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1.
Nat Commun ; 13(1): 171, 2022 01 10.
Article in English | MEDLINE | ID: mdl-35013284

ABSTRACT

The lack of tools to monitor the dynamics of (pseudo)hypohalous acids in live cells and tissues hinders a better understanding of inflammatory processes. Here we present a fluorescent genetically encoded biosensor, Hypocrates, for the visualization of (pseudo)hypohalous acids and their derivatives. Hypocrates consists of a circularly permuted yellow fluorescent protein integrated into the structure of the transcription repressor NemR from Escherichia coli. We show that Hypocrates is ratiometric, reversible, and responds to its analytes in the 106 M-1s-1 range. Solving the Hypocrates X-ray structure provided insights into its sensing mechanism, allowing determination of the spatial organization in this circularly permuted fluorescent protein-based redox probe. We exemplify its applicability by imaging hypohalous stress in bacteria phagocytosed by primary neutrophils. Finally, we demonstrate that Hypocrates can be utilized in combination with HyPerRed for the simultaneous visualization of (pseudo)hypohalous acids and hydrogen peroxide dynamics in a zebrafish tail fin injury model.


Subject(s)
Animal Fins/diagnostic imaging , Bacterial Proteins/genetics , Biosensing Techniques/methods , Fluorescent Dyes/chemistry , Hypochlorous Acid/analysis , Luminescent Proteins/genetics , Animal Fins/injuries , Animal Fins/metabolism , Animals , Bacterial Proteins/metabolism , Biosensing Techniques/instrumentation , Crystallography, X-Ray , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Genes, Reporter , Hydrogen Peroxide/chemistry , Hypochlorous Acid/chemical synthesis , Hypochlorous Acid/metabolism , Luminescent Proteins/metabolism , Neutrophils/cytology , Neutrophils/immunology , Oxidation-Reduction , Phagocytosis , Transcription Factors/genetics , Transcription Factors/metabolism , Zebrafish
2.
Anal Chem ; 92(12): 8285-8291, 2020 06 16.
Article in English | MEDLINE | ID: mdl-32456421

ABSTRACT

Biomolecule tracing with different imaging methods is of great significance for more accurately unravelling the fundamental processes in living systems. However, considering the different principles of each imaging method for probe design, it is still a great challenge to apply one molecular probe to achieve two or even more imaging analyses for biomarkers. In general, traditional oxime was reported as a recognition group for fluorescence imaging of HOCl. Herein, for the first time, we designed the oxime decorated iridium(III) complex, which can be directly used for chemiluminescence as well as two-photon luminescence and photoluminescence lifetime imaging of HOCl in living systems. Moreover, the novel chemiluminescence mechanism of Ir-CLFLPLIM for HOCl was also proposed and explored by continuously monitoring chemiluminescence peak shapes and mass spectra, inferring the reaction intermediate and calculating the chemical reaction energy range of the reaction process. This strategy could lead us to expand the chemiluminescence application of transition metal complexes and develop more multimodal imaging probes.


Subject(s)
Carcinoma, Hepatocellular/diagnostic imaging , Coordination Complexes/chemistry , Fluorescent Dyes/chemistry , Hypochlorous Acid/analysis , Iridium/chemistry , Liver Neoplasms/diagnostic imaging , Optical Imaging , Animals , Cell Line, Tumor , Coordination Complexes/chemical synthesis , Fluorescent Dyes/chemical synthesis , Humans , Hypochlorous Acid/chemical synthesis , Liver Neoplasms, Experimental/diagnostic imaging , Mice , Mice, Inbred BALB C , Mice, Nude , Molecular Structure
3.
J Phys Chem B ; 120(48): 12261-12271, 2016 12 08.
Article in English | MEDLINE | ID: mdl-27934228

ABSTRACT

The most abundant plasma protein, human serum albumin (HSA), plays a key part in the body's antioxidant defense against reactive species. This study was aimed at correlating oxidant-induced chemical and structural effects on HSA. Despite the chemical modification induced by the oxidant hypochlorite, the native shape is preserved up to oxidant/HSA molar ratio <80, above which a structural transition occurs in the critical range 80-120. This conformational variation involves the drifting of one of the end-domains from the rest of the protein and corresponds to the loss of one-third of the α-helix and a net increase of the protein negative charge. The transition is highly reproducible suggesting that it represents a well-defined structural response typical of this multidomain protein. The ability to tolerate high levels of chemical modification in a folded or only partially unfolded state, as well as the stability to aggregation, provides albumin with optimal features as a biological buffer for the local formation of oxidants.


