NADPH as a co-substrate for studies of the chlorinating activity of myeloperoxidase

The reinvestigation of the kinetics of myeloperoxidase (MPO) activity with the use of NADPH as a probe has allowed us to determine the effects of H2O2, Cl- ion and pH on the MPO-dependent production of HOCl. The chlorination rate of NADPH did not depend on NADPH concentration and was entirely related to the rate of production of HOCl by MPO. The overall oxidation of NADPH occurred similarly in the absence of O-2 and was insensitive to scavengers of the superoxide radical anion. Experiments performed on the direct oxidation of NADPH by MPO in the presence and the absence of H2O2 showed that neither the rate nor the stoichiometry of the reaction could interfere in the NADPH oxidation process involved in the steady-state chlorination cycle. The oxidation of NADPH was characterized by a decrease in the A(339) of the reduced nicotinamide with the concomitant appearance of a new chomophore with absorbance maximum at 274 nm, characterized by isosbestic points at 300 and 238 nm. The reaction product did not possess any enzymic properties with dehydrogenases and led to a metabolite other than NADP(+). Its amount accounted for a stoichiometric conversion of H2O2 into HOCl. Analyses of the NADPH reaction allowed the determination of both kinetic (k(cat) and K-m) and thermodynamic (K-d) parameters. When the values of kinetic parameters were compared with previously published ones, the main discrepancy was found with data obtained with the chlorination of monochlorodimedon and a better agreement with diethanolchloramine formation or H2O2 consumption. Variations in the extent of NADPH oxidation with Cl- concentration enabled us to determine the dissociation constant for the enzyme-Cl- complex. In the course of titration studies, the spectral properties of NADPH reacting with either HOCl or the MPO/H2O2/Cl- system were quantitatively similar in terms of stoichiometry and absorbance coefficient and thus led to identical chlorinated products. However, no spectral modification occurred with NADP(+) and adenine nucleotide analogues under the same conditions. A quantitative comparison of difference spectra obtained with NADPH and NMNH indicated that chlorination occurred on the nicotinamide part of the molecule.