N,N'-diethyldithiocarbamate as a stain for copper-zinc superoxide dismutase in polyacrylamide gels of red cell extracts.
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.
Jewett
S L
SL
Department of Chemistry, California State University, Northridge 91330.
Rocklin
A M
AM
D000180:Acrylic Resins; D000818:Animals; D002417:Cattle; D003300:Copper / Q000032:analysis / Q000737:chemistry; D004050:Ditiocarb; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004912:Erythrocytes / Q000201:enzymology*; D006454:Hemoglobins / Q000032:analysis; D013194:Staining and Labeling / Q000379:methods; D013482:Superoxide Dismutase / Q000097:blood*
D000180:Acrylic Resins; D006454:Hemoglobins; C016680:polyacrylamide gels; D003300:Copper; D004050:Ditiocarb; D013482:Superoxide Dismutase