A Spectroscopic Study of Nitric‐Oxide‐Treated Ceruloplasmin

An analysis of the absorbance difference (oxidized minus reduced) and circular dichroic (CD) spectrum of ceruloplasmin in Gaussian curves is presented. Above 330 nm, both spectra are composed of the same set of six Gaussian bands, Attributed to typr-1 copper. An Additional band in the absorbance difference spectrum at 320 nm is ascribed to typr-3 copper. The time dependence of thje effect of nitric oxide on the absorbance difference, circular dichroism and electron paramagnetic resonance (EPR) spectrum of ceruloplasmin has been studied. Upon incubation with nitric oxide for 5 min, the CD spectrum of ceruloplasmin shows a slight decrease of the ellipticity around 600 nm, and a marked decrease at 456 nm, indicative of the non-equivalence of the type-1 coppers in ceruloplasmin, Longer incubation (2 h) with nitric oxide leads to a decrease of the CD and absorbance spectrum of the ceruloplasmin-NO compound, due to reduction o0f the type-1 copper. Upon prolonged incubation with NO, the type-2 copper EPR spectrum (93 K) changes and a distinct superhyperfine pattern appears in the g, suggesting an interaction of the type-2 copper with nitrogen atoms from the protein moiety. At 15 K the EPR spectrum of oxidized ceruloplasmin exhibits a small signal at g=4. Upon incubsation with nitric oxide this signal increases in intensity, and shows a well-resolved hyperfine pattern of seven lines. In addintion ot hte type-2 copper signal, a broad signal is observed at g=2. It is proposed that these signals in NO-treated ceruloplasmin stem from magnetically coupled type-3 coppers.