Two Applications Using N,N'-Diethyldithiocarbamate as a Stain for Copper in Native Polyacrylamide Gels of Superoxide Dismutase.

ABSTRACT

N,N'-diethyldithiocarbamate has been shown to be an analytical stain for copper in native polyacrylamide gels of the copper-zinc superoxide dismutase from purified preparations as well as from crude red cell extracts, i.e. lysates from which hemoglogin has been removed (Jewett, S. L. and Rocklin, A. M. (1994). Analytical Biochemistry 217, 236-240).  Applying this methodology, it was found that the relative amounts of copper-containing forms of copper-zinc superoxide dismutase (EC 1.15.1.1.) from bovine red cell extracts did not change significantly with either the age or with hydrogen peroxide treatment of the red cells.  Furthermore, no significant changes were seen in the specific activity of the dismutase in either type of experiment.  These observations for both types of experiments are contrary to what was expected from similar studies  reported in the literature.  However, discrepencies may be accounted for by hemoglobin interference in indirect dismutase assays of the previous work.  In the case of the peroxide treatment of red cells, however, there is an additional factor in that the dismutase is protected from peroxide-mediated changes in copper content and heterogeneity by the hemoglobin present.  This protection was demonstrated in in vitro experiments using only a 24-fold excess of hemoglobin over the dismutase.

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