Tyrosinase-catalyzed hydroxylation of hydroquinone,a depigmenting agent,to hydroxyhydroquinone: A kinetic study

Hydroquinone (HQ) is used as a depigmenting agent. In this work we demonstrate that tyrosinase hydroxylates HQ to 2-hydroxyhydroquinone (HHQ). Oxy-tyrosinase hydroxylates HQ to HHQ forming the complex met-tyrosinase-HHQ, which can evolve in two different ways, forming deoxy-tyrosinase and p-hydroxy-o-quinone, which rapidly isomerizes to 2-hydroxy-p-benzoquinone or on the other way generating met-tyrosinase and HHQ. In the latter case, HHQ is rapidly oxidized by oxygen to generate 2-hydroxy-p-benzoquinone, and therefore, it cannot close the enzyme catalytic cycle for the lack of reductant (HHQ). However, in the presence of hydrogen peroxide, met-tyrosinase (inactive on hydroquinone) is transformed into oxy-tyrosinase, which is active on HQ. Similarly, in the presence of ascorbic acid, HQ is transformed into 2-hydroxy-p-benzoquinone by the action of tyrosinase; however, in this case, ascorbic acid reduces met-tyrosinase to deoxy-tyrosinase, which after binding to oxygen, originates oxy-tyrosinase. This enzymatic form is now capable of reacting with HQ to generate p-hydroxy-o-quinone, which rapidly isomerizes to 2-hydroxy-p-benzoquinone. The formation of HHQ during the action of tyrosinase on HQ is demonstrated by means of high performance liquid chromatography mass spectrometry (HPLC–MS) by using hydrogen peroxide and high ascorbic acid concentrations. We propose a kinetic mechanism for the tyrosinase oxidation of HQ which allows us the kinetic characterization of the process. A possible explanation of the cytotoxic effect of HQ is discussed.     

A colorimetric 96-well microtiter plate assay for the determination of urate oxidase activity and its kinetic parameters

Urate oxidase (E.C.1.7.3.3; uricase, urate oxygen oxidoreductase) is an enzyme of the purine breakdown pathway that catalyzes the oxidation of uric acid in the presence of oxygen to allantoin and hydrogen peroxide. A 96-well plate assay measurement of urate oxidase activity based on hydrogen peroxide quantitation was developed. The 96-well plate method included two steps: an incubation step for the urate oxidase reaction followed by a step in which the urate oxidase activity is stopped in the presence of 8-azaxanthine, a competitive inhibitor. Hydrogen peroxide is quantified during the second step by a horseradish peroxidase-dependent system. Under the defined conditions, uric acid, known as a radical scavenger, did not interfere with hydrogen peroxide quantification. The general advantages of such a colorimetric assay performed in microtiter plates, compared to other methods and in particular the classical UV method performed with cuvettes, are easy handling of large amounts of samples at the same time, the possibility of automation, and the need for less material. The method has been applied to the determination of the kinetic parameters of rasburicase, a recombinant therapeutic enzyme.     

Synthesis, characterization, X-ray molecular structure and catalase-like activity of a non-heme iron complex: Dichloro[N-propanoate-N,N-bis-(2-pyridylmethyl)amine]iron(III)

In this work we present the synthesis and characterization of the complex dichloro[N-propanoate-N,N-bis-(2-pyridylmethyl)amine]iron(III) [Fe^III(PBMPA)Cl₂]. The ligand LiPBMPA was synthesized through the Michael reaction of BMPA with methylacrylate, followed by alkaline hydrolysis. The complex [Fe^III(PBMPA)Cl₂] has been synthesized by the reaction of the ligand with FeCl₃ · H₂O and was mainly characterized by cyclic voltammetry, conductivimetry, and electronic, infrared and Mössbauer spectroscopies, and by X-ray structural analysis, which showed an iron center coordinated by one carboxylate oxygen in a monodentate way, one tertiary amine, two pyridine groups and two chloride ions. It has been proposed that in water the chloride ligands are shifted by the solvent molecules and the species [Fe^III(PBMPA)(H₂O)₂]Cl₂ is predominant. The catalase-like activity of the complex was tested in water, and it proved to be active in the hydrogen peroxide dismutation. Kinetics studies were conducted following the initial rates method. The reaction is first order in relation to both the complex and the hydrogen peroxide. Based on the presence of a lag phase that depends on the initial complex concentration, we propose that the active species that shows in situ catalase-like activity, is a binuclear complex.     

