Lighting up H2O2: The Molecule that Is a “Necessary Evil” in the Cell

A cunning plan : In the multifunctional fluorescent probe MitoPY1 the phosphonium head group (red) targets mitochondria and the boronate group (green) responds to hydrogen peroxide. MitoPY1 reacts selectively with mitochondrial H₂O₂ in living cells, and an increase in fluorescence is triggered by the conversion of MitoPY1 into MitoPY1ox (yellow).

     

An ICT-based approach to ratiometric fluorescence imaging of hydrogen peroxide produced in living cells

We present the synthesis, properties, and biological applications of Peroxy Lucifer 1 (PL1), a new fluorescent probe for imaging hydrogen peroxide produced in living cells by a ratiometric response. PL1 utilizes a chemoselective boronate-based switch to detect hydrogen peroxide by modulation of internal charge transfer (ICT) within a 1,8-naphthalimide dye. PL1 features high selectivity for hydrogen peroxide over similar reactive oxygen species, including superoxide, and nitric oxide, and a 65 nm shift in emission from blue-colored fluorescence to green-colored fluorescence upon reaction with peroxide. Two-photon confocal microscopy experiments in live macrophages show that PL1 can ratiometrically visualize localized hydrogen peroxide bursts generated in living cells at immune response levels.     

A FRET-based approach to ratiometric fluorescence detection of hydrogen peroxide

We report the synthesis, properties, and biological applications of Ratio-Peroxyfluor-1 (RPF1), a new ratiometric fluorescent reporter for hydrogen peroxide. RPF1 is comprised of a two-fluorophore cassette, where the spectral overlap between coumarin donor and fluoran/fluorescein acceptor partners can be controlled by the chemoselective peroxide-mediated deprotection of boronic ester pendants on the acceptor dye. RPF1 features good selectivity for hydrogen peroxide over a variety of reactive oxygen species, including superoxide and nitric oxide, a ca. 8-fold increase in fluorescence intensity ratio (lambda517/lambda464) upon H2O2 reaction, and excitation and emission profiles in the visible region. Experiments with viable yeast mitochondria show that RPF1 can monitor and quantify endogenous production of H2O2, establishing the potential utility of this approach for probing peroxide biology in living systems.     

Hydrogen Peroxide‐mediated Inactivation of Two Chloroplastic Peroxidases,Ascorbate Peroxidase and 2‐Cys Peroxiredoxin†

Reactive oxygen species (ROS), such as the superoxide anion and hydrogen peroxide, are generated by the photosystems because photoexcited electrons are often generated in excess of requirements for CO₂ fixation and used for reducing molecular oxygen, even under normal environmental conditions. Moreover, ROS generation is increased in chloroplasts if plants are subjected to stresses, such as drought, high salinity and chilling. Chloroplast‐localized isoforms of ascorbate peroxidase and possibly peroxiredoxins assume the principal role of scavenging hydrogen peroxide. However, in vitro studies revealed that both types of peroxidases are easily damaged by hydrogen peroxide and lose their catalytic activities. This is one contributing factor for cellular damage that occurs under severe oxidative stress. In this review, I describe mechanisms of hydrogen peroxide‐mediated inactivation of these two enzymes and discuss a reason why they became susceptible to damage by hydrogen peroxide.     

Functional modification of cytochrome c by peroxynitrite in an electron transfer reaction.

