Peroxide-mediated Oxidation Research Articles

Lipid Peroxide-Mediated Oxidative Rearrangement of the Pyrazinone Carboxamide Core of Neutrophil Elastase Inhibitor AZD9819 in Blood Plasma Samples.

This study focused on the mechanistic interpretation of ex vivo oxidation of a candidate drug in blood plasma samples. An unexpected lipid peroxide-mediated epoxidation followed by a dramatic rearrangement led to production of a five-membered oxazole derivative from the original six-membered pyrazinone-carboxamide core of a human neutrophil elastase inhibitor, 6-(1-(4-cyanophenyl)-1H-pyrazol-5-yl)-N-ethyl-5-methyl-3-oxo-4-(3-(trifluoromethyl)phenyl)-3,4-dihydropyrazine-2-carboxamide (AZD9819). The rearranged oxidation product 2-(1-(4-cyanophenyl)-1H-pyrazol-5-yl)-5-(N-ethylacetamido)-N-(3-(trifluoromethyl)phenyl)oxazole-4-carboxamide was characterized by accurate-mass tandem mass spectrometry fragmentations, by two-dimensional NMR and X-ray crystallography of an authentic standard, and by incorporation of an (18)O atom from molecular (18)O2 to the location predicted by our proposed mechanism. The lipid peroxide-mediated oxidation was demonstrated by using human low-density lipoprotein (LDL) in pH 7.4 phosphate buffer and by inhibiting the oxidation with ascorbic acid or l-glutathione, two antioxidants effective in both plasma and the LDL incubation. A nucleophilic mechanism for the epoxidation of AZD9819 by lipid hydroperoxides explains the prevention of its ex vivo oxidation by acidification of the plasma samples. The discovery of the lipid peroxide-dependent oxidation of an analyte and the means of prevention could provide valuable information for biotransformation and bioanalysis.

Peroxide-mediated oxidation and inhibition of the peptidyl-prolyl isomerase Pin1

Pin1 is a phosphorylation-dependent peptidyl-prolyl isomerase that plays a critical role in mediating protein conformational changes involved in signaling processes related to cell cycle control. Pin1 has also been implicated as being neuroprotective in aging-related neurodegenerative disorders including Alzheimer's disease where Pin1 activity is diminished. Notably, recent proteomic analysis of brain samples from patients with mild cognitive impairment revealed that Pin1 is oxidized and also displays reduced activity. Since the Pin1 active site contains a functionally critical cysteine residue (Cys113) with a low predicted pKa, we hypothesized that Cys113 is sensitive to oxidation. Consistent with this hypothesis, we observed that treatment of Pin1 with hydrogen peroxide results in a 32Da mass increase, likely resulting from the oxidation of Cys113 to sulfinic acid (Cys-SO2H). This modification results in loss of peptidyl-prolyl isomerase activity. Notably, Pin1 with Cys113 substituted by aspartic acid retains activity and is no longer sensitive to oxidation. Structural studies by X-ray crystallography revealed increased electron density surrounding Cys113 following hydrogen peroxide treatment. At lower concentrations of hydrogen peroxide, oxidative inhibition of Pin1 can be partially reversed by treatment with dithiothreitol, suggesting that oxidation could be a reversible modification with a regulatory role. We conclude that the loss of Pin1 activity upon oxidation results from oxidative modification of the Cys113 sulfhydryl to sulfenic (Cys-SOH) or sulfinic acid (Cys-SO2H). Given the involvement of Pin1 in pathological processes related to neurodegenerative diseases and to cancer, these findings could have implications for the prevention or treatment of disease.

Efficient and selective hydrogen peroxide-mediated oxidation of sulfides in batch and segmented and continuous flow using a peroxometalate-based polymer immobilised ionic liquid phase catalyst

Highly efficient sulfide oxidation has been achieved under segmented and continuous flow using a peroxometalate-based polymer immobilised ionic liquid phase catalyst.

Thermodynamic analysis of protein-ligand binding interactions in complex biological mixtures using the stability of proteins from rates of oxidation

The detection and quantification of protein-ligand binding interactions is crucial in a number of different areas of biochemical research from fundamental studies of biological processes to drug discovery efforts. Described here is a protocol that can be used to identify the protein targets of biologically relevant ligands (e.g., drugs such as tamoxifen or cyclosporin A) in complex protein mixtures such as cell lysates. The protocol utilizes quantitative, bottom-up, shotgun proteomics technologies (isobaric mass tags for relative and absolute quantification, or iTRAQ) with a covalent labeling technique, termed stability of proteins from rates of oxidation (SPROX). In SPROX, the thermodynamic properties of proteins and protein-ligand complexes are assessed using the hydrogen peroxide-mediated oxidation of methionine residues as a function of the chemical denaturant (e.g., guanidine hydrochloride or urea) concentration. The proteome-wide SPROX experiments described here enable the ligand-binding properties of hundreds of proteins to be simultaneously assayed in the context of complex biological samples. The proteomic capabilities of the protocol render it amenable to the detection of both the on- and off-target effects of ligand binding. The protocol can be completed in 5 d.

