Thiocyanate is the major substrate for eosinophil peroxidase in physiologic fluids. Implications for cytotoxicity.
The potent cytotoxic capacity of eosinophils for parasites and host tissue has in part been attributed to the catalytic action of eosinophil peroxidase (EPO), which preferentially oxidizes Br- to the powerful bleaching oxidant HOBr in buffers that mimic serum halide composition (100 mM Cl-, 20-100 microM Br-, less than 1 microM I-). However, serum also contains 20-120 microM SCN-, a pseudohalide whose peroxidative product, HOSCN, is a weak, primarily sulfhydryl-reactive oxidant. Because of its relative abundance and high oxidation potential, we hypothesized that SCN-, not Br- or I-, is the major substrate for EPO in physiologic fluids. We find that in Earle's buffer (100 mM Cl-) supplemented with 100 microM Br- and varying concentrations of SCN-, HOBr production by activated eosinophils and purified EPO, assayed by conversion of fluorescein to dibromofluorescein, was 50% inhibited (ID50) by only 1 microM SCN-. SCN- also blocked (ID50 10 microM) EPO oxidation of I- to HOI, assayed as iodofluorescein, despite the presence of 100 microM (i.e. grossly supraphysiologic) I-. Thionitrobenzoic acid oxidation kinetics indicate that SCN- is the initial species oxidized by EPO in equimolar mixtures of SCN- and Br- and in human serum. EPO also catalyzed the covalent incorporation of [14C]SCN- into proteins in buffers regardless of Br- concentration and in human serum. Comparing the cytotoxicity of HOSCN and HOBr for host cells, we find that even subphysiologic concentrations of SCN- (3.3-10 microM) nearly completely abrogate the potent Br(-)-dependent toxicity of EPO for 51Cr-labeled aortic endothelial cells and isolated working rat hearts, recently developed models of eosinophilic endocarditis. Thus, HOSCN, hitherto best known as a bacteriostatic agent in saliva and milk, is likely also the major oxidant produced by EPO in physiologic fluids, and the presence of SCN- averts damage to EPO-coated host tissues that might otherwise accrue as a result of HOBr generation. In view of these findings, the potential role of HOSCN in eosinophil killing of parasitic pathogens deserves close examination.
Slungaard
A
A
Department of Internal Medicine, University of Minnesota Medical School, Minneapolis 55455.
Mahoney
J R
JR
D000818:Animals; D001965:Bromides / Q000737:chemistry; D002021:Buffers; D002470:Cell Survival / Q000187:drug effects; D047209:Eosinophil Peroxidase; D006321:Heart / Q000187:drug effects / Q000502:physiology; D006801:Humans; D007454:Iodides / Q000737:chemistry; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000097:blood / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity; D013552:Swine; D013861:Thiocyanates / Q000378:metabolism* / Q000494:pharmacology
D001965:Bromides; D002021:Buffers; D007454:Iodides; D013861:Thiocyanates; D047209:Eosinophil Peroxidase; D010544:Peroxidases; C031760:thiocyanate