Anthony Kettle - Academia.edu (original) (raw)
Papers by Anthony Kettle
SUMMARY: Myeloperoxidase (MPO) uses hydrogen peroxide to oxidize chloride to hypochlorous acid. I... more SUMMARY: Myeloperoxidase (MPO) uses hydrogen peroxide to oxidize chloride to hypochlorous acid. It also converts numerous substrates to reactive free radicals. When released by neutrophils, the enzyme operates in the presence of a flux of superoxide. We show that superoxide has a profound influence on oxidative reactions catalysed by MPO. It reacts directly with the enzyme to modulate production of
![Research paper thumbnail of Corrigendum to “Rapid reaction of superoxide with insulin-tyrosyl radicals to generate a hydroperoxide with subsequent glutathione addition” [Free Radic. Biol. Med. 70 (2014) 86–95]](https://attachments.academia-assets.com/43914227/thumbnails/1.jpg)
](Free Radical Biology and Medicine, 2015
Corrigendum to "Rapid reaction of superoxide with insulin-tyrosyl radicals to generate a hydroper... more Corrigendum to "Rapid reaction of superoxide with insulin-tyrosyl radicals to generate a hydroperoxide with subsequent glutathione addition" [Free Radic. Biol. Med. 70 (2014) 86-95] Please cite this article as: Das, AB; et al. Corrigendum to "Rapid reaction of superoxide with insulin-tyrosyl radicals to generate a hydroperoxide with subsequent.... Free Radic. Biol. Med. (2014), http://dx.
Chemical research in toxicology, Jan 5, 2015
Urate hydroperoxide is a strong oxidant generated by the combination of urate free radical and su... more Urate hydroperoxide is a strong oxidant generated by the combination of urate free radical and superoxide. The formation of urate hydroperoxide as an intermediate in urate oxidation is potentially responsible for the pro-oxidant effects of urate in inflammatory disorders, protein degradation, and food decomposition. To understand the molecular mechanisms that sustain the harmful effects of urate in inflammatory and oxidative stress related conditions, we report a detailed structural characterization and reactivity of urate hydroperoxide toward biomolecules. Urate hydroperoxide was synthesized by photo-oxidation and by a myeloperoxidase/hydrogen peroxide/superoxide system. Multiple reaction monitoring (MRM) and MS(3) ion fragmentation revealed that urate hydroperoxide from both sources has the same chemical structure. Urate hydroperoxide has a maximum absorption at 308 nm, ε308nm = 6.54 ± 0.38 × 10(3) M(-1) cm(-1). This peroxide decays spontaneously with a rate constant of k = 2.80 ±...
We have used a quantitative assay that measures independent rate constants for phagocytosis and k... more We have used a quantitative assay that measures independent rate constants for phagocytosis and killing of Staphylococcus aureusto investigate the involvement of superoxide and myeloperoxidase in bacterial killing by human neutrophils. To inhibit superoxide-dependent processes, superoxide dismutase was cross-linked to immunoglobulin G and the conjugate was attached to the surface of S. aureus via protein A in its cell wall.
Journal of Biological Chemistry
Phagocytic neutrophils generate reactive oxygen species to kill microbes. Oxidant generation occu... more Phagocytic neutrophils generate reactive oxygen species to kill microbes. Oxidant generation occurs within an intracellular phagosome, but diffusible species can react with the neutrophil and surrounding tissue. To investigate the extent of oxidative modification we assessed the carbonylation of cytosolic proteins in phagocytic neutrophils. A four-fold increase in protein carbonylation was measured within 15 min of initiating phagocytosis. Carbonylation was dependent on NOX2 and myeloperoxidase activity, and was inhibited by butylated hydroxytoluene and Trolox, indicating a role for myeloperoxidase-dependent lipid peroxidation. Proteomic analysis of target proteins revealed significant carbonylation of the S100A9 subunit of calprotectin, a truncated form of Hsp70, actin, and hemoglobin from contaminating erythrocytes. Addition of the reactive aldehyde 4-hydroxynonenal (HNE) caused carbonylation, and HNE-glutathione adducts were detected in the cytosol of phagocytic neutrophils. The ...
