title: The reductive cleavage of myeloperoxidase in half, producing enzymically active hemi-myeloperoxidase.
abstract: Reduction and alkylation of human myeloperoxidase under nondenaturing conditions results in the cleavage of this enzyme. Sedimentation equilibrium data is presented which shows that the molecular weight of the cleavage product (78,000 +/- 2,000) is half that of the native enzyme (153,000 +/- 4,000). We conclude that the cleavage product is the half-enzyme hemi-myeloperoxidase. Hemi-myeloperoxidase retains both heme groups and contains both subunit types (Mr = 57,500 and 14,000) in the same ratio as native myeloperoxidase. The two halves of native myeloperoxidase are apparently not dependent upon one another for peroxidatic activity, as the specific activity of the half-enzyme is the same as that of the native enzyme. Analytical ultracentrifugation studies show native myeloperoxidase has a sedimentation coefficient of 8.0 and an axial ratio of 5:1, while hemi-myeloperoxidase has a sedimentation coefficient of 4.3 and an axial ratio of 10:1. When [3H]iodoacetic acid was used to prepare hemi-myeloperoxidase, the label incorporated with a stoichiometry of 1.2 [3H]carboxymethyl groups per hemi-myeloperoxidase, with 90% of this label associated with the heavy subunit. From these observations we conclude that native myeloperoxidase contains two heavy-light protomers, which are joined along their long axes by a single disulfide bond between the heavy subunits. Selective reduction of this disulfide bond by the use of nondenaturing conditions results in the formation of hemi-myeloperoxidase, a catalytically active heavy-light protomer of native myeloperoxidase.
mesh_terms: D000107:Acetylation; D004229:Dithiothreitol / Q000494:pharmacology; D006418:Heme / Q000032:analysis; D006801:Humans; D007460:Iodoacetamide / Q000494:pharmacology; D007962:Leukocytes / Q000201:enzymology; D008970:Molecular Weight; D010084:Oxidation-Reduction; D009195:Peroxidase / Q000302:isolation & purification / Q000378:metabolism*; D010544:Peroxidases / Q000378:metabolism*; D013053:Spectrophotometry
chemical_list: D006418:Heme; D010544:Peroxidases; D009195:Peroxidase; D004229:Dithiothreitol; D007460:Iodoacetamide