Catalase-like activity of bovine met-hemoglobin: Interaction with the pseudo-catalytic peroxidation of anthracene traces in aqueous medium

Hemoglobin is a member of the hemoprotein superfamily whose main role is to transport O₂ in vertebrate organisms. It has two known promiscuous enzymatic activities, peroxidase and oxygenase. Here we show for the first time that bovine hemoglobin also presents a catalase-like activity characterized by a V_max of 344 μM/min, a K_M of 24 mM and a k_cat equal to 115/min. For high anthracene and hemoglobin concentrations and low hydrogen peroxide concentrations, this activity inhibits the expected oxidation of anthracene, which occurs through a peroxidase-like mechanism. Anthracene belongs to the polycyclic aromatic hydrocarbon (PAH) family whose members are carcinogenic and persistent pollutants found in industrial waste waters. Our results show that anthracene oxidation by hemoglobin and hydrogen peroxide follows a typical bi-bi ping-pong mechanism with a V_max equal to 0.250 μM/min, K_M(H2O2) of 80 μM, K_M(ANT) of 1.1 μM and k_cat of 0.17/min. The oxidation of anthracene is shown to be pseudo-catalytic because an excess of hemoglobin and hydrogen peroxide is required to make PAH completely disappear. Thus, bovine hemoglobin presents, in different degrees, all the catalytic activities of the hemoprotein group, which makes it a very interesting protein for biotechnological processes and one with which structure-activity relationships can be studied.     

Pre-steady state kinetic characterization of human peroxiredoxin 5: taking advantage of Trp84 fluorescence increase upon oxidation

Human peroxiredoxin 5 (PRDX5) catalyzes different peroxides reduction by enzymatic substitution mechanisms. Enzyme oxidation caused an increase in Trp84 fluorescence, allowing performing pre-steady state kinetic measurements. The technique was validated by comparing with data available from the literature or obtained herein by alternative approaches. PRDX5 reacted with organic hydroperoxides with rate constants in the 10⁶-10⁷ M⁻¹ s⁻¹ range, similar to peroxynitrite-mediated PRDX5 oxidation, whereas its reaction with hydrogen peroxide was slower (10⁵ M⁻¹ s⁻¹). The method allowed determining the kinetics of intramolecular disulfide formation as well as thioredoxin 2-mediated reduction. The reactivities of PRDXs with peroxides were surprisingly high considering thiol pK_a, indicating that other protein determinants are involved in PRDXs specialization. The order of reactivities between PRDX5 towards oxidizing substrates differ from other PRDXs studied, pointing to a selective action of PRDXs with respect to peroxide detoxification, helping to rationalize the multiple enzyme isoforms present even in the same cellular compartment.     

Hydrogen peroxide- and nitric oxide-induced systemic antioxidant prime-like activity under NaCl-stress and stress-free conditions in citrus plants

We tested whether pre-treatments of roots with H₂O₂ (10 mM for 8 h) or sodium nitroprusside (SNP; 100 μM for 48 h), a donor of NO, could induce prime antioxidant defense responses in the leaves of citrus plants grown in the absence or presence of 150 mM NaCl for 16 d. Both root pre-treatments increased leaf superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) activities, and induced related-isoform(s) expression under non-NaCl-stress conditions. When followed by salinity, certain enzymatic activities also exhibited an up-regulation in response to H₂O₂ or SNP pre-exposure. An NaCl-stress-provoked decrease in the ascorbate redox state was partially prevented by both pre-treatments, whereas the glutathione redox state under normal and NaCl-stress conditions was increased by SNP. Real-time imaging of NO production was found in vascular tissues and epidermal cells. Furthermore, NaCl-induced inhibition in OH scavenging activity and promotion of OH-mediated DNA strand cleavage was partially prevented by SNP. Moreover, NaCl-dependent protein oxidation (carbonylation) was totally reversed by both pre-treatments as revealed by quantitative assay and protein blotting analysis. These results provide strong evidence that H₂O₂ and NO elicit long-lasting systemic primer-like antioxidant activity in citrus plants under physiological and NaCl-stress conditions.     

