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Hypochlorous acid and simple chloramines (RNHCl) are stable biologically derived chlorinating agents. In general, the chlorination potential of HOCl is much greater than that of RNHCl, allowing it to oxidize or chlorinate a much wider variety of reaction partners. However, in this study we demonstrate by kinetic analysis that the reactivity of RNHCl can be dramatically promoted by imidazole and histidyl model compounds via intermediary formation of the corresponding imidazole chloramines. Two biologically relevant reactions were investigated--loss of imidazole-catalyzed chlorinating capacity and phenolic ring chlorination using fluorescein and the tyrosine analog, 4-hydroxyphenylacetic acid (HPA). HOCl reacted stoichiometrically with imidazole, N-acetylhistidine (NAH), or imidazoleacetic acid to generate the corresponding imidazole chloramines which subsequently decomposed. Chloramine (NH2Cl) also underwent a markedly accelerated loss in chlorinating capacity when NAH was present, although in this case N-α-acetylhistidine chloramine (NAHCl) did not accumulate, indicating that the catalytic intermediate must be highly reactive. Mixing HOCl with 1-methylimidazole (MeIm) led to very rapid loss in chlorinating capacity via formation of a highly reactive chlorinium ion (MeImCl(+)) intermediate; this behavior suggests that the reactive forms of the analogous imidazole chloramines are their conjugate acids, e.g., the imidazolechlorinium ion (HImCl(+)). HOCl-generated imidazole chloramine (ImCl) reacted rapidly with fluorescein in a specific acid-catalyzed second-order reaction to give 3'-monochloro and 3',5'-dichloro products. Equilibrium constants for the transchlorination reactions HOCl + HIm = H2O + ImCl and NH2Cl + HIm = NH3 + ImCl were estimated from the dependence of the rate constants on [HIm]/[HOCl] and literature data. Acid catalysis again suggests that the actual chlorinating agent is HImCl(+); consistent with this interpretation, MeIm markedly catalyzed fluorescein chlorination by HOCl. Time-dependent imidazole-catalyzed HPA chlorination by NH2Cl was also demonstrated by product analyses. Quantitative assessment of the data suggests that physiological levels of histidyl groups will react with primary chloramines to generate a flux of imidazole chloramine sufficient to catalyze biological chlorination via HImCl(+), particularly in environments that generate high concentrations of HOCl such as the neutrophil phagosome., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Current viewpoints concerning the bactericidal mechanisms of neutrophils are reviewed from a perspective that emphasizes challenges presented by the inability to duplicate ex vivo the intracellular milieu. Among the challenges considered are the influences of confinement upon substrate availability and reaction dynamics, direct and indirect synergistic interactions between individual toxins, and bacterial responses to stressors. Approaches to gauging relative contributions of various oxidative and nonoxidative toxins within neutrophils using bacteria and bacterial mimics as intrinsic probes are also discussed., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The prospects for using bacterial DNA as an intrinsic probe for HOCl and secondary oxidants/chlorinating agents associated with it has been evaluated using both in vitro and in vivo studies. Single-strand and double-strand breaks occurred in bare plasmid DNA that had been exposed to high levels of HOCl, although these reactions were very inefficient compared to polynucleotide chain cleavage caused by the OH.-generating reagent, peroxynitrite. Plasmid nicking was not increased when intact Escherichia coli were exposed to HOCl; rather, the amount of recoverable plasmid diminished in a dose-dependent manner. At concentration levels of HOCl exceeding lethal doses, genomic bacterial DNA underwent extensive fragmentation and the amount of precipitable DNA-protein complexes increased several-fold. The 5-chlorocytosine content of plasmid and genomic DNA isolated from HOCl-exposed E. coli was also slightly elevated above controls, as measured by mass spectrometry of the deaminated product, 5-chlorouracil. However, the yields were not dose-dependent over the bactericidal concentration range. Genomic DNA recovered from E. coli that had been subjected to phagocytosis by human neutrophils occasionally showed small increases in 5-chlorocytosine content when compared to analogous cellular reactions where myeloperoxidase activity was inhibited by azide ion. Overall, the amount of isolable 5-chlorouracil from the HOCl-exposed bacterial cells was far less than the damage manifested in polynucleotide bond cleavage and cross-linking., (Copyright (c) 2009 Elsevier Inc. All rights reserved.)

