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 myeloperoxidase from HL60 promyelocytic cells in culture. Initial studies showed the bulk of peroxidase-positive myeloperoxidase activity to be located in the cetyltrimethylammonium bromide solubilized particulate fraction of cell homogenates. The myeloperoxidase was then chromatographically purified using concanavalin A followed by gel filtration. SDS-PAGE analysis of the final preparation showed the presence of only two proteins with molecular masses of approximately 55 and 15 kDa, corresponding to the large and small subunits 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 myeloperoxidase prepared by this method is apparently homogeneous. Preparations routinely yielded 12-20 mg of pure myeloperoxidase per 10 ml of cell pellet. The HL60 myeloperoxidase was shown to be indistinguishable from purified human neutrophil myeloperoxidase by size exclusion chromatography, analytical ultracentrifugation, SDS-PAGE, Western blot, and NH(2)-terminal sequence analysis. The activities of the two myeloperoxidase samples, as measured using either the tetramethylbenzidine or the taurine chloramine assay, were indistinguishable. Finally, both enzymes responded identically to dapsone and aminobenzoic acid hydrazide, known inhibitors of myeloperoxidase. A protocol is presented here for the rapid, large-scale purification of myeloperoxidase from cultured HL60 cells, as well as evidence for the interchangeability of this myeloperoxidase and that purified from human neutrophils.
Hope
H R
HR
Biochemistry and Molecular Biology Sciences, G.D. Searle/Monsanto Life Sciences Company, 700 North Chesterfield Parkway, St. Louis, Missouri, 63198, USA. Heidi.R.Hope@monsanto.com
Remsen
E E
EE
Lewis
C
C
Heuvelman
D M
DM
Walker
M C
MC
Jennings
M
M
Connolly
D T
DT
D001560:Benzidines / Q000737:chemistry; D015153:Blotting, Western; D002850:Chromatography, Gel; D003622:Dapsone / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000737:chemistry; D004795:Enzyme Stability; D018922:HL-60 Cells; D006801:Humans; D008970:Molecular Weight; D009504:Neutrophils / Q000737:chemistry*; D010084:Oxidation-Reduction; D009195:Peroxidase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D011489:Protein Denaturation; D013654:Taurine / Q000031:analogs & derivatives / Q000737:chemistry; D014461:Ultracentrifugation
D001560:Benzidines; D004791:Enzyme Inhibitors; D013654:Taurine; C021758:3,3',5,5'-tetramethylbenzidine; C043410:N-chlorotaurine; D003622:Dapsone; D009195:Peroxidase