Oxidative stress alters global histone modification and DNA methylation

The JmjC domain-containing histone demethylases can remove histone lysine methylation and thereby regulate gene expression. The JmjC domain uses iron Fe(II) and α-ketoglutarate (αKG) as cofactors in an oxidative demethylation reaction via hydroxymethyl lysine. We hypothesize that reactive oxygen species will oxidize Fe(II) to Fe(III), thereby attenuating the activity of JmjC domain-containing histone demethylases. To minimize secondary responses from cells, extremely short periods of oxidative stress (3 h) were used to investigate this question. Cells that were exposed to hydrogen peroxide (H₂O₂) for 3 h exhibited increases in several histone methylation marks including H3K4me3 and decreases of histone acetylation marks including H3K9ac and H4K8ac; preincubation with ascorbate attenuated these changes. The oxidative stress level was measured by generation of 2′,7′-dichlorofluorescein, GSH/GSSG ratio, and protein carbonyl content. A cell-free system indicated that H₂O₂ inhibited histone demethylase activity where increased Fe(II) rescued this inhibition. TET protein showed a decreased activity under oxidative stress. Cells exposed to a low-dose and long-term (3 weeks) oxidative stress also showed increased global levels of H3K4me3 and H3K27me3. However, these global methylation changes did not persist after washout. The cells exposed to short-term oxidative stress also appeared to have higher activity of class I/II histone deacetylase (HDAC) but not class III HDAC. In conclusion, we have found that oxidative stress transiently alters the epigenetic program process through modulating the activity of enzymes responsible for demethylation and deacetylation of histones.     

JNK1 activity lowers the cellular production of H2O2 and modulates the growth arrest response to scavenging of H2O2 by catalase

Hydrogen peroxide (H(2)O(2)) can interact with intracellular signaling pathways to regulate cell behavior. The c-Jun NH(2)-terminal kinase 1 (JNK1) signal, involved in diverse aspects of cellular functioning, is implicated as a cell sensor of redox stress. The growth-inhibitory effect of both high-level H(2)O(2) and H(2)O(2)-scavenging catalase treatments is accompanied by increased JNK1 activity. To investigate the role of this response in growth regulation, the JNK1 signal was increased by the introduction of ectopic HA-JNK1. HA-JNK1 expression correlated with increases in basal c-Jun phosphorylation in a dose-dependent manner. Transient expression of HA-JNK1 potentiated cell growth arrest by catalase; however, with stable expression a degree of resistance to this response was observed. Resistance was accompanied by a lowered endogenous production of H(2)O(2). Transient HA-JNK1 expression also reduced H(2)O(2) generation, and this effect was reversed by the JNK inhibitor SP600125. These results indicate that the JNK1 stress response contributes to growth inhibition by catalase treatment via inhibition of cellular H(2)O(2) production. Stable amplification of the JNK1 pathway leads to cellular adaptation to its signal, resulting in a diminished reliance upon H(2)O(2) for efficient growth.     

Mitochondria are required for ATM activation by extranuclear oxidative stress in cultured human hepatoblastoma cell line Hep G2 cells

Ataxia–telangiectasia mutated (ATM) is a serine/threonine protein kinase that plays a central role in DNA damage response (DDR). A recent study reported that oxidized ATM can be active in the absence of DDR. However, the issue of where ATM is activated by oxidative stress remains unclear. Regarding the localization of ATM, two possible locations, namely, mitochondria and peroxisomes are possible. We report herein that ATM can be activated when exposed to hydrogen peroxide without inducing nuclear DDR in Hep G2 cells, and the oxidized cells could be subjected to subcellular fractionation. The first detergent-based fractionation experiment revealed that active, phosphorylated ATM was located in the second fraction, which also contained both mitochondria and peroxisomes. An alternative fractionation method involving homogenization and differential centrifugation, which permits the light membrane fraction containing peroxisomes to be produced, but not mitochondria, revealed that the light membrane fraction contained only traces of ATM. In contrast, the heavy membrane fraction, which mainly contained mitochondrial components, was enriched in ATM and active ATM, suggesting that the oxidative activation of ATM occurs in mitochondria and not in peroxisomes. In Rho 0-Hep G2 cells, which lack mitochondrial DNA and functional mitochondria, ATM failed to respond to hydrogen peroxide, indicating that mitochondria are required for the oxidative activation of ATM. These findings strongly suggest that ATM can be activated in response to oxidative stress in mitochondria and that this occurs in a DDR-independent manner.     