The redox reaction of cytochrome c after modification with peroxynitrite under physiological conditions was investigated. Cytochrome c was treated with a bolus of synthetic peroxynitrite at a sub-millimolar concentration, and then subjected to reduction by superoxide and oxidation by hydrogen peroxide. The ability for the membrane potential formation in the mitochondrial respiratory chain was also evaluated. After the treatment with peroxynitrite, the cytochrome c molecule was mono-nitrated mainly at a tyrosine residue, using liquid chromatography-electrospray ionizing mass spectrometry (LC-ESI-MS) and HPLC. Although the redox capacity of cytochrome c was not affected by the peroxynitrite treatment, the oxidation of ferrocytochrome c to ferricytochrome c by hydrogen peroxide was accelerated. When cytochrome c was treated with peroxynitrite in the presence of 5-methoxytryptamine, an inhibitor for the tyrosine nitration by peroxynitrite, the acceleration of hydrogen peroxide-mediated oxidation was suppressed. It was also found that the formation of membrane potential in the rat liver mitochondria was suppressed when peroxynitrite-treated cytochrome c was used instead of the intact cytochrome c in vitro. From these results, we concluded that the peroxynitrite-treated cytochrome c was nitrated at a tyrosine residue and became more susceptible to oxidation by hydrogen peroxide, concomitantly losing the ability to transfer electrons in the mitochondrial respiratory chain. It is suggested that the peroxynitrite-induced modification of cytochrome c increases the susceptibility to non-physiological oxidants, and may cause dysfunction of mitochondria by suppressing of membrane potential.     

Localized surface plasmon resonance sensor for simultaneous kinetic determination of peroxyacetic acid and hydrogen peroxide

A new sensor for simultaneous determination of peroxyacetic acid and hydrogen peroxide using silver nanoparticles (Ag-NPs) as a chromogenic reagent is introduced. The silver nanoparticles have the catalytic ability for the decomposition of peroxyacetic acid and hydrogen peroxide; then the decomposition of them induces the degradation of silver nanoparticles. Hence, a remarkable change in the localized surface plasmon resonance absorbance strength could be observed. Spectra-kinetic approach and artificial neural network was applied for the simultaneous determination of peroxyacetic acid and hydrogen peroxide. Linear calibration graphs were obtained in the concentration range of (8.20 × 10⁻⁵ to 2.00 × 10⁻³ mol L⁻¹) for peroxyacetic acid and (2.00 × 10⁻⁵ to 4.80 × 10⁻³ mol L⁻¹) for hydrogen peroxide. The analytical performance of this sensor has been evaluated for the detection of simultaneous determination of peroxyacetic acid and hydrogen peroxide in real samples.     

Determination of superoxide dismutase and SOD-mimetic activities by a chemical system: Co2/H2O2/lucigenin

The bright chemiluminescence has been observed in the system: Co²⁺/hydrogen peroxide/lucigenin. The chemiluminescence intensity was directly proportional to either cobalt, hydrogen peroxide, or lucigenin concentrations. A procedure of determination of superoxide dismutase (SOD) activity by the chemiluminescence method in the cobalt–hydrogen peroxide–lucigenin system at pH 8.5 is suggested. A linear dependence was established between a relative chemiluminescence intensity and SOD concentration in the range of SOD concentrations between 0 and 4.5 nM, c _1/2 = 0.8 nM. The determination of SOD activity was performed in several tissue samples (rat plasma, erythrocyte hemolysate, and liver mitochondria). A technique of tissue sample preparation with the use of thermal inactivation of interfering proteins at 60 °C was used. The method was successfully applied for comparison of the efficiency of SOD mimetics.     

Quantitative analysis of hydrogen peroxide by <Superscript>1</Superscript>H NMR spectroscopy

A technique utilizing ¹H NMR spectroscopy has been developed to measure the concentration of hydrogen peroxide from 10^–3 to 10 M. Hydrogen peroxide produces a peak at around 10–11 ppm, depending upon the interaction between solvent molecules and hydrogen peroxide molecules. The intensity of this peak can be monitored once every 30 s, enabling the measurement of changes in hydrogen peroxide concentration as a function of time. ¹H NMR has several advantages over other techniques: (1) applicability to a broad range of solvents, (2) ability to quantify hydrogen peroxide rapidly, and (3) ability to follow reactions forming and/or consuming hydrogen peroxide as a function of time. As an example, this analytical technique has been used to measure the concentration of hydrogen peroxide as a function of time in a study of hydrogen peroxide decomposition catalyzed by iron(III) tetrakispentafluorophenyl porphyrin.Electronic Supplementary Material Supplementary material is available for this article at     