Free Radical Biology and Medicine, 2013
Free radical biology & medicine, Jan 22, 2015
Calprotectin provides nutritional immunity by sequestering manganese and zinc ions. It is abundan... more Calprotectin provides nutritional immunity by sequestering manganese and zinc ions. It is abundant in the lungs of patients with cystic fibrosis but fails to prevent their recurrent infections. Calprotectin is a major protein of neutrophils and composed of two monomers, S100A8 and S100A9. We show that the ability of calprotectin to limit growth of Staphylococcus aureus and Pseudomonas aeruginosa is exquisitely sensitive to oxidation by hypochlorous acid. The N-terminal cysteine residue on S100A9 was highly susceptible to oxidation which resulted in cross-linking of the protein monomers. The N-terminal methionine of S100A8 was also readily oxidized by hypochlorous acid, forming both methionine sulfoxide and the unique product dehydromethionine. Isolated human neutrophils formed these modifications on calprotectin when their myeloperoxidase generated hypochlorous acid. Up to 90% of the N-terminal amine on S100A8 in bronchoalveolar lavage fluid from young children with cystic fibrosis ...
Redox biology, Jan 22, 2014
Serotonin, 5-hydroxytryptamine, is a systemic bioactive amine that acts in the gut and brain. As ... more Serotonin, 5-hydroxytryptamine, is a systemic bioactive amine that acts in the gut and brain. As a substrate of myeloperoxidase in vitro, serotonin is oxidized to tryptamine-4,5-dione (TD), which is highly reactive with thiols. In this work, we successively prepared a monoclonal antibody to quinone-modified proteins and found that the antibody preferentially recognizes the TD-thiol adduct. Using the antibody, we observed that the chloride ion, the predominant physiological substrate for myeloperoxidase in vivo, is not competitive toward the enzyme catalyzed serotonin oxidation process, suggesting that serotonin is a plausible physiological substrate for the enzyme in vivo. Immunocytochemical analyses revealed that TD staining was observed in the cytosol of SH-SY5Y neuroblastoma cells while blot analyses showed that some cellular proteins were preferentially modified. Pull-down analyses confirmed that the cytoskeletal proteins tubulins, vimentin, and neurofilament-L were modified. Wh...
Free Radical Biology and Medicine, 2014
Neutrophils ingest and kill bacteria within phagocytic vacuoles. We investigated where they produ... more Neutrophils ingest and kill bacteria within phagocytic vacuoles. We investigated where they produce hypochlorous acid (HOCl) following phagocytosis by measuring conversion of protein tyrosine residues to 3-chlorotyrosine. We also examined how varying chloride availability affects the relationship between HOCl formation in the phagosome and bacterial killing. Phagosomal proteins, isolated following ingestion of opsonized magnetic beads, contained 11.4 Cl-Tyr per thousand tyrosine residues. This was 12 times higher than the level in proteins from the rest of the neutrophil and ~6 times higher than previously recorded for protein from ingested bacteria. These results indicate that HOCl production is largely localized to the phagosomes and a substantial proportion reacts with phagosomal protein before reaching the microbe. This will in part detoxify the oxidant but should also form chloramines which could contribute to the killing mechanism. Neutrophils were either suspended in chloride-free gluconate buffer or pretreated with formyl-Met-Leu-Phe, a procedure that has been reported to deplete intracellular chloride. These treatments, alone or in combination, decreased both chlorination in phagosomes and killing of Staphylococcus aureus by up to 50%. There was a strong positive correlation between the two effects. Killing was predominantly oxidant and myeloperoxidase dependent (88% inhibition by diphenylene iodonium and 78% by azide). These results imply that lowering the chloride concentration limits HOCl production and oxidative killing. They support a role for HOCl generation, rather than an alternative myeloperoxidase activity, in the killing process.