Subject(s)
Hypochlorous Acid/chemical synthesis , Serum Albumin/chemistry , Humans , Hypochlorous Acid/chemistry , Hypochlorous Acid/metabolism , Oxidation-Reduction , Protein Conformation , Serum Albumin/metabolism
4.
Free Radic Biol Med ; 63: 90-8, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23624305

ABSTRACT

Myeloperoxidase (MPO) is a heme-containing enzyme that generates hypochlorous acid (HOCl) from chloride (Cl(-)) and hydrogen peroxide (H2O2). It is implicated in the pathology of several chronic inflammatory conditions such as cardiovascular and pulmonary diseases and cancer. Recently we have shown that HOCl can destroy the heme prosthetic group of hemoproteins. Here, we investigated whether the HOCl formed during steady-state catalysis is able to destroy the MPO heme moiety and thereby function as a major source of free iron. UV-visible spectra and H2O2-specific electrode measurements recorded during steady-state HOCl synthesis by MPO showed that the degree of MPO heme destruction increased after multiple additions of H2O2 (10 µM), precluding the enzyme from functioning at maximum activity (80-90% inhibition). MPO heme destruction occurred only in the presence of Cl(-). Stopped-flow measurements revealed that the HOCl-mediated MPO heme destruction was complex and occurred through transient ferric species whose formation and decay kinetics indicated it participates in heme destruction along with subsequent free iron release. MPO heme depletion was confirmed by the buildup of free iron utilizing the ferrozine assay. Hypochlorous acid, once generated, first equilibrates in the solution as a whole before binding to the heme iron and initiating heme destruction. Eliminating HOCl from the MPO milieu by scavenging HOCl, destabilizing the MPO-Compound I-Cl complex that could be formed during catalysis, and/or inhibiting MPO catalytic activity partially or completely protects MPO from HOCl insults. Collectively, this study elucidates the bidirectional relationship between MPO and HOCl, which highlights the potential role of MPO as a source of free iron.


Subject(s)
Free Radicals/metabolism , Hydrogen Peroxide/metabolism , Iron/metabolism , Peroxidase/metabolism , Catalysis , Chlorides/metabolism , Feedback, Physiological , Ferrozine/metabolism , Heme/chemistry , Heme/metabolism , Humans , Hydrogen Peroxide/pharmacology , Hypochlorous Acid/chemical synthesis , Hypochlorous Acid/metabolism , Inflammation/metabolism , Inflammation/pathology , Kinetics , Metabolic Networks and Pathways , Oxidation-Reduction , Peroxidase/chemistry
5.
J Am Chem Soc ; 134(51): 20617-20, 2012 Dec 26.
Article in English | MEDLINE | ID: mdl-23214510

ABSTRACT

A hypochloritoiron(III) porphyrin species has been proposed as a key intermediate in an antimicrobial defense system in neutrophils and in heme-catalyzed chlorination reactions. We report herein the preparation, spectroscopic characterization, and reactivity of the bis(hypochlorito)iron(III) porphyrin complex [(TPFP)Fe(III)(OCl)(2)](-) (1) and the imidazole-hypochloritoiron complexes (TPFP)Fe(III)(OCl)(1-R-Im) [R = CH(3) (2), H (3), CH(2)CO(2)H (4)], in which TPFP is 5,10,15,20-tetrakis(pentafluorophenyl)porphyrinate. The structures of 1-4 were confirmed by absorption, (2)H and (19)F NMR, EPR, and resonance Raman spectroscopy and electrospray ionization mass spectrometry at low temperature. The reactions of 1 and 2 with various organic substrates show that 1 and 2 are capable of chlorination, sulfoxidation, and epoxidation reactions and that 1 is much more reactive with these substrates than 2.


Subject(s)
Ferric Compounds/chemistry , Hydrocarbons, Chlorinated/chemistry , Hypochlorous Acid/chemistry , Imidazoles/chemistry , Porphyrins/chemistry , Epoxy Compounds/chemistry , Ferric Compounds/chemical synthesis , Halogenation , Hydrocarbons, Chlorinated/chemical synthesis , Hypochlorous Acid/chemical synthesis , Imidazoles/chemical synthesis , Porphyrins/chemical synthesis , Spectrum Analysis , Sulfoxides/chemistry
6.
Bioorg Khim ; 37(4): 510-21, 2011.
Article in Russian | MEDLINE | ID: mdl-22096994

