Copper binding selectivity of N- and C-sites in serum (human)- and ovo-transferrin.

Copper binding selectivity of the N- and C-sites in serum (human)- and ovo-transferrin was investigated through copper binding constants, copper dissociation rate constants, and EPR spectra. At pH 7.4, stepwise copper binding constants of serum (human)-transferrin were K1 = 1.8 (+/- 0.6) x 10(12) M-1 and K2 = 1.2 (+/- 0.5) x 10(11) M-1, and those of ovo-transferrin were K1 = 1.9 (+/- 0.5) x 10(11) M-1 and K2 = 2.1 (+/- 0.4) x 10(11) M-1. Absorbance changes resulting from copper binding to the C- or N-site at various ratios of Cu2+/apo-transferrin were separated by a kinetic method. It was clearly indicated that, in serum (human)-transferrin, the copper binding affinity for the C-site was much larger than that for the N-site, whereas in ovo-transferrin, the C- and N-sites have almost the same affinity for copper ions. In the presence of anions (0.1 M KCl or 0.1 M NaClO4), the stepwise copper binding constants of serum (human)-transferrin were almost 10-times smaller than those in the absence of the anions. The selectivity in binding the copper ions to both sites of serum (human)-transferrin in the presence of 0.1 M NaClO4 is much smaller than that in the presence of 0.1 M KCl or in the absence of the anions (0.1 M KCl and 0.1 M NaClO4). EPR spectra of the copper ions of the N-site in dicupric serum-transferrin are dramatically changed respectively by the addition of 0.1 M KCl, 0.1 M NaCl, and 0.1 M NaClO4. This suggests that the change in the coordination geometry of the copper ions occurs at the N-site.