Proton stoichiometry associated with human neutrophil respiratory-burst reactions

Control of the intraphagosomal pH in neutrophils may be of importance in creating a microbicidal environment by regulating the activity of the O₂^.-generating NADPH oxidase and the lysosomal enzymes discharged into this comparment. In this study, we examined the proton stoichiometry associated with the primary enzymatic reaction underlying the respiratory burst. A preparation of the neutrophil-derived, membrane oxidase consumed NADPH and generated O₂^.- with a stoichiometry of 1 NADPH:2 O₂^.-. When the enzymatically produced ^.O₂^.- was prevented from undergoing dismutation, net protons were released in an approximate 1:2 stoichiometry with O₂^.- generated. In contrast, when O₂^.- was allowed to dismutate to H₂O₂, net protons were consumed in a 1:1 stoichiometry with the accumulated H₂O₂. Thus, the ΔpH associated with the NADPH oxidase-dependent production of O₂^.- was dictated by the fate of the generated radical. The consumption of the oxidase-generated H₂O₂ by the lysosomal enzyme myeloperoxidase resulted in the formation of HOCl which was trapped in the presence of taurine as the N-chloro derivative. The ratio of chlorinated product formed to H⁺ consumed was 1:1. The implications of these results are discussed in terms of the known intraphagosomal pH changes that occur following neutrophil stimulation. We conclude that the O₂^.-generating oxidase plays a dual role in the phagosome by simultaneously creating an oxidizing environment that optimizes pH-dependent microbicidal processes.