Preparation of selectively metal-free and metal-substituted derivatives by reaction of Cu--Zn superoxide dismutase with diethyldithiocarbamate.
Incubation of Cu--Zn superoxide dismutase with diethyldithiocarbamate at increasing ligand/protein ratios and subsequent high-speed centrifugation led to proportional removal of copper from the protein, at variance with previous results [Misra (1979) J. Biol. Chem. 254, 11623--11628]. No zinc was lost, even at very high excesses of chelating agent. In this way a copper-free protein could be readily prepared, with avoidance of the critical pH condition and the dialysis step required in a previous method employing cyanide. The holoprotein was fully reconstituted from the copper-free protein by stoicheiometric re-addition of copper. From the mixture of metal-depleted forms originated by treatment with slight diethyldithiocarbamate excess, the protein containing copper only on one subunit, [Cu1--Zn2], could be isolated by preparative column electrophoresis. This species reproducibly showed 25% more specific activity (catalytic constant per copper) than that of the native or reconstituted [Cu2--Zn2] protein. This may result from long-range conformational effects between the active sites. By adding Co2+ ions to the vacant copper site of [Cu1--Zn2] a hybrid molecule containing Cu(II) on one subunit and Co(II) in the homologous site of the other subunit was prepared. Its activity, referred to copper, was identical with that of the native protein.
D001665:Binding Sites; D003035:Cobalt / Q000378:metabolism; D003300:Copper / Q000378:metabolism*; D004050:Ditiocarb / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D011487:Protein Conformation; D013482:Superoxide Dismutase / Q000378:metabolism*; D013859:Thiocarbamates / Q000494:pharmacology*; D015032:Zinc / Q000378:metabolism
D013859:Thiocarbamates; D003035:Cobalt; D003300:Copper; D004050:Ditiocarb; D013482:Superoxide Dismutase; D015032:Zinc