Nebraska Redox Biology Center Educational Portal

Hydrogen Peroxide

Hydrogen peroxide (H2O2) is the nonradical product of 2-electron oxygen reduction. It is colorless liquid that is completely miscible in water and alcohol. It contain two hydrogens and two oxygens linked by covalent bond and known as a normal aerobic metabolite and strong oxydant [ 1, 2, 3 ]. This compound has diverse array of physiological and pathological effects within living cells depending on the extent, timing, and location of its production. H2O2 is a key molecule that is involved in redox regulation [ 2, 3, 4, 5, 6, 7 ]. It is both a toxic compound that can cause oxidative stress and a second messenger that is required for cell proliferation. Its signaling function is thought to result from direct or indirect oxidation of various cell signaling and regulatory components, and its toxicity from stochastic oxidative damage to proteins, lipids, and nucleic acids. [ 2, 3, 8, 9, 10, 11 ]. All known organisms are utilizing specialized enzymatic and non enzymatic systems for H2O2 detoxification. Thus, H2O2 concentration is finely regulated by peroxidates and catalases, and by low molecular weight compounds. [ 2, 3, 11 ].

Hydrogen peroxide is a weak acid. It forms a perhydroxyl ion at alkaline condition [ 1 ].

H2O2 ↔ H+ + HO2+, pKa=11.6;

Hydrogen peroxide is thermodynamically unstable and decomposes to form water and oxygen [ 1 ].

H2O2 → H2O + O2;

The rate of decomposition increases with rising temperature, concentration and pH.

The magor sources of metabolic H2O2

H2O2 is normal metabolite generated by all known organisms. The major sources of H2O2 are NADPH oxidases, mitochondrial respiratory chain and oxidative protein folding system in endoplasmic reticulum [ 12, 13, 14, 15, 16, 17, 18, 19 ].

1) NADPH Oxidases. The nicotinamide dinucleotide phosphate oxidases (NOX or NADPH Oxidases) proteins are integral membrane proteins sharing several conserved structural features. The C-terminal tails of all these proteins contain a catalytic subunit consisting of FAD and NADPH-binding sites, whereas the N-terminus is made up of six transmembrane domains and two haem groups which form a channel to allow successive transfer of electrons. [ 12, 13, 20 ]. ROS production is achieved through the removal and transfer of an electron from an NADPH substrate to FAD, then haem and finally to molecular oxygen, generating superoxide (O2.- ), which is rapidly converted into H2O2 by superoxide dismutase. H2O2 is then capable of freely diffusing across membranes due to the activity of aquaporin channel proteins [34]. [ 12, 13, 21 ].

NOX enzymes reduce molecular oxygen to superoxide as a primary product, and this is further converted to various reactive oxygen species. Different isoforms require additional subunits to form functional reactive oxygen species-generating NADPH Oxidase [ 22 ].

2) Mitochondrial ROS production. There are two respiratory chain complexes (I and III) involved in superoxide production. From quantitative data obtained on isolated mitochondria, it has been suggested that about 2-5% of oxygen consumption is due to superoxide anion generation and that about 70-80% of superoxide formation is is connected with the operation of the Q cycle in complex III. Another predictions estimate that, under physio- logic conditions, the superoxide production is around 0.1% of the respiratory rate [ 15, 16, 17 ]. Electrons (e-) donated from NADH and FADH2 pass through the electron transport chain and ultimately reduce O2 to form H2O at complex IV. ROS are produced from the leakage of e- to form superoxide (O2.-) at complex I and complex III. O2.- is produced within matrix at complex I, whereas at complex III O2.- is released towards both the matrix and the intermembrane space. Once generated, O2.- is dismutated to H2O + O2 by superoxide dismutase 1 (SOD1) in the intermembrane space and by SOD2 in the matrix. Afterwards, H2O2 is fully reduced to water by glutathione peroxidases (GPX). [ 15, 16, 17, 23 ].

