Ceruloplasmin–diethyldithiocarbamate (DTC) interaction Evidence of DTC chelation to two non enzymatic Cu(II)

Abstract

Ceruloplasmin (Cp) is a plasma copper oxidase containing several copper ions. Among them five are essential to enzymatic activity and are classified into three types [1]: two cupric ions of type I (I a and I b [2]) or ‘blue’, one cupric ion of type II or ‘non blue’ and two cupric ions of type III which are not EPR detectable. In this communication we report evidence suggesting the presence of two extra cupric ions that can bind chelating agents such as diethyldithiocarbamate (DTC). The addition of DTC to Cp (pH 5.5) yields an absorption at 445 nm in the Cp spectrum. When increasing equivalents of DTC are reacted with the protein no further spectral changes are appreciable at DTC/Cp > 4 and the absorptivity at 445 nm indicates the chelation of two Cu(II) forming two Cu(DTC) 2. The absorption at 610 nm is not modified and the circular dichroism (CD) spectrum exhibits only a slight decrease. On the other hand the band at 445 nm due to the DTC → Cu 2+ charge transfer transition is not optically active indicating that the Cu(DTC) 2 complex is not coordinated to the protein. Addition of an inhibitor, such as N − 3, to a Cp–DTC solution gives rise to the absorption at 380 nm and to the CD spectrum characteristics of N − 3 bound to type II Cu(II). Moreover, the Cp–DTC solution exhibits the same enzymatic activity as Cp free of DTC. From these results we can infer that the five cupric ions necessary to the enzymatic activity of Cp are not modified by the addition of DTC which instead chelates two extra cupric ions hereafter labeled X and Y. Both X and Y Cu(II) can be removed from Cp, by centrifugation, after addition of DTC, if this is performed within ten minutes following the addition of DTC. On the other hand, when Cp is incubated for several hours with DTC a very slight precipitate appears and the solution recovers the blue color of native Cp. If DTC is then added to the supernatant the absorption at 445 nm due to both DTC-bound X and Y reappears. These results suggest that as time elapses the Cu(DTC) 2 complexes dissociate and that X and Y Cu(II) reenter the protein.