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.
Hirose
J
J
Department of Food Science and Technology, Faculty of Engineering, Fukuyama University, Japan.
Fujiwara
H
H
Magarifuchi
T
T
Iguti
Y
Y
Iwamoto
H
H
Kominami
S
S
Hiromi
K
K
D000838:Anions / Q000737:chemistry / Q000494:pharmacology; D001665:Binding Sites; D003207:Conalbumin / Q000737:chemistry / Q000378:metabolism*; D003300:Copper / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D007700:Kinetics; D011487:Protein Conformation; D013379:Substrate Specificity; D014168:Transferrin / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*
D000838:Anions; D014168:Transferrin; D003207:Conalbumin; D003300:Copper