Binding of Cu (II), Tb (III) and Fe (III) to chicken ovotransferrin

Abstract

The kinetics of binding of Cu (II), Tb (III) and Fe(III) to ovotransferrin have been investigated using the stopped-flow technique. Rate constants for the second-order reaction, k ₊, were determined by monitoring the absorbance change upon formation of the metal-transferrin complex in time range of milliseconds to seconds. The N and C sites appeared to bind a particular metal ion with the same rate; thus, average formation rate constants k ₊ (average) were 2.4 × 10⁴ M⁻¹ s⁻¹ and 8.3 × 10⁴ M⁻¹ S ⁻¹ for Cu (II) and Tb (III) respectively. Site preference (N site for Cu (II) and C site for Tb (III)) is then mainly due to the difference in dissociation rate constant for the metals. Fe (III) binding from Fe-nitrilotriacetate complex to apo-ovotransferrin was found to be more rapid, giving an average formation rate constant k ₊ (average) of 5 × 10⁵ M⁻¹ s⁻¹, which was followed by a slow increase in absorbance at 465 nm. This slow process has an apparent rate constant in the range 3 s⁻¹ to 0.5 s⁻¹, depending upon the degree of Fe (III) saturation. The variation in the rate of the second phase is thought to reflect the difference in the rate of a conformational change for monoferric and diferric ovotransferrins. Monoferric ovotransferrin changes its conformation more rapidly (3.4s⁻¹) than diferric ovotransferrin (0.52 s⁻¹). A further absorbance decrease was observed over a period of several minutes; this could be assigned to release of NTA from the complex, as suggested by Honda et al. (1980).