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Preparation of selectively metal free and metal substituted derivatives by reaction of copper zinc super oxide dis mutase with di ethyl di thio carbamate


Biochemical Journal 199(3): 675-680
Preparation of selectively metal free and metal substituted derivatives by reaction of copper zinc super oxide dis mutase with di ethyl di thio carbamate
Incubation of Cu-Zn superoxide dismutase [bovine, horse, pig, yeast] with diethyldithiocarbamate at increasing ligand/protein ratios and subsequent high-speed centrifugation led to proportional removal of Cu from the protein, at variance with previous results. No Zn was lost, even at very high excesses of chelating agent. In this way a Cu-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 Cu-free protein by stoichiometric re-addition of Cu. From the mixture of metal-depleted forms originated by treatment with slight diethyldithiocarbamate excess, the protein containing Cu only on 1 subunit, [Cu1-Zn2], could be isolated by preparative column electrophoresis. This species reproducibly showed 25% more specific activity (catalytic constant per Cu) than that of the native or reconstituted [Cu2-Zn2] protein. This may result from long-rang conformational effects between the active sites. By adding Co2+ ions to the vacant Cu site of [Cu1-Zn2] a hybrid molecule containing Cu(II) on 1 subunit and Co(II) in the homologous site of the other subunit was prepared. Its activity, referred to Cu, was identical with that of the native protein.


Accession: 006173851



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