Figure 7. Equilibrium dialysis reveals two distinct forms of copper (II) co-ordination in PrP^91–115.

Scatchard plots of equilibrium dialysis data for PrP^91–115 show copper (II) binding with stoichiometries of 0.5:1 or 2:1, depending on the solution conditions. (A) In 50 mM Mops buffer at pH 7.4, with copper presented as a glycine complex, two sites (intercept=2.11) of equal affinity are observed, each with an apparent dissociation constant of 5.5±0.5 μM. (B and C)The formation of a 0.5:1 complex gives a non-linear Scatchard plot. The data were fitted numerically as described in the text, the results of which are represented as continuous lines. Dialysis against CuSO₄ in 5 mM Tris, pH 7.4 (B), showed formation of a Cu(PrP^91–115)₂ complex at low copper concentrations and a Cu₂PrP^91–115 complex at higher copper concentrations. In 10 mM acetate, pH 5.5, only the Cu(PrP^91–115)₂ complex was observed (C). (D) CD spectra could be observed for both the Cu(PrP^91–115)₂ and the Cu₂(PrP^91–115) complex. The spectra of the Cu(PrP^91–115)₂ complex formed in 5 mM Tris buffer, pH 7.4 (100 μM PrP^91–115, 60 μM CuSO₄, i.e. 0.5:1 CuSO₄ and 10 μM free CuSO₄) and the Cu(PrP^91–115)₂ complex formed in 10 mM acetate, pH 5.5, are indicated (100 μM PrP^91–115, 1 mM CuSO₄). The spectrum of the Cu₂PrP^91–115 complex formed at pH 7.4 in 50 mM Mops is also shown (60 μM PrP^91–115, 200 μM copper bisglycinate).