Inactivation of intracellular copper-zinc superoxide dismutase by copper chelating agents without glutathione depletion and methemoglobin formation.
The copper chelator N,N'-diethyldithiocarbamate (DDC), is often used to inactivate intracellular copper-zinc superoxide dismutase in erythrocytes. However, in studies with red cells we found that the compound also reacted with oxyhemoglobin to produce oxygen radicals in addition to generating lipid peroxidation products, oxidized N,N'-diethyldithiocarbamate, methemoglobin, and sulfhemoglobin. Moreover, intracellular glutathione was depleted and vital cellular enzymes were susceptible to inactivation. We, and others, have confirmed these findings in nonerythrocytic cell lines. Thus, cells exposed to DDC are severely damaged before studies on the effects of added putative superoxide producing compounds can be performed with them. In this report, we have systematically investigated other copper chelators for their ability to inactivate intracellular copper-zinc superoxide dismutase without producing the deleterious effects mentioned above. Catechol, triethylenetetramine, and tetraethylenepentamine were found to be such agents when erythrocytes were dialyzed in the cold against dilute solutions of these chelators. In addition, with a myeloid leukemic cell line (HL-60), triethylenetetramine inhibited SOD without causing significant GSH oxidation. Examination of the affinity constants of chelators active against purified copper-zinc superoxide dismutase indicated that an affinity binding constant (log K1) between 12.6 and 13.8 was required for the chelator to successfully remove copper from the enzyme.
Kelner
M J
MJ
Department of Pathology, University of California-San Diego, CA 92103.
Bagnell
R
R
Hale
B
B
Alexander
N M
NM
D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002614:Chelating Agents / Q000494:pharmacology*; D003300:Copper / Q000378:metabolism; D004050:Ditiocarb / Q000494:pharmacology*; D004912:Erythrocytes / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008706:Methemoglobin / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000037:antagonists & inhibitors / Q000097:blood*
D002614:Chelating Agents; D003300:Copper; D008706:Methemoglobin; D004050:Ditiocarb; D013482:Superoxide Dismutase; D005978:Glutathione