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 H(2)O(2) 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 H(2)O(2) and Cl(-) to produce HClO. The chemical mechanism behind the bactericidal action of the MPO-H(2)O(2)-Cl(-) system is unclear. Bactericidal action may result from (a) the direct reactions of HOCl with biological components (through amine chlorination) or (b) (1)O(2), formed non-enzymatically from HOCl and H(2)O(2), 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 (1)O(2) from a (1)O(2) source at pH 4.5-9.0. Using the MCLA-dependent chemiluminescence method, we found that the rate of formation of (1)O(2) from the MPO-H(2)O(2)-Cl(-) system peaked at pH 7.0. Segal et al. (28) reported that almost all Staphylococcus aureus is killed 2 min after phagocytosis by neutrophils 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-H(2)O(2)-Cl(-) system with Escherichia coli at pH 4.5 and 8.0 were paralleled by the rate of formation of (1)O(2). Combining these observations and the results reported by Segal et al., we concluded that (1)O(2) is a major chemical species in the killing of bacteria in neutrophil phagosomes.

[1]  J. Crowley,et al.  Human Neutrophils Use the Myeloperoxidase-Hydrogen Peroxide-Chloride System to Chlorinate but Not Nitrate Bacterial Proteins during Phagocytosis* , 2002, The Journal of Biological Chemistry.

[2]  Haruo Watanabe,et al.  Critical role of myeloperoxidase and nicotinamide adenine dinucleotide phosphate-oxidase in high-burden systemic infection of mice with Candida albicans. , 2002, The Journal of infectious diseases.

[3]  A. Kettle,et al.  Chlorination of Bacterial and Neutrophil Proteins during Phagocytosis and Killing of Staphylococcus aureus * , 2002, The Journal of Biological Chemistry.

[4]  J. Cadet,et al.  Chlorination of Guanosine and Other Nucleosides by Hypochlorous Acid and Myeloperoxidase of Activated Human Neutrophils , 2001, The Journal of Biological Chemistry.

[5]  A. Kettle,et al.  A kinetic analysis of the catalase activity of myeloperoxidase. , 2001, Biochemistry.

[6]  N. Misawa,et al.  Quenching of singlet oxygen by carotenoids produced in Escherichia coli – attenuation of singlet oxygen‐mediated bacterial killing by carotenoids , 2000, FEBS letters.

[7]  H. Koyama,et al.  Differential host susceptibility to pulmonary infections with bacteria and fungi in mice deficient in myeloperoxidase. , 2000, The Journal of infectious diseases.

[8]  C. Obinger,et al.  Mechanism of reaction of myeloperoxidase with hydrogen peroxide and chloride ion. , 2000, European journal of biochemistry.

[9]  J. P. Henderson,et al.  Molecular Chlorine Generated by the Myeloperoxidase-Hydrogen Peroxide-Chloride System of Phagocytes Produces 5-Chlorocytosine in Bacterial RNA* , 1999, The Journal of Biological Chemistry.

[10]  T. Maruyama,et al.  Singlet Oxygen (1ΔgO2) as the Principal Oxidant in Myeloperoxidase-Mediated Bacterial Killing in Neutrophil Phagosome ☆ , 1999 .

[11]  T. Fujinaga,et al.  Physiological production of singlet molecular oxygen in the myeloperoxidase‐H2O2‐chloride system , 1999, FEBS letters.

[12]  N. Misawa,et al.  Inactivation of bacterial respiratory chain enzymes by singlet oxygen , 1998, FEBS letters.

[13]  A. Kettle,et al.  Inside the neutrophil phagosome: oxidants, myeloperoxidase, and bacterial killing. , 1998, Blood.

[14]  Katsuya Ishiguro,et al.  Chemiluminescence of Cypridina Luciferin Analogs. Part 3. MCLA Chemiluminescence with Singlet Oxygen Generated by the Retro‐Diels‐Alder Reaction of a Naphthalene Endoperoxide , 1998 .

[15]  Y. Kambayashi,et al.  Useful 1O2 (1Δg) generator, 3‐(4′‐methyl‐1′‐naphthyl)‐propionic acid, 1′,4′‐endoperoxide (NEPO), for dioxygenation of squalene (a skin surface lipid) in an organic solvent and bacterial killing in aqueous medium , 1998, FEBS letters.

[16]  S. Hazen,et al.  Human Neutrophils Employ the Myeloperoxidase-Hydrogen Peroxide-Chloride System to Oxidize α-Amino Acids to a Family of Reactive Aldehydes , 1998, The Journal of Biological Chemistry.

[17]  A. Carr,et al.  Oxidation of neutrophil glutathione and protein thiols by myeloperoxidase-derived hypochlorous acid. , 1997, The Biochemical journal.

[18]  A. Kettle,et al.  Involvement of superoxide and myeloperoxidase in oxygen-dependent killing of Staphylococcus aureus by neutrophils , 1996, Infection and immunity.

