Re-examination of the reaction of diethyldithiocarbamate with the copper of superoxide dismutase.
The reaction of the copper of (Cu,Zn)-superoxide dismutase with diethyldithiocarbamate was studied at pH = 7.4 and the results obtained led to a reaction scheme basically different from the conclusion of a previous study (Misra, H. P. (1979) J. Biol. Chem. 254, 11623-11628). The analysis of optical and ESR spectra at 9 and 35 GHz, at different ligand/protein ratios and reaction times, showed that a ternary diethyldithiocarbamate. Cu(II).protein complex never formed in spectroscopically detectable amounts. The system is described in any condition as the mixture, in variable proportions, of only two components, that is the diethyldithiocarbamate-free (Cu(II) chelate and the copper-depleted protein. The formation of a catalytically active copper-diethyldithiocarbamate intermediate with distinct optical and ESR spectra was also ruled out by kinetic studies, which demonstrated that enzyme inactivation strictly parallels the binding of diethyldithiocarbamate as monitored by optical absorption and ESR. Separation of the copper complex from the protein was obtained for the first time, and the procedure was suitable for rapid preparation of reconstitutable copper-free superoxide dismutase.
D000818:Animals; D002417:Cattle; D003300:Copper / Q000097:blood*; D004050:Ditiocarb / Q000494:pharmacology*; D004578:Electron Spin Resonance Spectroscopy; D004912:Erythrocytes / Q000201:enzymology*; D007700:Kinetics; D011485:Protein Binding; D013482:Superoxide Dismutase / Q000097:blood*; D013859:Thiocarbamates / Q000494:pharmacology*
D013859:Thiocarbamates; D003300:Copper; D004050:Ditiocarb; D013482:Superoxide Dismutase