Large-scale purification of myeloperoxidase from HL60 promyelocytic cells:Characterization and comparison to human neutrophil myeloperoxidase

A large-scale purification procedure was developed for the isolation of mye loperoxidase from HL60 promyelocytic cells in culture. Initial studies show ed the bulk of peroxidase-positive myeloperoxidase activity to be located i n the cetyltrimethylammonium bromide solubilized particulate fraction of ce ll homogenates. The myeloperoxidase was then chromatographically purified u sing concanavalin A followed by gel filtration. SDS-PAGE analysis of the fi nal preparation showed the presence of only two proteins with molecular mas ses of approximately 55 and 15 kDa, corresponding to the large and small su bunits of myeloperoxidase. These data, along with Reinheit Zahl (RZ) values (A(430)/A(280)) of greater than or equal to 0.72, indicate that the myelop eroxidase prepared by this method is apparently homogeneous, Preparations r outinely yielded 12-20 mg of pure myeloperoxidase per 10 ml of cell pellet. The HL60 myeloperoxidase was shown to be indistinguishable from purified h uman neutrophil myeloperoxidase by size exclusion chromatography, analytica l ultracentrifugation, SDS-PAGE, Western blot, and NH2-terminal sequence an alysis, The activities of the two myeloperoxidase samples, as measured usin g either the tetramethylbenzidine or the taurine chloramine assay, were ind istinguishable. Finally, both enzymes responded identically to dapsone and aminobenzoic acid hydrazide, known inhibitors of myeloperoxidase. A protoco l is presented here for the rapid, large-scale purification of myeloperoxid ase from cultured HL60 cells, as well as evidence for the interchangeabilit y of this myeloperoxidase and that purified from human neutrophils. (C) 200 0 Academic Press.