[19]  L. Ellerby,et al.  Copper−Zinc Superoxide Dismutase: Why Not pH-Dependent? , 1996 .

[20]  M. Karnovsky,et al.  Intracellular singlet oxygen generation by phagocytosing neutrophils in response to particles coated with a chemical trap. , 1992, The Journal of biological chemistry.

[21]  S. Mashiko,et al.  Measurement of rate constants for quenching singlet oxygen with a Cypridina luciferin analog (2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazo [1,2-a]pyrazin-3-one) and sodium azide. , 1991, Journal of bioluminescence and chemiluminescence.

[22]  C. Bernofsky.  Nucleotide chloramines and neutrophil‐mediated cytotoxicity , 1991, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[23]  M. Wenzel,et al.  [Protective effect of D20 in bacteria (E. coli)]. , 1989, Zeitschrift fur Naturforschung. C, Journal of biosciences.

[24]  A. Kettle,et al.  Influence of superoxide on myeloperoxidase kinetics measured with a hydrogen peroxide electrode. , 1989, The Biochemical journal.

[25]  H. Iwamoto,et al.  Crystallization and properties of myeloperoxidase from normal human leukocytes. , 1986, Journal of biochemistry.

[26]  R. Bjerknes,et al.  Phagocytosis by human leukocytes, phagosomal pH and degradation of seven species of bacteria measured by flow cytometry. , 1985, Journal of medical microbiology.

[27]  A. Tauber,et al.  Biochemical requirements for singlet oxygen production by purified human myeloperoxidase. , 1984, The Journal of clinical investigation.

[28]  R. Lehrer,et al.  Assessment of chlorination by human neutrophils , 1983, Nature.

[29]  K. Kakinuma,et al.  Effects of neuraminidase on O2 consumption and release of O2 and H2O2 from phagocytosing human polymorphonuclear leukocytes. , 1982, Blood.

[30]  M. Geisow,et al.  The respiratory burst of phagocytic cells is associated with a rise in vacuolar pH , 1981, Nature.

[31]  J. K. Hurst,et al.  Mechanisms of chlorine oxidation of hydrogen peroxide , 1978 .

[32]  N. Krinsky.  Singlet Excited Oxygen as a Mediator of the Antibacterial Action of Leukocytes , 1974, Science.

[33]  T. Stelmaszyńska,et al.  Myeloperoxidase of human neutrophilic granulocytes as chlorinating enzyme. , 1974, European journal of biochemistry.

[34]  T. Kajiwara,et al.  Direct spectroscopic evidence for a deuterium solvent effect on the lifetime of singlet oxygen in water , 1973 .

[35]  D. Bainton,et al.  TEMPORAL CHANGES IN PH WITHIN THE PHAGOCYTIC VACUOLE OF THE POLYMORPHONUCLEAR NEUTROPHILIC LEUKOCYTE , 1973, The Journal of cell biology.

[36]  R. Allen,et al.  Evidence for the generation of an electronic excitation state(s) in human polymorphonuclear leukocytes and its participation in bactericidal activity. , 1972, Biochemical and biophysical research communications.

[37]  W. Ostrowski,et al.  Chloramines as intermediates of oxidation reaction of amino acids by myeloperoxidase. , 1971, Biochimica et biophysica acta.

[38]  C. Foote,et al.  Chemistry of singlet oxygen. X. Carotenoid quenching parallels biological protection. , 1970, Journal of the American Chemical Society.

[39]  S. Klebanoff.  Myeloperoxidase-Halide-Hydrogen Peroxide Antibacterial System , 1968, Journal of bacteriology.

[40]  W. Prütz.  Interactions of hypochlorous acid with pyrimidine nucleotides, and secondary reactions of chlorinated pyrimidines with GSH, NADH, and other substrates. , 1998, Archives of biochemistry and biophysics.

[41]  Ken Fujimori,et al.  Chemiluminescence of Cipridina luciferin analogues. Part 2. Kinetic studies on the reaction of 2-methyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one (CLA) with superoxide: hydroperoxyl radical is an actual active species used to initiate the reaction , 1995 .

[42]  M. Nakano.  Determination of superoxide radical and singlet oxygen based on chemiluminescence of luciferin analogs. , 1990, Methods in enzymology.

[43]  M. Grisham,et al.  Preparation and characterization of chloramines. , 1986, Methods in enzymology.

[44]  H. Rosen,et al.  [52] Antimicrobial activity of myeloperoxidase , 1984 .

[45]  R. Lehrer,et al.  Phagolysosomal pH of human neutrophils , 1984 .

[46]  J. K. Hurst,et al.  Biological reactivity of hypochlorous acid: implications for microbicidal mechanisms of leukocyte myeloperoxidase. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[47]  P. K. Glasoe,et al.  USE OF GLASS ELECTRODES TO MEASURE ACIDITIES IN DEUTERIUM OXIDE1,2 , 1960 .