Abstract
Stopped-flow and temperature-jump methods were used to determine the complexation constants for the reactions of polymyxin B with Ni2+ at 25° and 0.1 M ionic strength [N(CH3)4]. At pH 6.4-7.1 and with the ligand in excess, the formation of a 1:1 metal-ligand complex according to the reaction scheme Ni2+ + H3L3+ ⇄ NiH3L5 and Ni2+ + H4L4+ ⇄ NiH3 + H+ (k1 and k'1 are respective forward rate constants; k−1 and k'−1 are respective reverse rate constants) is consistent with the data. The determined rate constants are k1 = (3.8 ± 0.2) × 103 M−1 sec−1 and k−1 = (1.2 ± 0.1) × 10−1 sec−1. Upper and lower limits for k'1 of 2.4 and 0.6 M−1 sec−1, respectively, were set. The stability constant (K1 = k1/k−1) of the 1:1 complex determined by potentiometric titration is (3.3 ± 0.1) × 104 M; the four pKa values of polymyxin B agree with the literature values. At pH 7.1-7.9 and with the metal ion in excess, the described scheme and a binuclear complex reaction step, Ni2+ + NiHL3+ ⇄ Ni2HL5+ (k2 is the forward rate constant; k−2 is the reverse rate constant), fit the data. Upper and lower limits for k2 of 1 × 104 and 3 × 103 M−1 sec−1, respectively, were set for the formation of the binuclear complex. Comparison with model system complex formation rate constants indicates that the forward rate constants evaluated in this study are enhanced. This effect may be interpreted in terms of an internal conjugate base mechanism.