Use of cyanide and diethyldithiocarbamate in the assay of superoxide dismutases.
Eucaryotes have two major forms of superoxide dismutase (SOD), Cu,ZnSOD and MnSOD; in most tissues Cu,ZnSOD is present in higher amounts than MnSOD. To assay MnSOD, Cu,ZnSOD can be inhibited selectively by millimolar concentrations of cyanide ion. However, calculation of MnSOD activity from the differential cyanide inhibition assay is complex and small experimental errors can cause large errors in the calculated MnSOD activity. We have assessed how interaction of cyanide and hydrogen peroxide with cytochrome c can lead to further errors in the xanthine oxidase-cytochrome c assay for SOD. Alternatively, Cu,ZnSOD can be completely inactivated by 50 mM diethyldithiocarbamate (DDC) at 30 degrees C for 1 h without affecting the activity of MnSOD. Since DDC reduces cytochrome c, the treated samples must be thoroughly dialyzed or desalted before assay. In the case of lung homogenates, dialysis is not an extra step since fresh, untreated samples must also be dialyzed or desalted before assaying by the cytochrome c method. Cu,ZnSOD activity is equal to the activity in the untreated sample minus the activity in the DDC-treated portion of the sample. Another copper chelator, triethylenetetramine, did not inactivate Cu,ZnSOD and could not be used instead of DDC. For accurate measurement of both enzymes in samples where MnSOD contributes only a small fraction of the total SOD activity, the DDC method has the advantage that it provides a direct measure of the MnSOD activity without interference by Cu,ZnSOD.
Iqbal
J
J
Calvin and Flavia Oak Asthma Research and Treatment Facility, University of Miami School of Medicine, FL 33136.
Whitney
P
P
D000818:Animals; D003486:Cyanides / Q000494:pharmacology*; D003574:Cytochrome c Group / Q000378:metabolism; D004050:Ditiocarb / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D008099:Liver / Q000201:enzymology*; D008168:Lung / Q000201:enzymology*; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D013482:Superoxide Dismutase / Q000378:metabolism*; D014266:Trientine / Q000494:pharmacology; D014969:Xanthine Oxidase / Q000378:metabolism
D003486:Cyanides; D003574:Cytochrome c Group; D004050:Ditiocarb; D013482:Superoxide Dismutase; D014969:Xanthine Oxidase; D014266:Trientine