Purification of myeloperoxidase from human leucocytes

Myeloperoxidase (MPO), (transmitter H2O2 oxidoreductase, EC 1.11.1.7) is an enzyme found in mammalian neutrophil granules, play an important part in killing these bacteriums fagosited.Myeloperoxidase (MPO), enzym has been isolated from leukophoresis preparations and native mouse 32Dc13 cells and using CaCl2 from buffy coats by washing up leucocyte. Leucocyte has been obtained form horse blood containing anticoagulated with citrate, anticoagulated boold with EDTA and buffy coats on a ficoll density gradient.At that study; human Myeloperoxidase (MPO) enzyme has been purified with rank of CM Sefhadex İon Exchange, Sefhacryl S300 HR Gel filtration chromatography, Con A Sepharose 4B affinity gel chromatography and adjust to 80% ammonium sulfate by the addition of dry ammonium sulfate. In our study human myeloperoxidase had a specific activity of 1192,574 units per milligram protein when H2O2 used as a substrate with a purification fold of 71,45. The purity was controlled by using SDS-PAGE. The purified enzyme had a Km value of 0,369 µmol, which was calculated from Michaelis-Menten and Lineweaver-Burk plots, by using H2O2 as a substrate at the assay conditions of 370C in 50 mM KPO4 buffer (pH:5,4) containing 0,5% CETAB.