Analysis of the lifetime and spatial localization of hydrogen peroxide generated in the cytosol using a reduced kinetic model

Hydrogen peroxide (H₂O₂) acts as a signaling molecule via its reactions with particular cysteine residues of certain proteins. Determining the roles of direct oxidation by H₂O₂ versus disulfide exchange reactions (i.e. relay reactions) between oxidized and reduced proteins of different identities is a current focus. Here, we use kinetic modeling to estimate the spatial and temporal localization of H₂O₂ and its most likely oxidation targets during a sudden increase in H₂O₂ above the basal level in the cytosol. We updated a previous redox kinetic model with recently measured parameters for HeLa cells and used the model to estimate the length and time scales of H₂O₂ diffusion through the cytosol before it is consumed by reaction. These estimates were on the order of one micron and one millisecond, respectively. We found oxidation of peroxiredoxin by H₂O₂ to be the dominant reaction in the network and that the overall concentration of reduced peroxiredoxin is not significantly affected by physiological increases in intracellular H₂O₂ concentration. We used this information to reduce the model from 22 parameters and reactions and 21 species to a single analytical equation with only one dependent variable, i.e. the concentration of H₂O₂, and reproduced results from the complete model. The reduced kinetic model will facilitate future efforts to progress beyond estimates and precisely quantify how reactions and diffusion jointly influence the distribution of H₂O₂ within cells.     

The role of periplasmic antioxidant enzymes (superoxide dismutase and thiol peroxidase) of the Shiga toxin-producing Escherichia coli O157:H7 in the formation of biofilms

This study examined the role of the periplasmic oxidative defense proteins, copper, zinc superoxide dismutase (SodC), and thiol peroxidase (Tpx), from the Shiga toxin-producing Escherichia coli O157:H7 (STEC) in the formation of biofilms. Proteomic analyses have shown significantly higher expression levels of both periplasmic antioxidant systems (SodC and Tpx) in STEC cells grown under biofilm conditions than under planktonic conditions. An analysis of their growth phase-dependent gene expression indicated that a high level of the sodC expression occurred during the stationary phase and that the expression of the tpx gene was strongly induced only during the exponential growth phase. Exogenous hydrogen peroxide reduced the aerobic growth of the STEC sodC and tpx mutants by more than that of their parental strain. The two mutants also displayed significant reductions in their attachment to both biotic (HT-29 epithelial cell) and abiotic surfaces (polystyrene and polyvinyl chloride microplates) during static aerobic growth. However, the growth rates of both wild-type and mutants were similar under aerobic growth conditions. The formation of an STEC biofilm was only observed with the wild-type STEC cells in glass capillary tubes under continuous flow-culture conditions compared with the STEC sodC and tpx mutants. To the best of our knowledge, this is the first mutational study to show the contribution of sodC and tpx gene products to the formation of an E. coli O157:H7 biofilm. These results also suggest that these biofilms are physiologically heterogeneous and that oxidative stress defenses in both the exponential and stationary growth stages play important roles in the formation of STEC biofilms.     

Enhanced production of free trans-astaxanthin by oxidative stress in the cultures of the green microalga Chlorococcum sp

Free trans-astaxanthin accumulated in the alga Chlorococcum sp. was markedly enhanced from 3.664 mg g⁻¹ cell dry weight to 5.724 mg g⁻¹ cell dry weight when the culture was supplemented with hydrogen peroxide (0.1 mM) under mixotrophic conditions of growth. After saponification, a total of 7.086 mg astaxanthin per g cell dry weight was achieved. Similarly, in heterotrophic cultures, the total astaxanthin content was increased from 1.034 mg g⁻¹ cell dry weight without H₂O₂ to 1.782 mg g⁻¹ cell dry weight with 0.1mM H₂O₂. Results indicate that hydrogen peroxide effectively induces the formation of free trans-astaxanthin in Chlorococcum sp.     