Mitochondrial sourses of ROS. OM: outer membrane; IM: inner membrane [ 23 ].

3) ER associater ROS production. Oxidation of thiols to disulfide linkages during protein folding in the ER is achieved with the use of H2O2 produced by oxidant-generating sources such as Ero1, NADPH oxidase, and mitochondria. Ero1 produces H2O2 by transferring electrons from reduced PDI to O2. In the proposed model, CP-SH and CR-SH of Prx IV are first oxidized by H2O2 to form a disulfide. The oxidized state of Prx IV is then transferred to PDI thiols, thereby converting them to a disulfide and finally resulting in the formation of a disulfide in the protein to be folded. Prx IV in the ER physically interacts with PDI. It is thus thought to function as a sensor of H2O2 in PDI-mediated protein folding [ 18, 19 ].

Role of Prx IV as H2O2 sensor for protein folding in ER [ 18 ].

H2O2 and signaling

Although hydrogen peroxide is better known as a stong damaging compound, the eukaryotes, bacteria and archaea are utilize hydrogen peroxide as signaling molecule in the regulation of a variety of biological processes. Moreover, hydrogen peroxide detoxifying enzymes are also regulating peroxide-signal transduction. Several recent studies have identified peroxide-signaling mechanisms in which antioxidant enzymes are critically required as peroxide sensors [ 5, 6, 7, 8, 9, 10, 24, 25, 26, 27, 28, 29, 30 ].

The activation of OxyR, Yap1 and Pap1 upon H2O + O2 stress. Bacterial OxyR seems to respond directly to the oxidizing signal. The eukaryotic Yap1 and Pap1 transcription factors require the participation of upstream H2O + O2 sensors: the glutathione peroxidase Gpx3 and the peroxiredoxin Tpx1 [ 25 ].

They shown that peroxiredoxins and glutathione peroxidases (thiol peroxidases) are involved in H2O2 mediated signaling and suggested that the role of thiol peroxidases in oxidative stress defense may be overestimated [ 31, 32, 33, 34, 35 ]. They oxidize regulatory and signaling proteins by transferring of oxidative equvalent from peroxide to signaling proteins, resulting in transcriptional responses and signaling programs. The examples, the response to H2O2 was inhibited in Saccharomyces cerevisiae cells lacking multiple thiol peroxidases [ 35 ]. Tsa1 peroxiredoxin is shown to be involved in activation of a stress-activated MAP kinase in Schizosaccharomyces pombe. [ 36 ]. Mammalian ER peroxiredoxin 4 is involved in transferring oxidative equivalents to the proteins in the disulfide bond formation process [ 37 ]. Cytosolic peroxiredoxins 1 and 2 are involved in activation of apoptosis signaling kinase 1 (ASK1)/p38 signaling pathway by peroxide-induced mechanism [ 38 ]. Yeast peroxiredoxin Tpx1 can transfer a redox signal to transcription factor Pap1 at low hydrogen peroxide concentration, whereas higher concentrations of the oxidant inhibit the Tpx1-Pap1 redox pathway through the temporal inactivation of Tpx1 by oxidation of its catalytic cysteine to a sulfinic acid [ 39 ].

A model of redox regulation of gene expression in yeast. Schematic presentation of the current and proposed models of H2O2 mediated signaling. To activate hydroperoxide-dependent gene expression programs, H2O2 initially oxidizes peroxiredoxins (thioredoxin system example), which in turn oxidize transcription factors, kinases and other target proteins in yeast cells. Oxidation of these targets then elicits transcriptional response, redox regulation, signaling pathways and other programs. The model proposes that thiol peroxidases mediate gene expression, whereas a direct interaction between H2O2 and target proteins (dashed arrow) plays a secondary role. The involvement of multiple thiol peroxidases and their regulated interactions with target proteins could explain specificity of the system. Red arrows indicate the direction of electron flow, which is opposite to the direction of thiol peroxidase-mediated oxidative signals.

