Different binding thermodynamics of Ni2+, Cu2+, and Zn2+ to bacitracin A1 determined by capillary electrophoresis

Thermodynamics of the binding of Ni2+, Cu2+ and Zn2+ to bacitracin A1 was studied by capillary electrophoresis measuring the peptide effective mobility at different pH in the presence of increasing concentration of the three ligands. The affinity follows the order Ni2+ > Cu2+ > Zn2+, with association constant values of (2.3 ± 0.1)×104, (4.9 ± 0.2)×103, and (1.5 ± 0.1)×103 M−1, respectively. The only model able to rationalize mobility data implies that metal ion binds to the P0 peptide form. Moreover, mobility values indicated a change of bacitracin A1 acidic properties on Ni2+ and Cu2+ binding, with a shift of the pKa of N‐terminal Ile‐1 from 7.6 to about 5 and of the pKa of the δ‐amino group of D‐Orn‐7 from 9.7 to about 7. Even though on Zn2+ binding a shift of the N‐terminal Ile‐1 pKa was observed, restrictions in the pH range suitable for investigation, due to precipitation phenomena, did not allow establish if the shift of D‐Orn‐7 lateral chain pKa also occurred. Nonetheless, if present, the shift should be limited to the 7.8–9.7 range. Mobility data indicated that the Stokes radius of the complexes is ca. 3 Å lower than that of the free peptide. The present results indicate that metal‐ion binding to bacitracin A1 is more complex than previously assumed.