Evaluation of the carbonylation of filamentous fungi proteins by dry immune dot blotting

The development of mycological gerontology requires effective methods for assessing the biological age of fungal cells. This assessment is based on the analysis of a complex of aging and oxidative stress markers. One of the most powerful such markers is the protein carbonylation. In this study, the already known method of dry immune dot blotting is adapted for mycological studies of the content of protein carbonyl groups. After testing the method on a number of filamentous fungi species, some features of the accumulation of carbonylated proteins in mycelium were established. Among these features: (i) a weak effect of exogenous oxidative stress on the accumulation of carbonyls in a number of fungi, (ii) reversibility of the carbonyl accumulation, (iii) possibility of arbitrary regulation of carbonyl content by fungus itself and (iv) the influence of hormesis. In addition, two polar strategies for the accumulation of carbonyl modification were revealed, named Id-strategy (Indifferent) and Cn-strategy (Concern). Thus, even the analysis of one marker allows making some preliminary general assumptions and conclusions. For example, the idea that fungi can freely regulate their biological age is confirmed. This feature makes fungi very flexible in terms of responding to environmental influences and promising objects for gerontology.     

Redox reactions of the FAD-containing apoptosis-inducing factor (AIF) with quinoidal xenobiotics: a mechanistic study

Mitochondrial apoptosis-inducing factor (AIF) is a FAD-containing protein that under certain conditions translocates to the nucleus and causes a programmed cell death, apoptosis. The apoptogenic action of AIF is redox controlled as the NADH-reduced AIF dimer has lower affinity for DNA than the oxidized monomer. To gain further insights into the mechanism of AIF, we investigated its interaction with a series of quinone oxidants, including a number of anticancer quinones. Our data indicate that the NADH:quinone oxidoreduction catalyzed by AIF follows a “ping-pong” scheme, with the reductive half-reaction being rate-limiting and the FADH⁻–NAD⁺ charge-transfer complex serving as an electron donor. AIF is equally reactive toward benzo- and naphthoquinones, but may discriminate structures with a higher number of aromatic rings. The reactivity of quinones is mainly defined by their one-electron reduction potential, whereas the size and nature of the substituents play a minor role. AIF is unlikely to significantly contribute to bioreductive activation of low-potential quinoidal anticancer quinones. However, high-potential quinones, e.g. a toxic natural compound naphthazarin, maintain AIF in the oxidized state when a significant excess of NADH is present. Thus, these compounds may prevent the accumulation of the reduced form of AIF in vivo, and enhance AIF-mediated apoptosis.     

Adaptive mechanism of Echinochloa crus-galli Beauv. var. formosensis Ohwi under salt stress: Effect of salicylic acid on salt sensitivity

Our previous study revealed that salicylic acid (SA) accumulates in salt-stressed rice (Oryza sativa L. cv. Nipponbare) seedlings, and we hypothesized that the accumulation of SA might potentiate oxidative injury in rice seedlings since the inhibition of SA synthesis alleviated the growth inhibition under high salinity. To further clarify the action of SA under salt stress, we investigated the changes in the SA content, the activities of the antioxidative enzymes, and the effects of exogenous SA on barnyardgrass (Echinochloa crus-galli Beauv. var. formosensis Ohwi), a gramineous weed which shows lower SA content and is more salt tolerant than rice. In E. crus-galli seedlings exposed to high salinity, neither free nor conjugated SA content showed any increase, while the fresh weight of the shoot and chlorophyll fluorescence (Φ_PSII) slightly decreased. When E. crus-galli seedlings were treated with salt after foliar application of SA, the absorbed SA resulted in the enhancement of the salt-induced growth inhibition and a striking reduction of the Φ_PSII value. Catalase (CAT) and superoxide dismutase (SOD) activities of E. crus-galli seedlings were induced by the salt treatment. However, SA pre-treatment suppressed such an induction of CAT activity and further promoted SOD activity, both of which led to the elevation of the leaf hydrogen peroxide (H₂O₂) level. The present results suggested that enlargement of the cellular SA pool facilitates the generation of H₂O₂ through the suppression of CAT activity and through a remarkable promotion of SOD activity, and thereby enhances the oxidative injury caused by salt stress.     

