Ceruloplasmin-Anion Interactions: INDUCED SPECTRAL TRANSITIONS IN THE VISIBLE RANGE

Abstract

Changes in the visible spectrum of human ceruloplasmin resulting from the interaction of the protein-copper chromophore with azide, cyanide, thiocyanate, and cyanate have been examined. Absorption maxima determined for the various protein-anion complexes were the following: N3−, 375 mµ with a shoulder at 435 mµ; CN−, 378 mµ; NCS−, 375 mµ and 435 mµ; NCO−, 395 mµ. The spectral transitions induced by N3−, NCS−, and NCO− produced reductions in the absorption of the protein-copper chromophore of 75, 62, and 65%, respectively. Complex formation in the case of azide and cyanate was reversible, as indicated by a quantitative recovery of both chromophore absorption (610 mµ) and oxidase activity upon removal of the anion. Ceruloplasmin regenerated from the thiocyanate complex exhibited a 10% reduction in these two properties. Protein-bound copper was not removed by either azide, thiocyanate, or cyanate treatment. In the presence of 0.01 M CN− at pH 5.5, ceruloplasmin rapidly lost 1 g atom of copper per 160,000 g of protein. This resulted in the permanent loss of approximately one-third of the absorption at 610 mµ and 54% of the initial oxidase activity. Additional copper was slowly lost upon long term incubation with cyanide. The results of this investigation indicate that predominantly 1 chromophoric cupric copper atom interacts in a reversible manner with N3−, NCS−, and NCO− under the conditions studied. Furthermore, this cupric atom appears to be located on the surface of the molecule exposed to the external environment of the protein and is probably the copper atom readily removed by cyanide.