ABSTRACT

Broad prospects for the use of single-walled carbon nanotubes (SWNTs) in medicine and biotechnology raise the concerns about both their toxicity, and the mechanisms of biodegradation and excretion from the body. SWNTs biodegradation as a result of catalytic activity of myeloperoxidase (MPO) was shown in the isolated MPO system as well as in the suspension of neutrophils [Kagan V.E., et al., 2010]. In the present study we analyzed the ability of different MPO-produced oxidants to participate in the modification and degradation of SWNTs. The comparison of the ability of various peroxidases to degrade SWNTs in vitro revealed that myeloperoxidase, due to its ability to produce hypochlorite, and lactoperoxidase, due to its ability to produce hypobromite, are extremely efficient in the degradation of carbon nanotubes. The biodegradation of SWNTs in the model system can also be caused by free radicals generated as a result of heme degradation and, to a lesser extent, by active oxoferryl intermediates of peroxidases. Our experiments showed that in the presence of blood plasma, peroxidase intermediates or free radical products of heme degradation were unable to initiate biodegradation of carbon nanotubes, only the generation of hypochlorite by MPO can cause the biodegradation of carbon nanotubes in vivo. Titration of SWNTs suspension containing plasma with hypochlorite at high concentrations resulted in the decrease in the optical absorbance of the suspension indicating the degradation of nanotubes. Our results clearly indicate that hypochlorite can serve as a main oxidizing agent which is able to modify and degrade nanotubes in the sites of inflammation and in the phagosomes.


Subject(s)
Hypochlorous Acid/chemical synthesis , Nanotubes, Carbon/chemistry , Peroxidase/chemistry , Biodegradation, Environmental , Humans , Lactoperoxidase/chemistry , Oxidation-Reduction , Plasma/chemistry
7.
Environ Microbiol ; 11(6): 1330-9, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19453612

ABSTRACT

Several studies have demonstrated that extensive formation of organically bound chlorine occurs both in soil and in decaying plant material. Previous studies suggest that enzymatic formation of reactive chlorine outside cells is a major source. However, the ecological role of microbial-induced extracellular chlorination processes remains unclear. In the present paper, we assess whether or not the literature supports the hypothesis that extracellular chlorination is involved in direct antagonism against competitors for the same resources. Our review shows that it is by no means rare that biotic processes create conditions that render biocidal concentrations of reactive chlorine compounds, which suggest that extracellular production of reactive chlorine may have an important role in antagonistic microbial interactions. To test the validity, we searched the UniprotPK database for microorganisms that are known to produce haloperoxidases. It appeared that many of the identified haloperoxidases from terrestrial environments are originating from organisms that are associated with living plants or decomposing plant material. The results of the in silico screening were supported by various field and laboratory studies on natural chlorination. Hence, the ability to produce reactive chlorine seems to be especially common in environments that are known for antibiotic-mediated competition for resources (interference competition). Yet, the ability to produce haloperoxidases is also recorded, for example, for plant endosymbionts and parasites, and there is little or no empirical evidence that suggests that these organisms are antagonistic.


Subject(s)
Antibiosis , Chlorine/metabolism , Environmental Microbiology , Anti-Infective Agents/chemical synthesis , Anti-Infective Agents/metabolism , Bacteria/growth & development , Chloride Peroxidase/metabolism , Chlorine/analysis , Fungi/growth & development , Hypochlorous Acid/chemical synthesis , Hypochlorous Acid/metabolism , Organic Chemicals/chemistry , Plants/chemistry , Soil/analysis
8.
Chemistry ; 14(15): 4719-24, 2008.
Article in English | MEDLINE | ID: mdl-18386284

ABSTRACT

A new rhodamine B-based fluorescent probe for the hypochlorite anion (OCl(-)) has been designed, synthesized, and characterized. The probe comprises a spectroscopic unit of rhodamine B and an OCl(-)-specific reactive moiety of dibenzoylhydrazine. The probe itself is nearly nonfluorescent because of its spirolactam structure. Upon reaction with OCl(-), however, a largely enhanced fluorescence is produced due to the opening of the spirolactam ring by the oxidation of the exocyclic hydrazide and subsequently the formation of the hydrolytic product rhodamine B. Most notably, the fluorescence-on reaction shows high sensitivity and extremely high selectivity for OCl(-) over other common ions and oxidants, which makes it possible for OCl(-) to be detected directly in their presence. In addition, the reaction mechanism has been investigated and proposed. The OCl(-) anion selectively oxidizes the hydrazo group in the probe, and forms the analogue of dibenzoyl diimide, which in turn hydrolyzes and releases the fluorophore. The reaction mechanism that is described here might be useful in developing excellent spectroscopic probes with cleavable active bonds for other species.