An efficient recyclable peroxometalate-based polymer-immobilised ionic liquid phase (PIILP) catalyst for hydrogen peroxide-mediated oxidation

A linear cation-decorated polymeric support with tuneable surface properties and microstructure has been prepared by ring-opening metathesis polymerisation (ROMP) of a pyrrolidinium-functionalised norbornene-based monomer with cyclooctene. The derived peroxophosphotungstate-based polymer-immobilised ionic liquid phase (PIILP) catalyst is an efficient and recyclable system for the epoxidation of allylic alcohols and alkenes, with only a minor reduction in performance on successive cycles.

Neutrophil Myeloperoxidase: Soldier and Statesman

Myeloperoxidase (MPO) is a major protein constituent of the primary granules of vertebrate neutrophils. It catalyses the hydrogen peroxide-mediated oxidation of halide ions to hypohalous acids, especially HOCl. These reactive oxygen species can participate in a variety of secondary reactions, leading to modifications of amino acids and many types of biological macromolecules. The classic paradigm views MPO as a component of the phagocyte oxygen-dependent intracellular microbicidal system, and thus an important arm of the effector phase of innate immune responses. However, the limited immunodeficiency associated with lack of MPO in mouse and human models has challenged this paradigm. In this review we examine more recent information on the interaction between MPO, its bioreactive reaction products, and targets within the inflammatory microenvironment. We propose that two assumptions of the current model may require revisiting. First, many important targets of MPO modification are extracellular, rather than present only within the phagolysosome, such as various components of neutrophil extracellular traps. Second, we suggest that the pro-inflammatory pathological role of MPO may be a particular feature of chronic inflammation. In the physiological setting of acute neutrophil-mediated inflammation MPO may also form part of a negative feedback loop which down-regulates inflammation, limits tissue damage, and facilitates the switch from innate to adaptive immunity. This different perspective on this well-studied enzyme may usefully inform further research into its function in health and disease.

Thermodynamic analysis of protein-ligand interactions in complex biological mixtures using a shotgun proteomics approach.

Shotgun proteomics protocols are widely used for the identification and/or quantitation of proteins in complex biological samples. Described here is a shotgun proteomics protocol that can be used to identify the protein targets of biologically relevant ligands in complex protein mixtures. The protocol combines a quantitative proteomics platform with a covalent modification strategy, termed Stability of Proteins from Rates of Oxidation (SPROX), which utilizes the denaturant dependence of hydrogen peroxide-mediated oxidation of methionine side chains in proteins to assess the thermodynamic properties of proteins and protein-ligand complexes. The quantitative proteomics platform involves the use of isobaric mass tags and a methionine-containing peptide enhancement strategy. The protocol is evaluated in a ligand binding experiment designed to identify the proteins in a yeast cell lysate that bind the well-known enzyme cofactor, β-nicotinamide adenine dinucleotide (NAD+). The protocol is also used to investigate the protein targets of resveratrol, a biologically active ligand with less well-understood protein targets. A known protein target of resveratrol, cytosolic aldehyde dehydrogenase, was identified in addition to six other potential new proteins targets including four that are associated with the protein translation machinery, which has previously been implicated as a target of resveratrol.

Agonist- and Hydrogen Peroxide-Mediated Oxidation of the β2Adrenergic Receptor: Evidence of Receptor S-Sulfenation as Detected by a Modified Biotin-Switch Assay