Frecuently Used Methods for Hydrogen Peroxide Detection

Characterization of the cellular functions of H2O2 requires measurement of its concentration selectively in the presence of other oxygen metabolites and with spatial and temporal fidelity in live cells [ 40, 41, 42 ].

Amplex Red method: Hydrogen peroxide reacts with Amplex Red [N-acetyl-3,7- dihydroxyphenoxazine (Molecular Probes-Invitrogen, USA)] at a stoichiometry of 1:1 in a reaction catalyzed by horseradish peroxidase (HRP) to generate the highly fluorescent product resorufin. HRP catalyzes the decomposition of H2O2 to the hydroxyl radical, which is then reduced to water as a result of irreversible chemical oxidation of Amplex Red, a colorless and nonfluorescent derivative of resorufin, thereby generating fluo- rescent resorufin. Resorufin exhibits a maximum of fluorescence emission at a wavelength of 587 nm and maxi- mum excitation at 563 nm, and its extinction coefficient is ~54,000 M-1 cm-1. The method is highly sensitive, allowing measurement of H2O2 at concentrations as low as 50 nM [ 40, 43, 44 ].

Detection of H2O2 with Amplex Red. In the presence of HRP, H2O2 reacts stoichiometrically with Amplex Red to generate the red-fluorescent oxidation product, resorufin [ 40 ].

HRP-based spectrophotometric assay with 3,5,3'5'-tetramethylbenzidine (TMB): Exposure of TMB to Horse Redish Peroxidase and H2O2 results in the formation of a one-electron oxidation prod- uct, the TMB cation free radical The TMB cation free radical is in equilibrium with a charge- transfer complex, which is responsible for the blue color (absorbance maximum at 653 nm, with E=3.9x104 M-1 cm-1) that develops during TMB oxidation. Further oxidation with HRP and H2O2 or mild acidification of the radical yields a yellow two- electron oxidation product, the diimine (absorbance maximum of 450 nm, with E=5.9x104 M-1 cm-1) [ 40, 45 ].

Chemical structure of TMB and its oxidation products [ 40 ].

Spectrophotometric assay based on ferrithiocyanate generation: Hydrogen peroxide oxidizes the ferrous ion (Fe 2+) to the ferric ion (Fe3+), the latter of which forms a red ferrithiocyanate complex with thiocyanate (SCN-): The absorbance of the ferrithiocyanate complex can be measured spectrophotometrically at 480 nm [ 40 ].

Spectrophotometric assay based on the Ferrous Oxidation in the Presence of Xylenol Orange (FOX) method: In dilute acid, hydroperoxides oxidize the ferrous ion (Fe2+) to the ferric ion (Fe3+), the latter of which reacts with xylenol orange (XO) to form a blue-purple complex, o-cresolsulfone-phthalein 3',3''-bis(methylimino) diacetate, with an extinction coefficient of 1.5x104 M-1 cm-1 at 560 nm [ 40, 46, 47 ].

In addition, the hydroxyl radical generated by the Fenton reaction is scavenged by sorbitol, and the radical species so formed react with oxygen to yield a hydroperoxyl radical. The latter oxidizes the ferrous ion to the ferric ion, with the production of H2O2, which propagates the ferrous oxidation step:

The presence of sorbitol increases the yield of ferric ions to ~15 mol per mole of H2O2. The extinction coefficient of the ferric-xylenol orange complex at 560 nm is 1.5x4 M-1 cm-1, the apparent extinction coefficient obtained for H2O2 in an optimized reaction mixture containing sorbitol is ~2.25 x5 M-1 cm-1

