MCLA-dependent chemiluminescence suggests that singlet oxygen plays a pivotal role in myeloperoxidase-catalysed bactericidal action in neutrophil phagosomes

Bacteria ingested by a neutrophil are located in phagosomes in which H2O2 is produced through the NADPH oxidase-dependent respiratory burst. Myeloperoxidase (MPO) plays important role in the bactericidal action of phagosomes. MPO catalyses the reaction of H2O2 and Cl- to produce HClO. The chemical mechanism behind the bactericidal action of the MPO-H2O2-Cl system is unclear. Bactericidal action may result from (a) the direct reactions of HOCl with biological components (through amine chlorination) or (b) O-1(2), formed non-enzymatically from HOCl and H2O2, that mainly works to kill microorganisms through bacterial respiratory chain injury. To answer this question, we developed a Cypridina luciferin analogue (MCLA)-dependent chemiluminescence method to determine the rate of formation of O-1(2) from a O-1(2) source at pH 4.5-9.0. Using the MCLA-dependent chemiluminescence method, we found that the rate of formation of O-1(2) from the MPO-H2O2-C- system peaked at pH 7.0. Segal et al. (28) reported that almost all Staphylococcus aureus is killed 2 min after phagocytosis by neutophils where the phagosomal pH is 7.4-7.75. However, amine chlorination by HOCl did not proceed at pH > 7.0. Moreover, the bactericidal activities of the MPO-H2O2-Cl- system with Escherichia coli at pH 4.5 and 8.0 were paralleled by the rate of formation of O-1(2) Combining these observations and the results reported by Segal et A, we concluded that O-1(2) is a major chemical species in the killing of bacteria in neutrophil phagosomes. Copyright (C) 2003 John Wiley Sons, Ltd.