The bicarbonate-dependence of zinc(II)-transferrin binding

Abstract

The binding of zinc(II) to human serum transferrin has been studied as a function of the solution concentration of sodium bicarbonate in 100 mM, pH 7.4 hepes buffer at 25°C. The apparent molar absorptivity of the zinc-transferrin complex has been determined from the initial slopes of titration curves of Δϵ versus the ratio of [Zn] [Tf] . This absorptivity represents the difference between the positive absorbance of the ternary Zn-HCO 3-Tf species in the sample cuvette and the negative absorbance of binary HCO 3-Tf species in the reference cuvette. Higher concentrations of bicarbonate increase the degree of saturation of apo-Tf with bicarbonate and thus increase the apparent absorptivity of the zinc-Tf complex. Titrations of apo- and monoferric transferrins with bicarbonate indicate that there is little, if any, difference in the bicarbonate binding constants of the two specific transferrin binding sites. An equilibrium constant of log K = 2.49 has been used to calculate the degree of saturation of the C-terminal binding site with bicarbonate. The zinc-binding affinity of this site depends linearly on this degree of saturation. The scatter in the zinc-binding constants of the weaker N-terminal site precludes a similar analysis of the bicarbonate-dependence of binding at this site. The results strongly support the previous proposal that binding of the synergistic bicarbonate anion is responsible for the uv absorption observed upon addition of bicarbonate to apoTf.