Metal ion binding to human hemopexin

Binding of divalent metal ions to human hemopexin (Hx) purified by a new protocol has been characterized by metal ion affinity chromatography and potentiometric titration in the presence and absence of bound protoheme IX. ApoHx was retained by variously charged metal affinity chelate resins in the following order: Ni2+ > Cu2+ > Co2+ > Zn2+ > Mn2+. The Hx-heme complex exhibited similar behavior except the order of retention of the complex on Zn2+- and Co2+-charged columns was reversed. Onedimensional H-1 NMR of apoHx in the presence of Ni2+ implicates at least two His residues and possibly an Asp, Glu, or Met residue in Ni2+ binding. Potentiometric titrations establish that apoHx possesses more than two metal ion binding sites and that the capacity and/or affinity for metal ion binding is diminished when heme binds. For most metal ions that have been studied, potentiometric data did not fit to binding isotherms that assume one or two independent binding sites. For Mn2+, however, these data were consistent with a high-affinity site [K-A = (15 +/- 13) x 10(6) M-1] and a low-affinity site (K-A less than or equal to 2 x 10(3) M-1). Binding of CU2+ and Zn2+ to the Hx-heme complex produced significant changes in the SoretCD spectrum of the Hx-heme complex that were reversed with addition of EDTA. Possibly, these metal ions bind near the heme binding site and perturb the electronic environment of the heme, or their binding induces exchange of one axial His ligand to the heme iron with another adjacent His residue. A possible role for Hx in the maintenance of metal ion homeostasis is discussed.