–H2O (1:1, v/v). The stoichiometries of the complexes were determined using Job’s method, which revealed 1:2L–metal ion stoichiometries in the case of L–(Fe3+/Bi3+/Hg2+) complexes, while a 1:1L–metal ion stoichiometry was recognised for the L–Cu2+ complex. 1H NMR spectroscopy was applied to study the L–metal ion interactions in DMSO-d6 and results are presented.