Escherichia coli were transformed by electroporation to introduce a plasmid harboring a GFP gene-containing vector. The fluorescence of the purified GFP isolated from the transformant was quenched by myeloperoxidase (MPO)-generated HOCl, by peroxynitrous acid (ONOOH) and by enzymatically or radiolytically generated NO(2)(.) but not by other putative neutrophil-generated oxidants. Fluorescence from the bacterium was effectively quenched by HOCl but not peroxynitrite, oxidizing radicals derived from its O-O bond homolysis, or the other oxidants under study. Exposure of serum-opsonized bacteria to human neutrophils resulted in extensive loss of GFP fluorescence; fluorescence microscopy revealed that phagocytosed bacteria were completely quenched but that bacteria remaining in the external media were unquenched. Addition of sodium azide to the medium to inhibit MPO prevented neutrophil-mediated fluorescence quenching. Because the amount of HOCl required to inhibit bacterial fluorescence was an order of magnitude greater than required to inhibit colonial growth, these results imply that sufficient HOCl was formed within the neutrophil phagosome to kill the microbe.

The capabilities of stimulated neutrophils to initiate intraphagosomal and extracellular chlorination, nitration, and other oxidative reactions has been evaluated using a fluorescent particle and soluble phenolic compounds as target molecules. Neutrophils activated by the soluble stimulus, phorbol myristate acetate, both chlorinated fluorescein that was covalently attached to polyacrylamide microspheres and initiated tyrosine dimerization. When nitrite ion was present at millimolar concentration levels in the medium, nitration of the phenolic rings also occurred; the relative extent of nitration increased as the nitrite concentration was increased. Myeloperoxidase (MPO) also catalyzed nitration and chlorination of fluorescein and the fluorescein-conjugated particles in cell-free solutions; the relative nitration yields increased with increasing [NO2-]/[Cl-] ratios. Nitration did not involve intermediary formation of nitrating agents derived from reaction between MPO-generated HOCl and NO2- because this reaction also occurred in chloride-free media and direct addition of HOCl to solutions containing NO2- and fluorescein gave only chlorinated products. In marked contrast to these extracellular reactions, intraphagosomal nitration of the fluorescein-conjugated particles could not be detected (even at [NO2-] as high as 0.1 M), whereas chlorination of the probe was extensive. These data indicate that intraphagosomal aromatic nitration in neutrophils is negligible, although extracellular nitration of phenolic compounds by secreted MPO could occur at physiological concentration levels of NO2-.

Fluorescein was covalently attached through a cystamine linker group to carboxy-derivatized polyacrylamide microspheres to generate phagocytosable particles containing fluorescent reporter groups. A unique feature of these beads is that the dye was recoverable in near-quantitative yield from intracellular environments by thiol reduction of the cystamine disulfide bond. Fluorescence microscopy indicated that individual neutrophils could bind as many as approximately 20 serum-opsonized beads, although no appreciable cellular association was observed for unopsonized beads. By using methyl viologen to quench external fluorescence, it was demonstrated that 70-90% of the neutrophil-associated fluorescein on opsonized beads was inaccessible to the medium. The particle-bound fluorescein underwent near-stoichiometric conversion to chlorinated derivatives when reacted with HOCl or the cell-free myeloperoxidase (MPO)-H2O2-Cl- system; products were identified by HPLC separation and electrospray ionization mass spectrometry of the recovered dye. Fluorescence changes accompanying phagocytosis were consistent with chlorination of the dye; fluorescence spectrometric and chemical trapping measurements indicated that intraphagosomal chlorination was far more extensive than extracellular chlorination. Yields of recovered chlorofluoresceins determined by HPLC indicated that sufficient HOCl had been produced intracellularly to kill entrapped bacteria. Fluorescein chlorination coincided approximately with phagocytosis and stimulated uptake of O2 by the cells. Demonstration that HOCl is produced within phagosomes in sufficient concentrations to kill bacteria on a time scale associated with death constitutes strong evidence in support of a primary role for HOCl in the microbicidal action of neutrophils.

