Chemiluminescence assays of Cu2Zn2 superoxide dismutase mimicking Cu-complexes

The aqueous decay of K3CrO8 was used to compare the reactivity of Cu2Zn2 superoxide dismutase and two active centre analogues where the first shell atoms around the copper are four unsaturated nitrogens. Unlike the acetate or biuret type Cu(II) chelates these di-Schiff-base complexes had an identical reactivity compared to that of the intact enzyme. Nanomolar concentrations of copper coordinated in these complexes were sufficient to inhibit the K3CrO8 induced chemiluminescence by 50%. Furthermore, a lucigenin amplified chemiluminescence assay based on isolated polymorph nuclear leucocytes in the absence and presence of whole, unseparated blood was developed and successfully employed. CuPu(Im)2 and CuPu(Py)2 equivalent to 0.5 and 0.8 SOD units, only, were required to inhibit the photon emission by 50% in the absence of bovine serum albumin. Even in the presence of 600 microM albumin mimicking the competitive copper chelation in biological fluids Cu-Pu(Py)2 and CuPu(Im)2 remained active, whereas the carboxylate- and biuret type chelates Cu(Sal)2 and Cu(Ser)2 reacted like CuSO4. The same reactivity of these low M, SOD mimics was seen in human blood.