Fluorometric determination of hydrogen peroxide using resorufin and peroxidase

A fluorometric method for the determination of hydrogen peroxide using resorufin as a substrate for peroxidase is described. Two procedures were developed for the determination of hydrogen peroxide. One involves the addition of hydrogen peroxide sample to a solution of peroxidase and resorufin in phosphate buffer, pH 6.4. Fluorescence measurements are performed before and after hydrogen peroxide addition. The within-run CVs for final concentrations of hydrogen peroxide of 200 and 40 nmol/liter were 1.7 and 7.6%, respectively, and the limit of quantitation was 9 nmol/liter. The second procedure, in which the initial reaction of hydrogen peroxide with resorufin is performed in citrate buffer at pH 4.5, and then the fluorescence is measured after the pH is adjusted to 9.2 with borate buffer, has a limit of quantitation of 4.4 nmol/liter with a within-run CV of 6.5% for a final hydrogen peroxide concentration of 20 nmol/liter. The method is linear at least up to 1 μmol/liter.     

Electrochemical catalysis of styrene epoxidation with films of MnO(2) nanoparticles and H(2)O(2)

Films of polyions and octahedral layered manganese oxide (OL-1) nanoparticles on carbon electrodes made by layer-by-layer alternate electrostatic adsorption were active for electrochemical catalysis of styrene epoxidation in solution in the presence of hydrogen peroxide and oxygen. The highest catalytic turnover was obtained by using applied voltage -0.6 V vs SCE, O(2), and 100 mM H(2)O(2). (18)O isotope labeling experiments suggested oxygen incorporation from three different sources: molecular oxygen, hydrogen peroxide, and/or lattice oxygen from OL-1 depending on the potential applied and the oxygen and hydrogen peroxide concentrations. Oxygen and hydrogen peroxide activate the OL-1 catalyst for the epoxidation. The pathway for styrene epoxidation in the highest yields required oxygen, hydrogen peroxide, and a reducing voltage and may involve an activated oxygen species in the OL-1 matrix.     

Neuroprotective Bioorthogonal Catalysis in Mitochondria Using Protein-Integrated Hydrogen-Bonded Organic Frameworks

Mitochondria-targeted bioorthogonal catalysis holds promise for controlling cell function precisely, yet achieving selective and efficient chemical reactions within organelles is challenging. In this study, we introduce a new strategy using protein-integrated hydrogen-bonded organic frameworks (HOFs) to enable synergistic bioorthogonal chemical catalysis and enzymatic catalysis within mitochondria. Utilizing catalytically active tris(4,4′-dicarboxylicacid-2,2′-bipyridyl) ruthenium(II) to self-assemble with [1,1′-biphenyl]-4,4′-biscarboximidamide, we synthesized nanoscale RuB-HOFs that exhibit high photocatalytic reduction activity. Notably, RuB-HOFs efficiently enter cells and preferentially localize to mitochondria, where they facilitate bioorthogonal photoreduction reactions. Moreover, we show that RuB-HOFs encapsulating catalase can produce hydrogen sulfide (H₂S) in mitochondria through photocatalytic reduction of pro-H₂S and degrade hydrogen peroxide through enzymatic catalysis simultaneously, offering a significant neuroprotective effect against oxidative stress. Our findings not only introduce a versatile chemical toolset for mitochondria-targeted bioorthogonal catalysis for prodrug activation but also pave the way for potential therapeutic applications in treating diseases related to cellular oxidative stress.     

Determination of hydrogen peroxide by iron(III) complex of thiacalix[4]arenetetrasulfonate on a modified ion-exchanger with peroxidase-like catalytic activity.

An iron(III) complex of thiacalix[4]arenetetrasulfonate on a modified anion-exchanger (Fe3+-TCAS(A-500)) has shown high peroxidase-like activity at pH 5 - 6 for the reaction of quinoid-dye formation between 3-methyl-2-benzothiazolinone hydrazone and N-(3-sulfopropyl)aniline in the presence of hydrogen peroxide. Utilizing the peroxidase-like activity of Fe3+-TCAS(A-500) for this reaction, a method using Fe3+-TCAS(A-500) was applied for the spectrophotometric determination of hydrogen peroxide. The calibration curve by the method using Fe3+-TCAS(A-500) was linear over the range from 1 to 10 microg of hydrogen peroxide in a 1 ml sample solution. The apparent molar absorptivity for hydrogen peroxide was 2.4 x 10(4) l mol(-1) cm(-1). which was about 80% of that by peroxidase under the same conditions. This determination method of hydrogen peroxide using Fe3+-TCAS(A-500) was applied for the determination of glucose in diluted normal and abnormal control serum I and II.     

