The interaction of zinc, nickel and cadmium with serum albumin and histidine-rich glycoprotein assessed by equilibrium dialysis and immunoadsorbent chromatography

Abstract

Human serum albumin (HSA) has been shown to bind 2–3 mol of Zn 2+, Ni 2+, or Cd 2+ per mole of protein with apparent dissociation constants ( K d ) in the range of 10 μ m. Rabbit histidine-rich glycoprotein (HRG) binds 13, 9, and 6 mol of Zn 2+, Ni 2+, and Cd 2+ per mole of protein, respectively, with apparent K d s also near 10 μ m. However, the binding of metals by HRG exhibits positive cooperativity, so that the apparent K d s may underestimate HRGs true affinity for metal ions. The relative affinities of HSA and HRG for metal ions were found to be Zn 2+ > Ni 2+ > Cd 2+. In addition, histidine (a serum metal chelator) affected the binding of Ni 2+ by both proteins but not that of Zn 2+ or Cd 2+. At physiological concentrations of HSA (250 μ m), HRG (2.5 μ m), and histidine (100 μ m), HRG bound 36% of the Zn 2+, 9% of the Ni 2+, and 13% of the Cd 2+ at a total metal concentration of 25 μ m. Under the same conditions HSA held 37% of the Zn 2+, 14% of the Ni 2+, and 56% of the Cd 2+. Thus, HSA appears to have a lower intrinsic affinity for the three metals than HRG but would be expected to bind a higher proportion of these metals in serum. A specific immunoadsorbent column was prepared and used to study the metal binding by HRG in serum directly. Both 65Zn 2+ and 63Ni 2+ were associated with HRG in aliquots of rabbit serum after incubation with the corresponding metal ion. This evidence indicates that HRG must be considered as a metal binding component of serum.