Copper Depletion/Repletion of the Catalytic Centre of Ceruloplasmin as Monitored by Light Absorption, Fluorescence, EPR and Atomic Absorption Spectra

Abstract

0.1 mM human ceruloplasmin (CP) was dialyzed against 25 mM KCN in Tris/HCl, pH=7.0, at 4°C for 2,4,6,22 and 28 hrs, then 16 hrs against 10 mM EDTA 12-hr dialysis without EDTA followed. Copper in CP was measured by atomic absorption, its oxidase activity — in reaction with p-phenylene diamine. Light absorption, fluorescence and EPR spectra (77°K) were recorded (see Table 1). 2 hr of KCN treatment reduced CP oxidase activity to ≈0.1, though only about half of copper was lost. Concurrently fluorescence intensity increased, since copper ions are efficient fluorescence quenchers. Type I and II Cu2+ were the most vulnerable to KCN treatment judging by absorption at 610 nm (type I Cu2+) and EPR spectrum (type I and II Cu2+). The most resistant were type III Cu2+. Their absorption (330 nm) was the same after 6-hr dialysis, dropping to ≈0.5 after 28 hr. CP also retained its ability to dismutate O 2 - . Even 28-hr dialysis caused no total loss of copper and enzymatic functions of CP, which were achieved by reduction of Cu2+ in CP to Cu+ by ascorbate prior to dialysis. Re-constitution of the catalytic centre in CP then failed (see Table 1). Copper ions could be inserted into ascorbate non-treated CP. Then Cp regained a large part of its spectral properties and catalytic functions. For reconstitution CP samples (after 2–28 hr of dialysis against KCN) were dialyzed at 21°C in 0.1 M Na-acetate, pH=5.5, with 2.5 mM K2SO3 for 2 hr under pure argon flow. Then Cu(SCH)2 was added to 2xl0-5 M. 20 hr later samples were for 6 hr put into 0.1M K2HPO4/KH2PO4, pH=5.5. Intensive air bubbling followed for 1 hr.