Journal of Biological Chemistry, 2015
Phagocytic neutrophils generate reactive oxygen species to kill microbes. Oxidant generation occu... more Phagocytic neutrophils generate reactive oxygen species to kill microbes. Oxidant generation occurs within an intracellular phagosome, but diffusible species can react with the neutrophil and surrounding tissue. To investigate the extent of oxidative modification we assessed the carbonylation of cytosolic proteins in phagocytic neutrophils. A four-fold increase in protein carbonylation was measured within 15 min of initiating phagocytosis. Carbonylation was dependent on NOX2 and myeloperoxidase activity, and was inhibited by butylated hydroxytoluene and Trolox, indicating a role for myeloperoxidase-dependent lipid peroxidation. Proteomic analysis of target proteins revealed significant carbonylation of the S100A9 subunit of calprotectin, a truncated form of Hsp70, actin, and hemoglobin from contaminating erythrocytes. Addition of the reactive aldehyde 4-hydroxynonenal (HNE) caused carbonylation, and HNE-glutathione adducts were detected in the cytosol of phagocytic neutrophils. The post-translational modification of neutrophil proteins will influence the functioning and fate of these immune cells in the period following phagocytic activation, and provides a marker of neutrophil activation during infection and inflammation.
Blood, 1998
IN THE 1880s Elie Metchnikoff observed specialized phagocytic cells ingesting bacteria, and recog... more IN THE 1880s Elie Metchnikoff observed specialized phagocytic cells ingesting bacteria, and recognized the importance of phagocytosis as a defense mechanism in multicellular organisms. 1 Neutrophils are one of the professional phagocytes in humans. They ingest ...
Atmospheric Chemistry in a Changing World, 2003
Despite its importance for particle formation and climate, relatively little effort has been spen... more Despite its importance for particle formation and climate, relatively little effort has been spent on understanding the sources and removal processes of NH 3 • Most work on atmospheric ammonia has been performed with respect to eutrophication and acidification close to the terrestrial sources; large scale transport and chemistry of NH 3 and ammonium (NHt) have received much less attention, especially over remote marine regions. The global source strength of ammonia is about megan@igacproject.org
The Biochemical journal, 2014
MIF (macrophage migration inhibitory factor) plays a central role in the promotion and maintenanc... more MIF (macrophage migration inhibitory factor) plays a central role in the promotion and maintenance of the inflammatory response. It is implicated in a number of inflammatory diseases including sepsis, arthritis and colitis, and in diseases with an inflammatory component, such as atherosclerosis, diabetes and cancer. MIF has an unusual N-terminal proline with catalytic activity, and targeting of this residue by small-molecule inhibitors has been shown to interfere with the biological activity of MIF. The objective of the present study was to determine if MIF was susceptible to modification by epicatechins, a group of dietary flavonoids with known anti-inflammatory properties. Epicatechins are substrates for peroxidases including neutrophil-derived MPO (myeloperoxidase). In the present study we show that oxidation of the catechol moiety of epicatechins to an ο-quinone by MPO generates potent MIF inhibitors. Near complete inhibition of MIF by the MPO/H2O2/epicatechin system was achieve...
Redox Report, 2006
Exhaled breath condensate (EBC) analysis has been proposed as a non-invasive method of assessing ... more Exhaled breath condensate (EBC) analysis has been proposed as a non-invasive method of assessing airway pathology. A number of substances, including hydrogen peroxide (H2O2), have been measured in EBC, without adequate published details of validation and optimisation. To explore factors that affect accurate quantitation of H2O2 in EBC. H2O2 was measured in EBC samples using fluorometry with 4-hydroxyphenylacetic acid. A number of factors that might alter quantitation were studied including pH and buffering conditions, reagent storage, and assay temperature. Standard curve slope was significantly altered by pH, leading to a potential difference in H2O2 quantification of up to 42%. These differences were resolved by increasing the buffering capacity of the reaction mix. H2O2 added to EBC remained stable for 1 h when stored on ice. The assay was unaffected by freezing assay reagents. The limit of detection for H2O2 ranged from 3.4 nM to 8.8 nM depending on the buffer used. The reagents required for this assay can be stored for several months allowing valuable consistency in longitudinal studies. The quantitation of H2O2 in EBC is pH-dependent but increasing assay buffering reduces this effect. Sensitive reproducible quantitation of H2O2 in EBC requires rigorous optimisation.
Journal of Biological Chemistry, 2014
Background: Lactoperoxidase plays a key role in host defence by oxidizing thiocyanate to the bact... more Background: Lactoperoxidase plays a key role in host defence by oxidizing thiocyanate to the bactericidal agent hypothiocyanite. Results: Urate is a good substrate for lactoperoxidase and competes with thiocyanate for oxidation in vitro. Conclusion: Urate is a likely physiological substrate for lactoperoxidase. Significance: Urate may influence the bactericidal activity of lactoperoxidase.
