The ratio of type-1 and -2 Cu(II) in human ceruloplasmin

Abstract

1. 1 Spectroscopic titrations of ceruloplasmin with NADH, in the presence of trace amounts of phenazine methosulphate, demonstrate that at pH 7.0 the protein is capable of accepting only four electron equivalents. Changing the ambient pH to 5.2 or preincubation with neocuproin or azide at pH 7.0 does not alter the number of equivalents that can be accepted. 2. 2 Titration curves at pH 5.2 show a different character from those obtained at pH 7.0 and this difference is due to the species being reduced, namely the Cu(II) atoms. 3. 3 Nernst plots of the tritration data demonstrate an equilibrium between type-1 and type-2 Cu and suggest that ceruloplasmin contains one type-1 Cu and three type-2 Cu atoms. 4. 4 The concave character of the titration curve at pH 5.2 and 25°C is due to a difference in redox potential of 36 mV between type-1 and type-2 Cu, together with the lack of absorbence of type-2 Cu. At pH 7.0 there is no difference in redox potential. 5. 5 Neocuproin and azide both cause a difference in redox potential between the two types at pH 7.0. The data also support the idea that cerulosplasmin contains one type-1 and three type-2 Cu atoms. 6. 6 Increasing concentrations of azide produce an increasing difference in redox potential between the two types of Cu(II). 7. 7 Titrations followed at 330 nm suggest that the changes observed at this wavelength are induced by a conformational changes of the protein.