Detection of Intracellular H2O2 with 5-(and 6-) Chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-DCFH-DA): 2',7'-Dichlorodihydrofluorescein (DCFH) is a colorless and non-fluorescent "dihydro" derivative of fluorescein. However, its oxidation to the parent dye provides the basis for it to serve as a fluorogenic probe for the detection of ROS including H2O2. DCFH is not cell permeable because of its polarity, but its diacetate ester, DCFH-DA, passively diffuses into cells, where the two acetate groups are cleaved by intracellular esterases to yield DCFH. Subsequent two-electron oxidation of this trapped nonfluorescent molecule by ROS results in formation of the highly fluorescent product DCF. DCFH-DA has been used widely to measure intracellular ROS. Unlike DCFH, DCF is membrane permeable and can leak out of cells. Detection of slow H2O2 production over time can thus be difficult with DCFH-DA. To improve cellular retention of the oxidation product, a chloromethyl derivative of DCFH-DA, 5-(and 6-)chloromethyl-2',7'-dichlorodihydrofluore-scein diacetate (CM-DCFH DA), was introduced. The chloromethyl group of CM-DCFH- DA allows for covalent binding to intracellular thiol components, resulting in retention of CM-DCF within the cell for longer time intervals. Accumulation of CM-DCF in cells can be measured on the basis of an increase in fluorescence at ~530 nm on excitation at ~488 nm. The fluorescence can be detected with a fluorometer, flow cytometer, microplate reader, or fluorescence microscope and is thought to be proportional to the concentration of ROS in cells [ 40, 48, 49, 50 ].

Basis for the detection of intracellular H2O2 with CM-DCFH-DA. CM- DCFH-DA diffuses into cells and becomes trapped as a result of its deacetylation by intracellular esterases. The nonfluorescent CM-DCFH is then oxidized by various oxidants including H2O2 to yield the intensely fluorescent CM-DCF. In the absence of the CM (chloromethyl) group attached to the DCF moiety, oxidized DCF molecules leakout of the cell over time [ 40 ].

Measurement of intracellular H2O2 with deprotection-based fluorescent probes: To overcome the drawbacks of dihydro derivatives of fluoresce- in such as CM-DCFH-DA for the fluorometric detection of H2O2, which include their sensitivity to various physiological oxidants other than H2O2 and their susceptibility to autoxidation and photo-oxidation, research has focused on the development of an H2O2-specific probe whose function is not dependent on probe oxidation. Two such probes, mono-boronated 2-methyl-4-methoxy Tokyo Green (Peroxy Green 1, PG1) and monoboronated resorufin (Peroxy Crimson 1, PC1), have been designed. PG1 and PC1 show high selectivity for H2O2 over other oxidants such as peroxynitrite, the hydroxyl radical, lipid peroxides, nitric oxide, and hypochloride. PG1 features one prominent absorption band in the visible region centered at 460 nm (E=5500 M-1 cm-1) and shows weak fluorescence with an emission maximum at 510 nm. PC1 has one major absorption maximum at 480 nm (E=4800 M-1 cm-1), with a corresponding weak emission band centered at 584 nm. H2O2-mediated "deprotection" of PG1 and PC1 results in marked increases in green (10-fold) and red (40-fold) fluores- cence, respectively, as the result of generation of 2-methyl-4- O-methyl Tokyo Green and resorufin, respectively. PG1 and PC1 are able to enter cells, whereas the deprotection products remain restricted to the intracellular environment. PG1 and PC1 were successfully applied to the measurement of epidermal growth factor (EGF)-induced H2O2 generation in A431 cells and in hippocampal neurons by confocal microscopy [ 40, 51 ].

Activation of the nonfluorescent probes PG1 and PC1 via an H2O2-dependent deprotection reaction [ 40 ].