Free Radical Biology and Medicine, 2013
Free Radical Biology and Medicine, 2013
Free Radical Biology and Medicine, 2014
SUMMARY: Myeloperoxidase (MPO) uses hydrogen peroxide to oxidize chloride to hypochlorous acid. I... more SUMMARY: Myeloperoxidase (MPO) uses hydrogen peroxide to oxidize chloride to hypochlorous acid. It also converts numerous substrates to reactive free radicals. When released by neutrophils, the enzyme operates in the presence of a flux of superoxide. We show that superoxide has a profound influence on oxidative reactions catalysed by MPO. It reacts directly with the enzyme to modulate production of
Free Radical Biology and Medicine, 2015
Corrigendum to "Rapid reaction of superoxide with insulin-tyrosyl radicals to generate a hydroper... more Corrigendum to "Rapid reaction of superoxide with insulin-tyrosyl radicals to generate a hydroperoxide with subsequent glutathione addition" [Free Radic. Biol. Med. 70 (2014) 86-95] Please cite this article as: Das, AB; et al. Corrigendum to "Rapid reaction of superoxide with insulin-tyrosyl radicals to generate a hydroperoxide with subsequent.... Free Radic. Biol. Med. (2014), http://dx.
Chemical research in toxicology, Jan 5, 2015
Urate hydroperoxide is a strong oxidant generated by the combination of urate free radical and su... more Urate hydroperoxide is a strong oxidant generated by the combination of urate free radical and superoxide. The formation of urate hydroperoxide as an intermediate in urate oxidation is potentially responsible for the pro-oxidant effects of urate in inflammatory disorders, protein degradation, and food decomposition. To understand the molecular mechanisms that sustain the harmful effects of urate in inflammatory and oxidative stress related conditions, we report a detailed structural characterization and reactivity of urate hydroperoxide toward biomolecules. Urate hydroperoxide was synthesized by photo-oxidation and by a myeloperoxidase/hydrogen peroxide/superoxide system. Multiple reaction monitoring (MRM) and MS(3) ion fragmentation revealed that urate hydroperoxide from both sources has the same chemical structure. Urate hydroperoxide has a maximum absorption at 308 nm, ε308nm = 6.54 ± 0.38 × 10(3) M(-1) cm(-1). This peroxide decays spontaneously with a rate constant of k = 2.80 ±...
We have used a quantitative assay that measures independent rate constants for phagocytosis and k... more We have used a quantitative assay that measures independent rate constants for phagocytosis and killing of Staphylococcus aureusto investigate the involvement of superoxide and myeloperoxidase in bacterial killing by human neutrophils. To inhibit superoxide-dependent processes, superoxide dismutase was cross-linked to immunoglobulin G and the conjugate was attached to the surface of S. aureus via protein A in its cell wall.
Journal of Biological Chemistry
Phagocytic neutrophils generate reactive oxygen species to kill microbes. Oxidant generation occu... more Phagocytic neutrophils generate reactive oxygen species to kill microbes. Oxidant generation occurs within an intracellular phagosome, but diffusible species can react with the neutrophil and surrounding tissue. To investigate the extent of oxidative modification we assessed the carbonylation of cytosolic proteins in phagocytic neutrophils. A four-fold increase in protein carbonylation was measured within 15 min of initiating phagocytosis. Carbonylation was dependent on NOX2 and myeloperoxidase activity, and was inhibited by butylated hydroxytoluene and Trolox, indicating a role for myeloperoxidase-dependent lipid peroxidation. Proteomic analysis of target proteins revealed significant carbonylation of the S100A9 subunit of calprotectin, a truncated form of Hsp70, actin, and hemoglobin from contaminating erythrocytes. Addition of the reactive aldehyde 4-hydroxynonenal (HNE) caused carbonylation, and HNE-glutathione adducts were detected in the cytosol of phagocytic neutrophils. The ...