Subject(s)
Fluorescent Dyes/chemistry , Fluorescent Dyes/chemical synthesis , Hypochlorous Acid/chemistry , Hypochlorous Acid/chemical synthesis , Rhodamines/chemistry , Rhodamines/chemical synthesis , Anions/chemistry , Crystallography, X-Ray , Hypochlorous Acid/analysis , Models, Molecular , Molecular Structure , Time Factors
9.
Bioorg Med Chem ; 14(18): 6409-13, 2006 Sep 15.
Article in English | MEDLINE | ID: mdl-16759870

ABSTRACT

The gallic acid and several n-alkyl gallates, with the same number of hydroxyl substituents, varying only in the side carbonic chain length, with respective lipophilicity defined through the C log P, were studied. It evidenced the structure-activity relationship of the myeloperoxidase activity inhibition and the hypochlorous acid scavenger property, as well as its low toxicity in rat hepatic tissue. The gallates with C log P below 3.0 (compounds 2-7) were more active against the enzyme activity, what means that the addition of 1-6 carbons (C log P between 0.92 and 2.92) at the side chain increased approximately 50% the gallic acid effect. However, a relationship between the HOCl scavenging capability and the lipophilicity was not observed. With these results it is possible to suggest that the gallates protect the HOCl targets through two mechanisms: inhibiting its production by the enzyme and scavenging the reactive specie.


Subject(s)
Esters/chemistry , Gallic Acid/analogs & derivatives , Gallic Acid/chemistry , Hypochlorous Acid/chemical synthesis , Peroxidase/chemistry , Animals , Cell Survival/drug effects , Chemical Phenomena , Chemistry, Physical , Chlorocebus aethiops , Enzyme Activation/drug effects , Esters/pharmacology , Free Radical Scavengers/chemistry , Gallic Acid/pharmacology , Hydrophobic and Hydrophilic Interactions , Hypochlorous Acid/chemistry , Liver/chemistry , Liver/drug effects , Liver/metabolism , Lung/chemistry , Lung/enzymology , Male , Peroxidase/antagonists & inhibitors , Rats , Rats, Wistar , Software , Solubility , Structure-Activity Relationship , Vero Cells
10.
Free Radic Biol Med ; 39(11): 1468-77, 2005 Dec 01.
Article in English | MEDLINE | ID: mdl-16274882

ABSTRACT

We describe a new assay for the chlorination activity of myeloperoxidase and detection of chloramines. Chloramines were detected by using iodide to catalyze the oxidation of either 3,3',5,5'-tetramethylbenzidine (TMB) or dihydrorhodamine to form strongly absorbing or fluorescent products, respectively. With TMB as little as 1 muM taurine chloramine could be detected. The sensitivity of the dihydrorhodamine assay was about 10-fold greater. The chlorination activity of myeloperoxidase was measured by trapping hypochlorous acid with taurine and subsequently using iodide to promote the oxidation reactions of the accumulated taurine chloramine. A similar approach was used to detect hypochlorous acid production by stimulated human neutrophils. Iodide-dependent catalysis distinguished N-chloramines from N-bromamines. This allows for discrimination between heme peroxidases that generate either hypochlorous acid or hypobromous acid. The assay has distinct advantages over existing assays for myeloperoxidase with regard to sensitivity, specificity, and its ease and versatility of use.


Subject(s)
Chloramines/analysis , Peroxidase/metabolism , Benzidines/chemistry , Bromides/analysis , Chloramines/metabolism , Humans , Hypochlorous Acid/chemical synthesis , Iodides/chemistry , Neutrophils/metabolism , Rhodamines/chemistry , Sensitivity and Specificity , Taurine/analogs & derivatives , Taurine/chemistry
11.
Environ Sci Technol ; 38(11): 3203-8, 2004 Jun 01.
Article in English | MEDLINE | ID: mdl-15224756

ABSTRACT

The production of chlorine and hypochlorite is of great economical and technological interest due to their large-scale use in many kinds of commercial applications. Yet, the current processes are not without problems such as inevitable side reactions and the high cost of production. This work reports the photoelectrocatalytic oxidation of chloride ions to free chlorine as it has been investigated by using titanium dioxide (TiO2) and several metal-doped titanium dioxide (M-TiO2) material electrodes. An average concentration of 800 mg L(-1) of free chlorine was obtained in an open-air reactor using a TiO2 thin-film electrode biased at +1.0 V (SCE) and illuminated by UV light. The M-doped electrodes have performed poorly compared with the pure TiO2 counterpart. Test solutions containing 0.05 mol L(-1) NaCl pH 2.0-4.0 were found to be the best conditions for fast production of free chlorine. A complete investigation of all parameters that influence the global process of chlorine production by the photo electrocatalytic method such as applied potential, concentration of NaCl, pH solution, and time is presented in detail. In addition, photocurrent vs potential curves and the reaction order are also discussed.


Subject(s)
Chlorine/analysis , Hypochlorous Acid/chemical synthesis , Nanotechnology , Chlorides/chemistry , Conservation of Natural Resources , Crystallization , Electrochemistry , Electrodes , Ions , Oxidation-Reduction , Photochemistry
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