Imaging intracellular H2O2 with organelle-targetable, deprotection-based fluorescent probes based on SNAP-tag protein labeling: SNAP-tag (Covalys Biosciences, Switzerland) is a 20-kDa mutant of the DNA repair protein O6-alkylguanine-DNA alkyl-transferase (AGT) that reacts specifically and rapidly with benzylguanine and benzylchloropyrimidine derivatives, thus allowing the specific covalent attachment of synthetic ligands (SNAP-tag substrates) to a fusion protein containing this tag. For example, the gene for an organelle- targeting protein (OTP) can be cloned into a SNAP-express plasmid for expression of the targeting protein as a SNAP-tag fusion (SNAP-OTP). SNAP-tag has thus been fused to the carboxyl terminus of histone H2B (SNAP-H2B) for localization to the nucleus as well as to the carboxyl terminus of cytochrome c oxidase subunit 8 (SNAP-Cox8A) for mitochondrial localization. The addition of the amino acid sequence KDEL, a signal sequence for protein retention in the endoplasmic reticulum, to the carboxyl terminus of SNAP-tag also resulted in localization of the fusion product (SNAP-KDEL) to this organelle. The parent SNAP-tag without a targeting sequence (pSNAP) is expressed uniformly in the cytoplasm and nucleus. A SNAP-tag substrate has been synthesized by conjugation of the deprotection-based H2O2 probe PG1 to benzyl-2-chloro-6-aminopyrimidine. The hybrid molecule (SPG) was found to be membrane permeable. Furthermore, incubation of mammalian cells expressing a SNAP-OTP fusion protein with SPG led to the intracellular generation of an organelle-targeted fluorescent probe, SNAP-PG1 [ 40, 52, 53 ].

Generation of organelle-specific H2O2 fluorescent probes by SNAP-tag conjugation chemistry. A fusion protein (SNAP-OTP) of an organelle-targeting protein (OTP) and SNAP-tag (AGT mutant) is transiently expressed in cells by transfection with an appropriate SNAP-express plasmid.Incubation of the transfected cells with the SNAP-tag substrate SPG (PG1 conjugated to benzyl-2-chloro-6-aminopyrimidine) results in its specific binding to SNAP-tag and its covalent attachment via the benzylic carbon to the thiol group of cysteine 145, with elimination of the 2-chloro-6-aminopyrimidine moiety. This bioconjugation results in localization of the monoboronated fluorophore to a specific subcellular compartment determined by the OTP. Deprotection of the fluorophore by H2O2 results in a marked increase in green fluorescence at the target organelle [ 40 ].

Imaging of H2O2 with genetically encoded redox-sensitive green fluorescent proteins: the deprotection-based probes PG1 and PC1 are selective for H2O2 but their reaction with H2O2 is irreversible, with the result that they are not suitable for detection of dynamic changes in H2O2 concentration. To overcome this drawback, redox-sensitive green fluorescent proteins (roGFPs), including roGFP1 (GFP with the mutations C48S, S147C, and Q204C) and roGFP2 (GFP with the same mutations as roGFP1 plus S65T), have been prepared by site-directed mutagenesis of the GFP gene. In an oxidizing environment, a disulfide bond is formed between Cys 147 and Cys 204 on adjacent beta strands close to the chromophore of roGFP, and the resulting conformational change leads to an increase in the size of the excitation spectrum peak near 400 nm at the expense of that near 490 nm. The ratio of fluorescence elicited by excitation at 400 nm to that elicited at 490 nm (400/490 nm excitation ratio) is thus indicative of the extent of oxidation-that is, of the oxidant level [ 40, 54 ].

Detection of H2O2 with HyPer. (A) Domain structure of HyPer-C (cytosolic HyPer). The conformational change attributable to the H2O2-induced formation of a disulfide bond between Cys 199 and Cys 208 located in the amino (N) and carboxyl (C) portions of the regulatory domain of OxyR (OxyR-RD), respectively, drives a ratiometric fluorescence change of cpYFP. The reverse reaction is catalyzed by glutaredoxin (Grx). (B) Excitation spectra of HyPer at neutral pH before and after oxidation by H2O2. Emission should be measured at 530 nm [ 40 ].

Summary of Hystorical Methods of Hydrogen Peroxide Detection [ 40 ].

Summary of Hystorical Methods of Hydrogen Peroxide Detection

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