Determination of hydrogen peroxide by xenon trioxide oxidation

Aqueous xenon trioxide solution has been used as the oxidizing agent in three precise methods of analysis for hydrogen peroxide. A catalytic method, which utilizes hydrogen peroxide to initiate the reaction between t-butanol and xenon trioxide, is described for determining amounts of hydrogen peroxide as low as 0.9 microg or 36 parts per milliard (ppM). A direct spectrophotometric titration was found to have a lower limit of about 50 microg, or 20 ppM. An indirect titrimetric method was also used to determine hydrogen peroxide in amounts as low as 50 microg with a relative standard deviation of 4% which decreased to 1 % for amounts over 200 microg.     

The reduction of ferricyanides and a sensitive test for the detection of hydrogen peroxide

Yellow zinc ferricyanide is reduced by heating to white zinc ferrocyanide by hydrogen peroxide in the presence of zinc sulphate and sodium, acetate. Copper ferricyanide, however, is reduced to brown copper ferrocyanide at room temperature, by means of hydrogen peroxide in the presence of copper sulphate and sodium acetate. The latter reaction can be applied for the detection of extremely small quantities of hydrogen peroxide both in a test tube (2.5 γ in 1 ml) and as a spot test (0.5 to 1 γ).     

Magnesium hydroperoxyacetate (MHPA) and magnesium dihydroperoxide (MDHP): New antibacterial agents for fibrous substrates

Water-dispersable products have been prepared from the reaction of magnesium acetate tetrahydrate with hydrogen peroxide at mole ratios of 1 : 2 to 1 : 40 to produce compositions with active oxygen or peroxide contents of 1–30%. The products are believed to be stoichiometric mixtures of HOO Mg OAc and HOO Mg OOH that vary in composition with the molar ratios used. These new compounds are hydrolytically stable at ambient temperatures for extended periods (at least 60 days) and thermally stable below 300°C. Pad-cure processes are described for applying the above reaction products as a dispersion in water or aqueous hydrogen peroxide or as a foam in aqueous hydrogen peroxide to impart antibacterial activity to celulosics, synthetic fibers and fiber blends. The textiles are treated with dispersions or foam containing 10–17% of the reaction products derived from mole ratios of 1 : 2 to 1 : 40 magnesium acetate tetrahydrate: hydrogen peroxide. On subsequent heating for 2–4 min at 120–150°C, washing and drying, the modified textiles contain durably bound active oxygen or peroxide (0.1–1.7%) that has activity against representative gram-positive and gramnegative bacteria for up to 50 launderings.     

Sensitive Flow-Injection Electrochemical Determination of Hydrogen Peroxide at a Palladium Nanoparticle-Modified Pencil Graphite Electrode

A novel flow-injection amperometric method was proposed for the sensitive and enzymeless determination of hydrogen peroxide based on its electrocatalytic reduction at a palladium nanoparticle-modified pretreated pencil graphite electrode in a laboratory-constructed electrochemical flow cell. Cyclic voltammograms of the unmodified and modified electrodes were recorded in pH 7.0 phosphate buffer containing 0.10 M KCl at a scan rate of 50?mV s^?1 for the investigation of electrocatalytic reduction of hydrogen peroxide at the palladium nanoparticle-modified pretreated pencil graphite electrode. Cyclic voltammograms of the pretreated pencil graphite electrode revealed an irreversible oxidation peak and a weak reduction peak of hydrogen peroxide at +1100?mV and –450?mV vs. an Ag/AgCl/KCl saturated reference electrode. However, the reduction of hydrogen peroxide was observed at –100?mV with an increase in current in the cyclic voltammograms of the palladium nanoparticle-modified pretreated pencil graphite electrode compared to the unmodified electrode. These results indicate that the palladium nanoparticle-modified pretreated pencil graphite electrode exhibits efficient electrocatalytic activity for the reduction of hydrogen peroxide. A linear concentration range was obtained between .01 and 10.0?mM hydrogen peroxide with a detection limit of 3.0 µM from flow injection amperometric current–time curves recorded in pH 7.0 phosphate buffer at –100?mV and a 2.0?mL min^?1 flow rate. The novelty of this work relies on its use of a laboratory-constructed flow cell constructed for the pencil graphite electrode using these inexpensive, disposable, and electrochemically reactive modified electrodes for the amperometric determination of hydrogen peroxide in a flow injection analysis system.     