Reactive oxygen species (ROS), including hydrogen peroxide (H(2)O(2)), have recently been shown to be generated upon agonism of several members of the G protein-coupled receptor (GPCR) superfamily, including β(2)-adrenergic receptors (β(2)ARs). Previously, we have demonstrated that inhibition of intracellular ROS generation mitigates β(2)AR signaling, suggesting that β(2)AR-mediated ROS generation is capable of feeding back to regulate receptor function. Given that ROS, specifically H(2)O(2), are able to post-translationally oxidize protein cysteine sulfhydryls to cysteine-sulfenic acids, the goal of the current study was to assess whether ROS are capable of S-sulfenating β(2)AR. Using a modified biotin-switch assay that is selective for cysteine-sulfenic acids, our results demonstrate for the first time that H(2)O(2) treatment facilitates S-sulfenation of transiently overexpressed β(2)AR in human embryonic kidney 293 cells. It is noteworthy that stimulation of cells with the β-agonist isoproterenol produces both dose- and time-dependent S-sulfenation of β(2)AR, an effect that is receptor-dependent, and demonstrates that receptor-generated ROS are also capable of oxidizing the β(2)AR. Receptor-dependent S-sulfenation was inhibited by the chemoselective sulfenic acid alkylator dimedone and the cysteine antioxidant N-acetyl-l-cysteine. Moreover, our results reveal that receptor oxidation occurs in cells that endogenously express physiologically relevant levels of β(2)AR, because treatment of human alveolar epithelial A549 cells with either H(2)O(2) or the β(2)-selective agonist formoterol promoted receptor S-sulfenation. These findings provide the first evidence, to our knowledge, that a mammalian GPCR can be oxidized by S-sulfenation and signify an important first step toward shedding light on the overlooked role of ROS in the regulation of β(2)AR function.

Pro-oxidant activity of apocynin radical

Apocynin has been widely used as an NADPH oxidase inhibitor in many experimental models. However, concern regarding the efficacy, selectivity, and oxidative side effects of the inhibitor is increasing. In this study, our aim was to characterize the pro-oxidant properties of apocynin and the structurally-related compounds vanillin and vanillic acid. Glutathione (GSH), cysteine, ovalbumin, and the coenzyme NADPH were chosen as potential target biomolecules that could be affected by transient free radicals from apocynin, vanillin and vanillic acid. Additionally, trolox and rifampicin were used as models of hydroquinone moieties, which are particularly susceptible to oxidation. Transient radicals were generated by horseradish peroxidase/hydrogen peroxide-mediated oxidation. In the presence of apocynin, oxidation of GSH was increased seven-fold, and the product of this reaction was identified as GSSG. Similar results were obtained for oxidation of cysteine and ovalbumin. Oxidation of the coenzyme NADPH increased more than 100-fold in the presence of apocynin. Apocynin also caused rapid oxidation of trolox and rifampicin to their quinone derivatives. In conclusion, the pro-oxidant activity of apocynin is related to its previous oxidation leading to transient free radicals. This characteristic may underlie some of the recent findings regarding beneficial or deleterious effects of the phytochemical.

Electrogeneration of hydrogen peroxide applied to the peroxide-mediated oxidation of (R)-limonene in organic media

Horse radish peroxidase (HRP) from Armoracia rusticana catalyses the oxidation of ( R )-limonene into the oxidized derivatives carveol and carvone. This study compares the direct addition (DA) of hydrogen peroxide with its continuous electrogeneration (EG) during the enzymatic oxidation of ( R )-limonene. Reaction mixtures containing HRP, ( R )-limonene as substrate, and hydrogen peroxide, added directly or electrogenerated, in 100 mM sodium-potassium phosphate buffer pH 7.0, at 25oC were studied. Two electrochemical systems for the hydrogen peroxide electrogeneration were evaluated, both containing as auxiliary electrode (AE) a platinum wire and saturated calomel electrode (SCE) as reference. Reticulated vitreous carbon foam (RVCF) and an electrolytic copper web (CW) were evaluated as working electrodes (WE). Results were compared in terms of hydrogen peroxide electrogeneration, ( R )-limonene residual concentration or conversion and product selectivity. Best results in terms of maximum H 2 O 2 concentration (1.2 mM) were obtained using the CW electrode at -620 mV SCE , and continuous aeration. Use of the EG system under optimized conditions, which included the use of acetone (30% v/v) as a cosolvent in a 3 hrs enzymatic reaction, lead to a 45% conversion of ( R )-limonene into carveol and carvone (2:1). In comparison to the results obtained with DA, the use of EG also improved the half-life of the enzyme.

