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 K-1 = 1.8 (+/-0.6) X 10(12) M(-1) and K-2 = 1.2 (+/-0.5) X 10(11) M(-1), and those of ovo-transferrin were K-1 = 1.9 (+/-0.5) X 10(11) M(-1) and K-2 = 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 NI KCl or in the absence of anions. The copper dissociation rate constants from the N- and C-sites of dicupric serum-Tf were slightly changed by the addition 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.