Detection of hydrogen peroxide by lactoperoxidase-mediated dityrosine formation

The aim of this work was to study the dityrosine-forming activity of lactoperoxidase (LPO) and its potential application for measuring hydrogen peroxide (H₂O₂). It was observed that LPO was able to form dityrosine at low H₂O₂ concentrations. Since dityrosine concentration could be measured in a simple fluorimetric reaction, this activity of the enzyme was utilized for the measurement of H₂O₂ production in different systems. These experiments successfully measured the activity of NADPH oxidase 4 (Nox4) by this method. It was concluded that LPO-mediated dityrosine formation offers a simple way for H₂O₂ measurement.     

Effects of peroxidase and hydrogen peroxide on the dityrosine formation and the mixing characteristics of wheat-flour dough

The effects of adding hydrogen peroxide and peroxidase to wheat-flour dough on dityrosine formation and mixing characteristics were investigated. Dityrosine in wheat-flour dough was identified by HPLC with a fluorescence detector and by LC/MS/MS. Formation of dityrosine increased with the addition of hydrogen peroxide, and hydrogen peroxide plus peroxidase, to wheat-flour dough, while the addition of peroxidase had no effect on the amount of dityrosine formed. The mixing curve obtained by a doughgraph changed with the addition of hydrogen peroxide, and hydrogen peroxide plus peroxidase; the peak time was significantly delayed and the dough development time was extended. We found that dityrosine cross-links in wheat-flour dough increased with the addition of peroxidase plus hydrogen peroxide. It is thought that these cross-links can lead to polymerization of the proteins in wheat-flour dough.     

Decomposition of hydrogen peroxide by metmyoglobin: A cyclic formation of the ferryl intermediate

• 1.1. Metmyoglobin reacted with H₂O₂ to form ferryl-myoglobin, which reverted back spontaneously to the met-form.
• 2.2. Through this cyclic reaction of myoglobin between metMb(III) and ferryl-Mb(IV), we propose that H₂O₂, one of the potent oxidants in vivo, can be decomposed continuously in cardiac and skeletal muscle tissues in the absence of catalase and peroxidase.

     

A long-lived tyrosyl radical from the reaction between horse metmyoglobin and hydrogen peroxide

The reaction between metmyoglobin (metMb) and hydrogen peroxide has been known since the 1950s to produce globin-centered free radicals. The direct electron spin resonance spectrum of a solution of horse metMb and hydrogen peroxide at room temperature consists of a multilined signal that decays in minutes at room temperature. Comparison of the direct ESR spectra obtained from the system under N(2)- and O(2)-saturated conditions demonstrates the presence of a peroxyl radical, identified by its g-value of 2.014. Computer simulations of the spectra recorded 3 s after the mixture of metMb and H(2)O(2) were calculated using hyperfine coupling constants of a(H2,6) = 1.3 G and a(H3,5) = 7.0 G for the ring and a(beta)(H1) = 16.7 G and a(beta)(H2) = 14.2 G for the methylene protons, and are consistent with a highly constrained, conformationally unstable tyrosyl radical. Spectra obtained at later time points contained a mixture of the 3 s signal and another signal that was insufficiently resolved for simulation. Efficient spin trapping with 3, 5-dibromo-4-nitrosobenzenesulfonic acid was observed only when the spin trap was present at the time of H(2)O(2) addition. Spin trapping experiments with either 5,5-dimethyl-1-pyrroline N-oxide (DMPO) or perdeuterated 2-methyl-2-nitrosopropane (MNP-d(9)), which have been shown to trap tyrosyl radicals, were nearly equally effective when the spin trap was added before or 10 min after the addition of H(2)O(2). The superhyperfine structure of the ESR spectra obtained from Pronase-treated MNP-d(9)/*metMb confirmed the assignment to a tyrosyl radical. Delayed spin trapping experiments with site-directed mutant myoglobins in which either Tyr-103 or Tyr-146 was replaced by phenylalanine indicated that radical adduct formation with either DMPO or MNP-d(9) requires the presence of Tyr-103 at all time points, implicating that residue as the radical site.     

