Jewett SL, Rocklin,
AM (1994). Anal. Biochem. 217: 236-240.
N,N'-Diethyldithiocarbamate as a Stain for Copper-Zinc
Superoxide Dismutase in Polyacrylamide Gels of Red Cell Extracts.
ABSTRACT
N,N'-diethyldithiocarbamate reacts with copper in
the copper-zinc superoxide dismutase (EC 1.15.1.1.) in polyacrylamide gels
to form stable yellow-brown bands that are quantifiable at 448 nm.
This method of examining superoxide dismutase has been applied to crude
extracts of the enzyme obtained from red cell lysates from which hemoglobin
has been removed by chloroform-ethanol precipitation. This treatment
did not affect the activity and heterogeneity of purified dismutase added
to lysates and recovered by the same method. The bands that develop
in the dithiocarbamate stained gels of the extracts correspond exactly
to the bands of dismutase activity obtained with a positive activity stain
using dianisidine, indicating that the dismutase is the only copper protein
that gives rise to these bands. The amount of superoxide dismutase
in the bands, determined by comparing the areas under unknown peaks to
areas obtained with a standard dismutase sample, agrees with the amount
predicted from indirect superoxide dismutase activity measurements.
Any color in gels due to trace hemoglobin or hemoglobin degradation products
is bleached overnight during staining with the dithiocarbamate.
For application of this work see Jewett, SL, Rocklin, AM (1996).
Anal. Biochem. 237:
65-69.
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