Hydrogen peroxide-induced apoptosis in pc12 cells and the protective effect of puerarin

In this study, the effect of puerarin on hydrogen peroxide-induced apoptosis in PC12 cells was studied. Exposure of cells to 0.5mM H(2)O(2)may cause significant viability loss and apoptotic rate increase. When c-Myc, Bcl-2 and Bax expression and caspase-3 activity were measured, using Ac-DEVD-AMC as a substrate, the changes in these apoptosis regulatory and effector proteins suggested that the elevation of c-Myc, decrease in Bcl-2:Bax protein ratio, and caspase-3 activation all play a key role in apoptosis. When cells were treated with puerarin prior to 0.5 mM H(2)O(2)treatment, a reduction in viability loss and apoptotic rate was seen. In addition, c-Myc expression decreased and Bcl-2:Bax ratio increased. Puerarin also reduced the H(2)O(2)-induced elevation of caspase-3 activation. These results suggest that puerarin can protect neurons against oxidative stress. It can block apoptosis in its early stages via the regulation of anti- and pro-apoptotic proteins, as well as by the attenuation of caspase-3 activation in H(2)O(2)-induced PC12 cells.     

Pro-oxidative synergic bactericidal effect of NO: kinetics and inhibition by nitroxides

NO plays diverse roles in physiological and pathological processes, occasionally resulting in opposing effects, particularly in cells subjected to oxidative stress. NO mostly protects eukaryotes against oxidative injury, but was demonstrated to kill prokaryotes synergistically with H₂O₂. This could be a promising therapeutic avenue. However, recent conflicting findings were reported describing dramatic protective activity of NO. The previous studies of NO effects on prokaryotes applied a transient oxidative stress while arbitrarily checking the residual bacterial viability after 30 or 60 min and ignoring the process kinetics. If NO-induced synergy and the oxidative stress are time-dependent, the elucidation of the cell killing kinetics is essential, particularly for survival curves exhibiting a “shoulder” sometimes reflecting sublethal damage as in the linear-quadratic survival models. We studied the kinetics of NO synergic effects on H₂O₂-induced killing of microbial pathogens. A synergic pro-oxidative activity toward gram-negative and gram-positive cells is demonstrated even at sub-μM/min flux of NO. For certain strains, the synergic effect progressively increased with the duration of cell exposure, and the linear-quadratic survival model best fit the observed survival data. In contrast to the failure of SOD to affect the bactericidal process, nitroxide SOD mimics abrogated the pro-oxidative synergy of NO/H₂O₂. These cell-permeative antioxidants, which hardly react with diamagnetic species and react neither with NO nor with H₂O₂, can detoxify redox-active transition metals and catalytically remove intracellular superoxide and nitrogen-derived reactive species such as ^•NO₂ or peroxynitrite. The possible mechanism underlying the bactericidal NO synergy under oxidative stress and the potential therapeutic gain are discussed.     

Morphological and enzymatic responses of a recombinant Aspergillus niger to oxidative stressors in chemostat cultures

Continuous chemostat cultures of a recombinant strain of Aspergillus niger (B1-D), engineered to produce the marker protein hen egg white lysozyme, were investigated with regard to their susceptibility to oxidative stress. The culture response to oxidative stress, produced either by addition of exogenous hydrogen peroxide (H₂O₂) or by high dissolved oxygen tension (DOT), was characterised in terms of the activities of two key defensive enzymes: catalase (CAT) and superoxide dismutase (SOD). Since the morphology is so critical in submerged fungal bioprocesses, the key morphological indices were analysed using a semi-automated image analysis system. Both oxidant stressors, H₂O₂ and elevated DOT, increased both enzyme activities, however, the extent was different: exogenous H₂O₂ led mainly to increased CAT activity, whereas gassing with O₂ enriched air, which resulted in a DOT of 165% of air saturation, increased both enzyme activities more than 2-fold compared with the control steady state culture. Addition of exogenous H₂O₂ resulted in shorter hyphae compared with control steady state cultures. These findings indicate that it is unsound to use exogenous H₂O₂ to simulate oxidative stress induced by elevated dissolved oxygen levels since the response to each might be quite different, both in terms of enzymatic (defensive) responses and in terms of culture morphology.