title: Thiocyanate is the major substrate for eosinophil peroxidase in physiologic fluids. Implications for cytotoxicity.
abstract: 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.
authors.affiliation: Department of Internal Medicine, University of Minnesota Medical School, Minneapolis 55455.
authors.forename: A
authors.initials: A
authors.lastname: Slungaard
authors.forename: J R
authors.initials: JR
authors.lastname: Mahoney
mesh_terms: 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
chemical_list: D001965:Bromides; D002021:Buffers; D007454:Iodides; D013861:Thiocyanates; D047209:Eosinophil Peroxidase; D010544:Peroxidases; C031760:thiocyanate