Protein nitrotryptophan: formation, significance and identification

Reactive nitrogen species are formed during a variety of disease states and have been shown to modify several amino acids on proteins. To date, the majority of research in this area has focused on the nitration of tyrosine residues to form 3-nitrotyrosine. However, emerging evidence suggests that another modification, nitration of tryptophan residues, to form nitrotryptophan (NO(2)-Trp), may also play a significant role in the biology of nitrosative stress. This review takes an in-depth look at NO(2)-Trp, presenting the current research about its formation, prevalence and biological significance, as well as the methods used to identify NO(2)-Trp-modified proteins. Although more research is needed to understand the full biological role of NO(2)-Trp, the data presented herein suggest a contribution to nitrosative stress-induced cell dysregulation and perhaps even in physiological cell processes.     

The hydrogen peroxide/copper ion system, but not other metal-catalyzed oxidation systems, produces protein-bound dityrosine

Dityrosine formation leads to the cross-linking of proteins intra- or intermolecularly. The formation of dityrosine in lens proteins oxidized by metal-catalyzed oxidation (MCO) systems was estimated by chemical and immunochemical methods. Among the four MCO systems examined (H(2)O(2)/Cu, H(2)O(2)/Fe-ethylenediaminetetraacetic acid (Fe-EDTA), ascorbate/Cu, ascorbate/Fe-EDTA), the treatment with H(2)O(2)/Cu preferentially caused dityrosine formation in the lens proteins. The success of oxidative protein modification with all the MCO systems was confirmed by carbonyl formation estimated using 2,4-dinitrophenylhydrazine. The loss of tyrosine by the MCO systems was partly due to the formation of protein-bound 3,4-dihydroxyphenylalanine. The formation of dityrosine specific to H(2)O(2)/Cu was confirmed by using poly-(Glu, Ala, Tyr) and N-acetyl-tyrosine as a substrate. The dissolved oxygen concentration in the H(2)O(2)/Cu system hardly affected the amount of dityrosine formation, suggesting that dityrosine generation by the H(2)O(2)/Cu system is independent of oxygen concentration. Moreover, the combination of copper ion with H(2)O(2) is the most effective system for dityrosine formation among various metal ions examined. The addition of reducing agents, glutathione or ascorbic acid, into the H(2)O(2)/Cu system suppressed the generation of the dityrosine moiety, suggesting effective quench of tyrosyl radicals by the reducing agents.     

Peroxidation of linoleate at physiological pH: hemichrome formation by substrate binding protects against metmyoglobin activation by hydrogen peroxide

Peroxidation by metmyoglobin, MbFe(III), by metmyoglobin/hydrogen peroxide, MbFe(III)/H(2)O(2), to yield the myoglobin ferryl radical (*MbFe(IV)=O), or by ferrylmyoglobin, MbFe(IV)=O, was investigated at physiological pH (7.4) in oil-in-water linoleate emulsions. Linoleate peroxidation was followed using second derivative ultraviolet (UV)-spectroscopy for monitoring formation of conjugated dienes and quantitative determination of specific linoleate hydroperoxides by liquid chromatography with photodiode absorption detection. Modifications of myoglobins during lipid peroxidation were followed simultaneously by changes in the Soret absorption band (410 or 424 nm), and in the visible absorption region (from 450 to 700 nm), combined with electron spin resonance (ESR) spectroscopy for direct detection of changes in the spin state of the iron center. In contrast to MbFe(IV)=O, MbFe(III) and MbFe(III)/H(2)O(2) were not able to initiate linoleate peroxidation in oil-in-water emulsions, and MbFe(III) was converted, by binding of linoleate (but not methyl linoleate), to a low-spin hemichrome derivate, HMbFe(III), with the distal histidine reversibly bound to the iron center. HMbFe(III) is ineffective in initiating lipid peroxidation and cannot be activated to *MbFe(IV)=O or MbFe(IV)=O by addition of moderate amounts of H(2)O(2). Addition of MbFe(III) to linoleate emulsions containing H(2)O(2) results in the competitive formation of *MbFe(IV)=O and HMbFe(III) in favor of HMbFe(III), and little linoleate peroxidation is detected, demonstrating the inherent protection, at physiologic pH, against peroxidation by reversible binding of the substrate to the potential myoglobin catalyst.     