Electrogeneration of hydrogen peroxide applied to the peroxide-mediated oxidation of (R)-limonene in organic media

Electrogeneration of hydrogen peroxide applied to the peroxide-mediated oxidation of (R)-limonene in organic media

Mutational Analysis of Active Site Residues Essential for Sensing of Organic Hydroperoxides by Bacillus subtilis OhrR

Bacillus subtilis OhrR is the prototype for the one-Cys family of organic peroxide-sensing regulatory proteins. Mutational analyses indicate that the high sensitivity of the active site cysteine (C15) to peroxidation requires three Tyr residues. Y29 and Y40 from the opposing subunit of the functional dimer hydrogen bond with the reactive Cys thiolate, and substitutions at these positions reduce or eliminate the ability of OhrR to respond to organic peroxides. Y19 is also critical for peroxide sensing, and the Ala substitution mutant (OhrR Y19A) is less susceptible to oxidation at the active site C15 in vivo. The Y19A protein also displays decreased sensitivity to peroxide-mediated oxidation in vitro. Y19 is in van der Waals contact with two residues critical for protein function, F16 and R23. The latter residue makes critical contact with the DNA backbone in the OhrR-operator complex. These results indicate that the high sensitivity of the OhrR C15 residue to oxidation requires interactions with the opposed Tyr residues. Oxidative modification of C15 likely disrupts the C15-Y29'-Y40' hydrogen bond network and thereby initiates conformational changes that reduce the ability of OhrR to bind to its operator site.

Development and Semicommercial Implementation of an Environmentally Benign, Hydrogen Peroxide Mediated Oxidation of Aliphatic Alcohols

A process for the oxidation of an aliphatic alcohol (primary alcohol in C12−C15 region, substrate selected on the basis of a customer requirement) was developed and scaled up from laboratory (100 mL and 1 L scale) to bench (CR26 reactor) and to semicommercial scale (1000 litre) for toll manufacturing purposes. This oxidation was performed using hydrogen peroxide and a transition metal catalyst under phase transfer conditions. Although hydrogen peroxide mediated oxidation is a potentially hazardous process, the process was successfully scaled up using additional experimental data (from laboratory and bench scale), simple mathematical modelling, and simulation of the hydrodynamics of the multiphase system. The product acid from a C13 primary alcohol could be obtained at alcohol conversions exceeding 95% in selectivities approaching 100%.

Effects of Antioxidants on the Hydrogen Peroxide-Mediated Oxidation of Methionine Residues in Granulocyte Colony-Stimulating Factor and Human Parathyroid Hormone Fragment 13-34

The effects and mechanisms of different antioxidants, methionine, glutathione, acetylcysteine, and ascorbic acid (AscH2), on the oxidation of methionine residues in granulocyte colony-stimulating factor (G-CSF) and human parathyroid hormone fragment 13-34 (hPTH 13-34) by hydrogen peroxide (H2O2) were quantified and analyzed. The rates of oxidation of methionine residues in G-CSF were determined by peptide mapping analyses, and the oxidation of methionine residue in hPTH 13-34 was quantified by reverse-phase HPLC. At pH 4.5, free methionine reduces, glutathione and acetylcysteine have no obvious effect on, and AscH2 promotes the rates of oxidation of methionine residues in G-CSF. The H2O2-induced oxidation rate constants for free methionine, acetylcysteine, and glutathione at pH 4.5 were measured to be 32.07, 1.00, and 1.63 M(-1)h(-1), respectively, while the oxidation rate constant for Met1, the most readily oxidizable methionine residue in G-CSF, is 13.95 M(-1)h(-1). Therefore, the different effects of free methionine, acetylcysteine, and glutathione on the rates of oxidation of methionine residues in G-CSF are consistent with their different reactivity toward oxidation by H2O2. By using hPTH 13-34, the effect of AscH2 on the H2O2-induced oxidation of methionine residue was quantified, and the mechanisms involved were proposed. Because of the presence of trace transition metal ions in solution, at low concentrations, AscH2 is prone to be a prooxidant, increasing the hydroxyl radical (.OH) production rate via Fenton-type reactions. In addition to peroxide oxidation, these radicals lead to the degradation of hPTH 13-34 to smaller peptide fragments. At high concentrations, AscH2 tends to act as an OH scavenger. EDTA inhibits OH production and thus eliminates the degradation of hPTH 13-34 by forming complexes with transition metal ions. However, the rate of oxidation of the methionine residue in hPTH 13-34 increases as the concentration of AscH2 is increased from 0 to 200 mM, and the reason for this is still not clear. Our results demonstrate that free methionine is an effective antioxidant to protect G-CSF against methionine oxidation at pH 4.5. Acetylcysteine and glutathione are not effective antioxidants at pH 4.5. Their oxidation rates at different pH values imply that they would be much more effective antioxidants than free methionine at alkaline conditions. AscH2 is a powerful electron donor. It acts as a prooxidant in the conditions in this study and is unlikely to prevent oxidation by H2O2 in protein formulation, whether or not EDTA is present.