Free Radical Biology and Medicine, 2013
Free radical biology & medicine, Jan 22, 2015
Calprotectin provides nutritional immunity by sequestering manganese and zinc ions. It is abundan... more Calprotectin provides nutritional immunity by sequestering manganese and zinc ions. It is abundant in the lungs of patients with cystic fibrosis but fails to prevent their recurrent infections. Calprotectin is a major protein of neutrophils and composed of two monomers, S100A8 and S100A9. We show that the ability of calprotectin to limit growth of Staphylococcus aureus and Pseudomonas aeruginosa is exquisitely sensitive to oxidation by hypochlorous acid. The N-terminal cysteine residue on S100A9 was highly susceptible to oxidation which resulted in cross-linking of the protein monomers. The N-terminal methionine of S100A8 was also readily oxidized by hypochlorous acid, forming both methionine sulfoxide and the unique product dehydromethionine. Isolated human neutrophils formed these modifications on calprotectin when their myeloperoxidase generated hypochlorous acid. Up to 90% of the N-terminal amine on S100A8 in bronchoalveolar lavage fluid from young children with cystic fibrosis ...
Redox biology, Jan 22, 2014
Serotonin, 5-hydroxytryptamine, is a systemic bioactive amine that acts in the gut and brain. As ... more Serotonin, 5-hydroxytryptamine, is a systemic bioactive amine that acts in the gut and brain. As a substrate of myeloperoxidase in vitro, serotonin is oxidized to tryptamine-4,5-dione (TD), which is highly reactive with thiols. In this work, we successively prepared a monoclonal antibody to quinone-modified proteins and found that the antibody preferentially recognizes the TD-thiol adduct. Using the antibody, we observed that the chloride ion, the predominant physiological substrate for myeloperoxidase in vivo, is not competitive toward the enzyme catalyzed serotonin oxidation process, suggesting that serotonin is a plausible physiological substrate for the enzyme in vivo. Immunocytochemical analyses revealed that TD staining was observed in the cytosol of SH-SY5Y neuroblastoma cells while blot analyses showed that some cellular proteins were preferentially modified. Pull-down analyses confirmed that the cytoskeletal proteins tubulins, vimentin, and neurofilament-L were modified. Wh...
Free Radical Biology and Medicine, 2014
Neutrophils ingest and kill bacteria within phagocytic vacuoles. We investigated where they produ... more Neutrophils ingest and kill bacteria within phagocytic vacuoles. We investigated where they produce hypochlorous acid (HOCl) following phagocytosis by measuring conversion of protein tyrosine residues to 3-chlorotyrosine. We also examined how varying chloride availability affects the relationship between HOCl formation in the phagosome and bacterial killing. Phagosomal proteins, isolated following ingestion of opsonized magnetic beads, contained 11.4 Cl-Tyr per thousand tyrosine residues. This was 12 times higher than the level in proteins from the rest of the neutrophil and ~6 times higher than previously recorded for protein from ingested bacteria. These results indicate that HOCl production is largely localized to the phagosomes and a substantial proportion reacts with phagosomal protein before reaching the microbe. This will in part detoxify the oxidant but should also form chloramines which could contribute to the killing mechanism. Neutrophils were either suspended in chloride-free gluconate buffer or pretreated with formyl-Met-Leu-Phe, a procedure that has been reported to deplete intracellular chloride. These treatments, alone or in combination, decreased both chlorination in phagosomes and killing of Staphylococcus aureus by up to 50%. There was a strong positive correlation between the two effects. Killing was predominantly oxidant and myeloperoxidase dependent (88% inhibition by diphenylene iodonium and 78% by azide). These results imply that lowering the chloride concentration limits HOCl production and oxidative killing. They support a role for HOCl generation, rather than an alternative myeloperoxidase activity, in the killing process.
Journal of Biological Chemistry, 2015
Phagocytic neutrophils generate reactive oxygen species to kill microbes. Oxidant generation occu... more Phagocytic neutrophils generate reactive oxygen species to kill microbes. Oxidant generation occurs within an intracellular phagosome, but diffusible species can react with the neutrophil and surrounding tissue. To investigate the extent of oxidative modification we assessed the carbonylation of cytosolic proteins in phagocytic neutrophils. A four-fold increase in protein carbonylation was measured within 15 min of initiating phagocytosis. Carbonylation was dependent on NOX2 and myeloperoxidase activity, and was inhibited by butylated hydroxytoluene and Trolox, indicating a role for myeloperoxidase-dependent lipid peroxidation. Proteomic analysis of target proteins revealed significant carbonylation of the S100A9 subunit of calprotectin, a truncated form of Hsp70, actin, and hemoglobin from contaminating erythrocytes. Addition of the reactive aldehyde 4-hydroxynonenal (HNE) caused carbonylation, and HNE-glutathione adducts were detected in the cytosol of phagocytic neutrophils. The post-translational modification of neutrophil proteins will influence the functioning and fate of these immune cells in the period following phagocytic activation, and provides a marker of neutrophil activation during infection and inflammation.
