Lökosit Miyeloperoksidaz Enziminin Saflaştırılması, Kinetik Özellikleri ile Reaktif Türlerin Oluşumundaki Etkilerinin Araştırılması

Abstract

Myeloperoxidase (MPO) has been purified from human leucocytes obtained by leukapheresis, and kinetic properties were determined, nitrite oxidation and tyrosine nitration catalyzed by the purified enzyme have been studied. MPO was solubilized and extracted from saline-washed leucocytes using phosphate buffer containing 1 % (w/v) HETAB. MPO from the extract was purified by chromatography on Concanavalin A-Sepharose, and CM-Sephadex ion-exchange chromatography at pH 8 with a yield of 70.3. Under reducing and denaturing conditions on polyacrylamide-gel electrophoresis (SDS-PAGE), purified enzyme gave rise to protein bands of M, 57000 and 15500 Dalton. Spectral analysis of the enzyme gave a Reinheit Zahl (RZ) values (A430/A280) of 86, indicating that the enzyme was pure at least 99 % pure. Kinetic properties of MPO were determined using hydrogen peroxide and tetramethylbenzidine as co-substrates. Km and Vmax values for H2O2 were 0.727mM and 273±8 IU/mg protein; for tetramethylbenzidine were 0.111 mM and 283.04±39 IU/mg protein, respectively. Myeloperoxidase was found to catalyze the oxidation of nitrite in the presence of H2O2. The oxidation of nitrate was pH and time-dependent, and half of nitrite disappeared in the reaction medium was recovered as nitrate. MPO-catalyzed nitrite oxidation was observed between pH 4.5-6, and no spontaneous oxidation could be detected within this pH range. Above pH 6.5. Myeloperoxidase can utilize nitrite and hydrogen peroxide as substrates to catalyze tyrosine nitration. Both free tyrosine and tyrosine residues on proteins are subject to nitration. MPO was found to catalyze free tyrosine nitration around intra-phagosom pH (between pH 5-6). The extent of nitration was dependent on concentration of substrates (nitrite, hydrogen peroxide and tyrosine). Two physiological antioxidant compounds, reduced glutathione and ascorbic acid were found to inhibit MPO-catalyzed tyrosine nitration by a mixed-type inhibition mechanism.