Catalase catalyzes nitrotyrosine formation from sodium azide and hydrogen peroxide

Sodium azide (NaN₃) is known as an inhibitor of catalase, and a nitric oxide (NO) donor in the presence of catalase and H₂O₂. We showed here that catalase-catalyzed oxidation of NaN₃ can generate reactive nitrogen species which contribute to tyrosine nitration in the presence of H₂O₂. The formation of free-tyrosine nitration and protein-bound tyrosine nitration by the NaN₃/catalase/H₂O₂ system showed a maximum level at pH 6.0. Free-tyrosine nitration induced by peroxynitrite was inhibited by ethanol and dimethyl-sulfoxide (DMSO), and augmented by superoxide dismutase (SOD). However, free-tyrosine nitration induced by the NaN₃/catalase/H₂O₂ system was not affected by ethanol, DMSO and SOD. NO^-₂ and NO donating agents did not affect free-tyrosine nitration by the NaN₃/catalase/H₂O₂ system. The reaction of NaN₃ with hydroxyl radical generating system showed free-tyrosine nitration, but no formation of nitrite and nitrate. The generation of nitrite (NO^-₂) and nitrate (NO^-₃) by the NaN₃/catalase/H₂O₂ system was maximal at pH 5.0. These results suggested that the oxidation of NaN₃ by the catalase/H₂O₂ system generates unknown peroxynitrite-like reactive nitrogen intermediates, which contribute to tyrosine nitration.     

Catalase catalyzes nitrotyrosine formation from sodium azide and hydrogen peroxide

Sodium azide (NaN₃) is known as an inhibitor of catalase, and a nitric oxide (NO) donor in the presence of catalase and H₂O₂. We showed here that catalase-catalyzed oxidation of NaN₃ can generate reactive nitrogen species which contribute to tyrosine nitration in the presence of H₂O₂. The formation of free-tyrosine nitration and protein-bound tyrosine nitration by the NaN₃/catalase/H₂O₂ system showed a maximum level at pH 6.0. Free-tyrosine nitration induced by peroxynitrite was inhibited by ethanol and dimethyl-sulfoxide (DMSO), and augmented by superoxide dismutase (SOD). However, free-tyrosine nitration induced by the NaN₃/catalase/H₂O₂ system was not affected by ethanol, DMSO and SOD. NO^-₂ and NO donating agents did not affect free-tyrosine nitration by the NaN₃/catalase/H₂O₂ system. The reaction of NaN₃ with hydroxyl radical generating system showed free-tyrosine nitration, but no formation of nitrite and nitrate. The generation of nitrite (NO^-₂) and nitrate (NO^-₃) by the NaN₃/catalase/H₂O₂ system was maximal at pH 5.0. These results suggested that the oxidation of NaN₃ by the catalase/H₂O₂ system generates unknown peroxynitrite-like reactive nitrogen intermediates, which contribute to tyrosine nitration.     

Haemichrome formation from haemoglobin subunits by hydrogen peroxide.

The effect of H2O2 on ferrous human haemoglobin subunits (alphash-, betash-, alphapmb- and betapmb-chains) was studied. These chains were easily transformed to haemichrome by the addition of H2O2 or H2O2-generating systems, including glucose oxidase (EC 1.1.3.4) AND XANTHINE OXIDASE (EC 1.2.3.2), and this was ascertained by e.p.r. measurements and by absorption spectra. The changes in these haemoglobin subunits were not inhibited by superoxide dismutase (EC 1.15.1.1), but were decreased by catalase (EC 1.11.1.6). The rate of oxidation of alphapmb-chains was higher than that of alphash-chains, and the rate of oxidation of betapmb-chains was higher than that of betash-chains. Haemichrome was demonstrated to be formed directly from these ferrous chains by the attack by H2O2, and this process did not involve formation of methaemoglobin. On the basis of these findings the kinetics of the reaction between the haemoglobin subunits and H2O2 was studied, and the pathological significance of H2O2 in disorders of erythrocytes such as thalassaemia was discussed.     