A specific bio-electrochemical sensor for hydrogen peroxide

A bio-electrochemical sensor specific for hydrogen peroxide is described. The sensor consists of two membranes—a catalase-collagen membrane and a teflon membrane—an alkaline solution, a platinum cathode and a lead anode. The catalase-collagen membrane is prepared electrochemically, the thickness being 1 μ; the enzyme activity is similar to that of native catalase. The sensor responds to hydrogen peroxide with a response time of only 1–2 min. The calibration curve is quite linear over a concentration range of 0–1.5 mmol l^-1 for hydrogen peroxide. The utility of the sensor in continuous usage is discussed.     

An Unmediated Hydrogen Peroxide Sensor Based on a Hemoglobin-sds Film Modified Electrode

ABSTRACT

An unmediated hydrogen peroxide sensor is designed in this paper by employing a hemoglobin-SDS film modified electrode. Hemoglobin exhibits direct (unmediated) electrochemistry at the modified electrode. The protein also shows elegant catalytic activity towards the electrochemical reduction of hydrogen peroxide. Consequently, a prototype hydrogen peroxide sensor is prepared. Under optimum conditions, this sensor provides a linear response over the hydrogen peroxide concentrations in the range of 1×10^-5～1×10^-4 mol/L. The detection limit was 2×10^-6 mol/L The relative standard deviation was 4.2% for 6 successive determinations of the hydrogen peroxide at 1×10^-5 mol/L. This configuration is shown to be sensitive, stable and easily fabricated. It might be useful in the biological and industrial fields.     

Reaction of H with H2O2 as observed by optical absorption of perhydroxyl radicals or aliphatic alcohol radicals and of OH with H2O2. A pulse radiolysis study

Two new procedures were employed for studying the reaction of hydrogen atoms with hydrogen peroxide. The absorption in the UV-range was observed either for an acidic aqueous solution containing only hydrogen peroxide or for a similar solution but also containing an aliphatic alcohol. From the increase in absorption of various alcohol radicals, a rate constant of 3.5×10⁷ dm³ mol⁻¹ s⁻¹ was determined. In addition, the rate constant for the reaction of hydroxyl radicals with hydrogen peroxide was determined to be 3.0×10⁷ dm³ mol⁻¹ s⁻¹.     

Colorimetric Determination Of Hydrogen Peroxide Based on Localized Surface Plasmon Resonance of Silver Nanoprisms Using a Microchannel Chip

Silver nanoprisms (AgNPrs) have unique optical phenomena due to their localized surface plasmon resonance that results in the extinction of light from the visible to the near-infrared spectral region. In this study, we propose the colorimetric determination of silver nanoprisms in microchannels using a smartphone camera. Image acquisition was performed by capturing an image of the colloidal solution of the silver nanoprisms in the microchannel using the transmitted light. Red, green, and blue chromaticity levels were extracted from the recorded images for further quantification of the silver nanoprisms. This technique was employed for the detection and colorimetric determination of hydrogen peroxide (H₂O₂). Good linearity between the change in the green chromaticity level and concentration of hydrogen peroxide was observed for values from 10 to 300?μM with an R² value of 0.9670. We anticipate that the developed methodology for the quantification of silver nanoprisms and hydrogen peroxide by monitoring the change in color in the images of transmitted light will enhance the development of simple, rapid, and reliable detection systems for quality control in the production of silver nanoprisms as well as in chemical sensor applications.