The resonance Raman spectra of neutrophil cytochrome b558 obtained upon Soret excitation indicate that the heme is low spin six-coordinate in both ferric and ferrous oxidation states; comparison with the spectra of bis-imidazole hemin suggests imidazole or imidazolate axial ligation. Minor bands attributable to vibrational motions of ring-conjugated vinyl substituents were also observed, consistent with a heme assignment of protoporphyrin IX. The spectra of deoxycholate-solubilized cytochrome b558 were indistinguishable from neutrophil plasma membranes or specific granules, as were spectra from unstimulated and phorbol myristate acetate-stimulated cells, indicating that the hemes are structurally identical in various subcellular environments and cellular physiological states. However, structural complexity was suggested by biphasic ferric-ferrous photoreduction under 413-nm illumination and the absence of an EPR spectrum for the ferric heme under conditions where simple bis-imidazole heme-containing cytochromes are expected to give detectable signals. Midpoint reduction potentials and resonance Raman spectra of the soluble cytochrome b558 from an individual with cytochrome b558 positive (type IA.2) chronic granulomatous disease were nearly identical to normal oxidase, with the exception that the deficient oxidase did not undergo heme photoreduction. Possible structural models are discussed in relation to other physical properties (ligand binding, thermodynamic potentials) exhibited by the cytochrome.

Hypochlorous acid (HOC1) rapidly chlorinates fluorescein compounds forming, sequentially, the corresponding 4'-chlorofluorescein and 4',5'-dichlorofluoresceins. Chlorination by cell-free myeloperoxidase-catalyzed chloride peroxidation systems gives rise to these compounds as well as variable amounts of isomeric compounds chlorinated in the 2'- and 2',7'-positions. The fluorescence intensity of the dianionic form of the dye is partially quenched upon chlorination, and its proton equilibrium constants are shifted to more acidic values. Fluorescein covalently bound to zymosan (5-isothiocyanatofluorescein-zymosan) also formed these products when the unopsonized particles were incubated with phorbol myristate acetate- or N-formyl-methionyl-leucyl-phenylalanine-stimulated human neutrophils. This reaction was associated with a fall in fluorescence intensity, which was not observed when cells from individuals with chronic granulomatous disease or myeloperoxidase deficiency were used or when azide or catalase were added to the reaction medium. Fluorescent changes accompanying phagocytosis of serum-opsonized 5-isothiocyanatofluorescein-zymosan were also consistent with chlorination of the label; the changes were shown to be myeloperoxidase-dependent by use of myeloperoxidase-deficient or azide-treated cells. Oxidative bleaching of the structurally similar sulfonphthalein dyes by HOCl also occurs at rates which parallel the dye basicities. Results are discussed in relation to the use of fluoresceinated particles and sulfonphthalein dyes in the measurement of intraphagosomal acidification.

Titrimetric addition of hypochlorous acid (HOCl) or chloramine (NH2Cl) to suspensions of Escherichia coli decreases their ability to accumulate 14C-labeled glutamine, proline, thiomethylgalactoside, and leucine in a manner that approximately coincides with loss of cell viability; quantitative differences in cellular response are observed with the two oxidants. Inhibition of beta-galactosidase activity in E. coli ML-35, a strain lacking functional lactose permease, is complex and also depends upon the identity of the oxidant. Membrane proton conductivities and glycerol permeabilities are unchanged by addition of HOCl or NH2Cl in excess of that required for inactivation. The combined results are interpreted to indicate that the locus of HOCl attack is the cell envelope, that HOCl inactivation does not occur by loss of membrane structural integrity, that loss of transport function can be identified with either selective oxidative inhibition of the transport proteins or loss of cellular metabolic energy, and that different mechanisms of inactivation may exist for HOCl and NH2Cl.

Following a brief introduction of cellular response to stimulation comprising leukocyte activation, three major areas are discussed: (1) the neutrophil oxidase; (2) myeloperoxidase (MPO)-dependent oxidative microbicidal reactions; and (3) MPO-independent oxidative reactions. Topics included in section (A) are current views on the activation mechanism, redox composition, structural and topographic organization of the oxidase, and its respiratory products. In section (B), emphasis is placed on recent research on cidal mechanisms of HOCl, including the oxidative biochemistry of active chlorine compounds, identification of sites of lesions in bacteria, and attendant metabolic consequences. In section (C), we review the (bio)chemistry of H2O2 and .OH microbicidal reactions, with particular attention being given to addressing the controversial issue of probe methods to identify .OH radical and critical assessment of the recent proposal that MPO-independent killing arises from site-specific metal-catalyzed Fenton-type chemistry.