Oxidation of oxymyoglobin to metmyoglobin with hydrogen peroxide: involvement of ferryl intermediate

Hydrogen peroxide, one of the potent oxidants in muscle tissues, can induce very rapid oxidation of oxymyoglobin (MbO2) to metmyoglobin (metMb) with an apparent rate constant of 7.5 X 10(4) h-1 M-1 (i.e., 20.8 s-1 M-1) over the wide pH range of 5.5-10.2 in 0.1 M buffer at 25 degrees C. Its molecular mechanism, however, is quite different from that of the autoxidation of MbO2 to metMb. Kinetic analysis has revealed that the hydrogen peroxide oxidation proceeds through the formation of ferryl-Mb(IV) from deoxy-Mb(II), which is in equilibrium with MbO2, by a two-equivalent oxidation with H2O2. Once the ferryl species is formed, it reacts rapidly with another deoxy-Mb(II) in a bimolecular fashion so as to yield 2 mol of metMb(III). Under physiological conditions, the rate-determining step was the oxidation of the deoxy species by H2O2, its rate constant being estimated to be on the order of 3.6 X 10(3) s-1 M-1 at 25 degrees C. These findings leads us to the view that a good supply of dioxygen provides rather an important defense against the oxidation of myoglobin with hydrogen peroxide in cardiac and skeletal muscle tissues.     

The mechanism of oxyperoxidase formation from ferryl peroxidase and hydrogen peroxide

Formation of oxyperoxidase from the reaction of ferryl horseradish peroxidase with H2O2 is inhibited by a small amount of tetranitromethane (TNM), a powerful scavenger of superoxide anion radical. The inhibition by TNM, however, does not exceed 35% as the TNM concentration is increased above 5 microM. The stoichiometry of the reaction in the presence of TNM suggests the following equation for TNM-sensitive formation of oxyperoxidase. Ferryl peroxidase + H2O2----(ferric peroxidase + O2- + H+)----oxyperoxidase The kinetic study on the TNM-resistant formation of oxyperoxidase suggests that the displacement of the oxygen with H2O2 takes place at the sixth coordination position at maximal rates of 0.048 and 0.054 s-1 for peroxidases A and C, respectively, at 5 degrees C. The TNM-sensitive and -resistant reactions are concluded to occur in parallel, and both yield oxyperoxidase. In either mechanism, the protonated form of ferryl peroxidase is active and the pK alpha value is 7.1 for peroxidase A and 8.6 for peroxidase C. Oxyperoxidase decomposes spontaneously with a large activation energy (23.0 kcal/mol), and the reaction of ferryl peroxidase with H2O2 reaches a steady level of oxyperoxidase, which depends on pH and the concentration of H2O2.     

Investigation of hydrogen peroxide formation in plants

A convenient and simple electrophoretic procedure was used to study the NAD(P)H-dependent generation of the hydrogen peroxide needed for the polymerization of coniferyl alcohol by peroxidases from the wood of Ailanthus glandulosa. The results showed that an NAD(P)H-dependent generation of hydrogen peroxide could be brought about by either: a FMN or riboflavin-dependent system; or a Mn²⁺ -dependent system. The most active system was the one incorporating Mn²⁺, followed closely by that incorporating riboflavin. In nature it appears that the method of hydrogen peroxide formation is determined by the amounts of cofactors present in the lignifying tissue. Because no quantitative data are available in the literature, further studies of the concentrations of these cofactors in the plant cell-wall are needed.     

Interrelationship between hydrogen peroxide,ammonia, glutamine,hydroxylamine and nitrite metabolism in the cyanobacterium Phormidium uncinatum

On transition from nitrogen starvation to ammonia or ammonia/glutamine sufficiency Phormidium uncinatum produces high amounts of H₂O₂, which is consumed by several oxidative reactions catalyzed by thylakoid membrane bound enzymes. These include: oxidation of glutamine to free hydroxylamine, of ammonia to nitrite, of bound hydroxylamine to nitrite, and dismutation of free hydroxylamine to ammonia and nitrite. A possible role of these transformations for detoxification is discussed.Non-standard abbreviations FCCP p-trifluormethoxy carbonylcyanide phenylhydrazone - DCMU dichloromethyl urea