MECHANISM OF THE OXIDATION OF 3,5,3',5'-TETRAMETHYLBENZIDINE BY MYELOPEROXIDASE DETERMINED BY TRANSIENT-STATE AND STEADY-STATE KINETICS

Earlier investigations of the oxidation of 3,5,3',5'-tetramethylbenzid ine (TMB) using horseradish peroxidase and prostaglandin H-synthase ha ve shown the formation of a cation free radical of TMB in equilibrium with a charge-transfer complex, consistent with either a two- or a one -electron initial oxidation. In this work, we exploited the distinct s pectroscopic properties of myeloperoxidase and its oxidized intermedia tes, compounds I and II, to establish two successive one-electron oxid ations of TMB. By employing stopped-flow techniques under transient-st ate and steady-state conditions, we also determined the rate constants for the elementary steps of the myeloperoxidase-catalyzed oxidation o f TMB at pH 5.4 and 20 degrees C. The second-order rate constant for c ompound I formation from the reaction of native enzyme with H2O2 is 2. 6 x 10(7) M-1 s(-1). Compound I undergoes a one-electron reduction to compound II in the presence of TMB, and the rate constant for this rea ction was determined to be (3.6 +/- 0.1) x 10(6) M-1 s(-1). The spectr al scans show that compound HT accumulates in the steady state. The ra te constant for compound II reduction to native enzyme by TMB obtained under steady-state conditions is (9.4 +/- 0.6) x 10(5) M-1 s(-1). The results are applied to a new, more accurate assay for myeloperoxidase based upon the formation of the charge-transfer complex between TMB a nd its diimine final product.