Novel Stabilization of a Metalloprotein-Ternary Complex: Reaction of Copper-Zinc Superoxide Dismutase with  Diethyldithiocarbamate in Polyacrylamide Gels and its Application to the Study of the Reaction of the Dismutase with Hydrogen Peroxide

ABSTRACT

Polyacrylamide gels of bovine liver copper-zinc superoxide dismutase electrophoresed at pH 8.5 in TRIS-glycine develop a stable yellow color at the dismutase bands upon incubation with diethyldithiocarbamate dissolved in the electrophoresis buffer.  The area under scans of the peaks at 448 nm is unchanged after storage of the gels at 4 oC for a least one month and probably longer.  This indicates that copper is not lost from the protein in the gel.  The implication of these observations is that the ternary dithiocarbamate complex is stabilized in the polyacrylamide matrix.¹  The 448 nm absorption corresponds to the absorbance maximum of the copper-dithiocarbamate complex in solution.  The area under the peaks in scans of the gels is a linear function of the amount of dismutase in the peak.  This method is applied to the study of the reaction of the dismutase with hydrogen peroxide, a reaction which causes a loss of copper and an increase in heterogeneity.²  The application of this procedure to other metalloproteins³ will depend upon the stability of the copper-dithiocarbamate complex and the resistance of the copper to removal during the incubation of the gels with the dithiocarbamate.

1.  An alternate explanation is that the copper is lost from the SOD but is retained in the gel in a polymer with the dithiocarbamate.

2.  See also Jewett, SL, Cushing. S, Gillespie, F, Smith D, Sparks S. (1989).  Reaction of Bovine Liver Copper-Zinc Superoxide Dismutase with Hydrogen Peroxide: Evidence for Reaction of H₂O₂ and HO₂- Leading to Loss of Copper. Eur. J. Biochem. 180: 569-575.

3.  For example, we know that copper is lost from galactose oxidase during electrophoresis and that copper in azurin is not accessible to the dithiocarbamate.