Preparation of selectively metal-free and metal-substituted derivatives by reaction of Cu-Zn superoxide dismutase with diethyldithiocarbamate

Abstract

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.

Targeting of Nuclear Factor-κB and Proteasome by Dithiocarbamate Complexes with Metals

“…This reaction apparently leads to a cooperative binding of two DDTC to copper ion (complex of two DDTC with Cu(II) is well described in [110]) with consequent removal of the metal from the protein. Copper depleted SOD can be easily and rapidly prepared by this way [111]. DDTC inhibits also endogenous SOD in vivo, this inhibition is not reversed by dialysis, but it is reversed by incubation with …”

Superoxide Dismutase, A Potential Theranostics Against Oxidative Stress Caused by Nanomaterials

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“…The electron transfer of the Cu, Zn-SOD promoted by the SAM of cysteine on an Au electrode shown in Fig. CuESOD) were prepared by removing Cu (II) and Zn (II) from the native Cu, Zn-SOD, respectively, with a method described previously [127]. This was conducted by comparing the electrochemical responses between Cu-free derivative EZnSOD (E ϭ empty), Zn-free derivative CuESOD, and the reconstituted Cu, Zn-SOD from EZnSOD and Cu 2ϩ , as shown in Fig.…”

“…This was conducted by comparing the electrochemical responses between Cu-free derivative EZnSOD (E ϭ empty), Zn-free derivative CuESOD, and the reconstituted Cu, Zn-SOD from EZnSOD and Cu 2ϩ , as shown in Fig. 6.2) and that of Cu-free SOD (EZnSOD) reported by Cocco [127]. The Cu-free derivative SOD (i.e.…”