Blood, 1998
IN THE 1880s Elie Metchnikoff observed specialized phagocytic cells ingesting bacteria, and recog... more IN THE 1880s Elie Metchnikoff observed specialized phagocytic cells ingesting bacteria, and recognized the importance of phagocytosis as a defense mechanism in multicellular organisms. 1 Neutrophils are one of the professional phagocytes in humans. They ingest ...
Atmospheric Chemistry in a Changing World, 2003
Despite its importance for particle formation and climate, relatively little effort has been spen... more Despite its importance for particle formation and climate, relatively little effort has been spent on understanding the sources and removal processes of NH 3 • Most work on atmospheric ammonia has been performed with respect to eutrophication and acidification close to the terrestrial sources; large scale transport and chemistry of NH 3 and ammonium (NHt) have received much less attention, especially over remote marine regions. The global source strength of ammonia is about megan@igacproject.org
The Biochemical journal, 2014
MIF (macrophage migration inhibitory factor) plays a central role in the promotion and maintenanc... more MIF (macrophage migration inhibitory factor) plays a central role in the promotion and maintenance of the inflammatory response. It is implicated in a number of inflammatory diseases including sepsis, arthritis and colitis, and in diseases with an inflammatory component, such as atherosclerosis, diabetes and cancer. MIF has an unusual N-terminal proline with catalytic activity, and targeting of this residue by small-molecule inhibitors has been shown to interfere with the biological activity of MIF. The objective of the present study was to determine if MIF was susceptible to modification by epicatechins, a group of dietary flavonoids with known anti-inflammatory properties. Epicatechins are substrates for peroxidases including neutrophil-derived MPO (myeloperoxidase). In the present study we show that oxidation of the catechol moiety of epicatechins to an ο-quinone by MPO generates potent MIF inhibitors. Near complete inhibition of MIF by the MPO/H2O2/epicatechin system was achieve...
Redox Report, 2006
Exhaled breath condensate (EBC) analysis has been proposed as a non-invasive method of assessing ... more Exhaled breath condensate (EBC) analysis has been proposed as a non-invasive method of assessing airway pathology. A number of substances, including hydrogen peroxide (H2O2), have been measured in EBC, without adequate published details of validation and optimisation. To explore factors that affect accurate quantitation of H2O2 in EBC. H2O2 was measured in EBC samples using fluorometry with 4-hydroxyphenylacetic acid. A number of factors that might alter quantitation were studied including pH and buffering conditions, reagent storage, and assay temperature. Standard curve slope was significantly altered by pH, leading to a potential difference in H2O2 quantification of up to 42%. These differences were resolved by increasing the buffering capacity of the reaction mix. H2O2 added to EBC remained stable for 1 h when stored on ice. The assay was unaffected by freezing assay reagents. The limit of detection for H2O2 ranged from 3.4 nM to 8.8 nM depending on the buffer used. The reagents required for this assay can be stored for several months allowing valuable consistency in longitudinal studies. The quantitation of H2O2 in EBC is pH-dependent but increasing assay buffering reduces this effect. Sensitive reproducible quantitation of H2O2 in EBC requires rigorous optimisation.
Journal of Biological Chemistry, 2014
Background: Lactoperoxidase plays a key role in host defence by oxidizing thiocyanate to the bact... more Background: Lactoperoxidase plays a key role in host defence by oxidizing thiocyanate to the bactericidal agent hypothiocyanite. Results: Urate is a good substrate for lactoperoxidase and competes with thiocyanate for oxidation in vitro. Conclusion: Urate is a likely physiological substrate for lactoperoxidase. Significance: Urate may influence the bactericidal activity of lactoperoxidase.
Free Radical Biology and Medicine, 2013
Free Radical Biology and Medicine, 2013
Free Radical Biology and Medicine, 2014