Oxidation of guaiacol by myeloperoxidase: a two-electron-oxidized guaiacol transient species as a mediator of NADPH oxidation

Abstract

The present study was first aimed at a complete steady-state kinetic analysis of the reaction between guaiacol (2-methoxyphenol) and the myeloperoxidase (MPO)/H2O2 system, including a description of the isolation and purification of MPO from human polymorphonuclear neutrophil cells. Secondly, the overall reaction of the oxidation of NADPH, mediated by the reactive intermediates formed from the oxidation of guaiacol in the MPO/H2O2 system, was analysed kinetically. The presence of guaiacol stimulates the oxidation of NADPH by the MPO/H2O2 system in a concentration-dependent manner. Concomitantly, the accumulation of biphenoquinone (BQ), the final steady-state product of guaiacol oxidation, is lowered, and even inhibited completely, at high concentrations of NADPH. Under these conditions, the stoichiometry of NADPH:H2O2 is 1, and the oxidation rate of NADPH approximates to that of the rate of guaiacol oxidation by MPO. The effects of the presence of superoxide dismutase, catalase and of anaerobic conditions on the overall oxidation of NADPH have also been examined, and the data indicated that superoxide formation did not occur. The final product of NADPH oxidation was shown to be enzymically active NADP+, while guaiacol was generated continuously from the reaction between NADPH and oxidized guaiacol product. In contrast, similar experiments performed on the indirect, tyrosine-mediated oxidation of NADPH by MPO showed that a propagation of the free radical chain was occurring, with generation of both O2(-.) and H2O2. BQ, in itself, was able to spontaneously oxidize NADPH, but neither the rate nor the stoichiometry of the reaction could account for the NADPH-oxidation process involved in the steady-state peroxidation cycle. These results provide evidence that the oxidation of NADPH does not involve a free nucleotide radical intermediate, but that this is probably due to a direct electron-transfer reaction between NADPH and a two-electron-oxidized guaiacol intermediate.

Myeloperoxidase-derived Hypochlorous Acid Antagonizes the Oxidative Stress-mediated Activation of Iron Regulatory Protein 1

“…The rate constants k 1MPO and k 2MPO for H 2 O 2 decomposition by MPO were obtained by using linear fit software (Microcal Origin 6.0, Microcal Software Inc.). To better compare our results with those reported in the literature, MPO activity was also determined by means of the conventional guaiacol assay (39,40). The standard molar activity of MPO was defined reaction mix of 10 M H 2 O 2 , 88.7 mM guaiacol, and 10 nM MPO in 10 mM phosphate buffer, pH 7.4.…”

Estrogen, neutrophils and oxidation

“…Myeloperoxidase activity was determined by measuring the peroxidase-mediated oxidation of guaiacol in the presence of H 2 O 2 (Caravaca and May, 1964;Capeillere-Blandin, 1998). 250 Al of the supernatant from activated neutrophils was added to a 40 mM solution of potassium phosphate buffer (pH 7.4) containing 4 Amol of H 2 O 2 and 6 Amol of guaiacol.…”

Antioxidant supplementation influences the neutrophil tocopherol associated protein expression, but not the inflammatory response to exercise

“…• C for 2.7 MPO activity and protein levels MPO activity of neutrophils was measured by guaiacol oxidation [34]. The reaction mixture contained sodium phosphate buffer (pH 7) and 13.5 mM guaiacol.…”