Mechanisms of Alpha Hemoglobin Stabilizing Protein (AHSP) Actions.

Alpha hemoglobin stabilizing protein (AHSP) is an abundant erythroid factor that specifically binds and stabilizes free α hemoglobin (Kihm et al., Nature 417:758, Gell et al., JBC, 277:40602). In mice, AHSP deficiency causes oxidative stress, mild anemia, and reticulocytosis. Furthermore, loss of AHSP exacerbates α globin precipitation and anemia in a murine model for β thalassemia. These data suggest that AHSP operates as a molecular chaperone to stabilize free α hemoglobin and inhibit its ability to catalyze redox chemistry. We investigated how AHSP associates with α hemoglobin and thereby reduces its oxidative capacity. By NMR and mutational analysis, we determined the structure of AHSP and defined its α globin-interacting surfaces. AHSP consists of three linked α helices. Key α globin-binding residues lie along helices 1 and 2 in a region that resembles the G and H helical fold of β globin. Hence, AHSP presents a surface resembling that of β hemoglobin and thus promotes the formation of a “pseudo α/β interface”. To investigate how formation of this complex affects α hemoglobin-mediated redox chemistry, we examined the ability of AHSP to inhibit peroxide-mediated oxidation of the redox-sensitive dye TMPD. Oxy-α hemoglobin alone catalyzed TMPD oxidation at a rate of 140 μMmin−1; preincubation with AHSP reduced this rate significantly, to 65 μMmin−1. Furthermore, AHSP reduced both heme loss from α hemoglobin and its ability to oxidize hemoglobin A. AHSP binding to oxy- α hemoglobin induced a spectral shift in the UV/visible range, suggesting that stabilization might be associated with structural alterations in the heme moiety. Specifically, the Soret peak was reduced and shifted leftward, while the 541 and 576 nm peaks of the visible region were reduced with concomitant rises at 500 nm and at the region above 580 nm. These alterations suggest that heme-bound iron is converted to a ferric (Fe3+) form that is liganded at all 6 coordinate positions, and therefore, can no longer catalyze redox chemistry. Consistent with this possibility, addition of KCN to the α hemoglobin-AHSP complex produced cyanomethemoglobin-AHSP, but at a slower rate than reaction of KCN with ferric-α hemoglobin alone. In addition, electron paramagnetic resonance and resonance raman spectroscopy showed that the heme iron exists in a low spin state within the α hemoglobin-AHSP complex. The oxidized complex was insensitive to pH alteration, indicating that iron was not bound by hydroxyl ion, which typically occurs at alkaline conditions. Together, our findings illustrate a potential mechanism whereby AHSP renders α hemoglobin chemically inert by inhibiting the reactivity of heme-bound iron. We propose that within the AHSP-α hemoglobin dimer, the oxidized heme is held within a bi-histidyl form, thus blocking its ability to act as a redox catalyst.

Cysteine-106 of DJ-1 is the most sensitive cysteine residue to hydrogen peroxide-mediated oxidation in vivo in human umbilical vein endothelial cells

Mutation in DJ-1 gene is the cause of autosomal recessive Parkinson’s disease, however, its physiological function remains unclear. The isoelectric point of DJ-1 shows an acidic shift after cells are treated with hydrogen peroxide. This suggests that DJ-1 is modified in response to oxidative stress. Here we report the structural characterization of an acidic isoform of DJ-1 using a proteomic approach with nanospray interface liquid chromatography-electrospray ionization/linear ion trap mass spectrometer. When human umbilical vein endothelial cells were exposed to hydrogen peroxide, all three cysteines in DJ-1 were oxidized to cysteine sulphonic acid. Although a small part of the Cys-46 and Cys-53 were oxidized, Cys-106 was oxidized completely at any hydrogen peroxide concentration used here. These results suggest that Cys-106 is the most sensitive among three cysteine residues to oxidative stress, and that DJ-1 function is regulated, in terms of the intracellular redox state, by oxidation of Cys-106.

New heterogeneous polyoxometalate based mesoporous catalysts for hydrogen peroxide mediated oxidation reactions.

Inorganic-organic hybrid mesoporous materials were prepared by cocrystallization of a "sandwich" type polyoxometalate, [ZnWZn2(H2O)2(ZnW9O34)2]12-, and branched tripodal organic polyammonium salts, tris[2-(trimethylammonium)ethyl]-1,3,5-benzenetricarboxylate or 1,3,5-tris[4-(N,N,N-trimethylammoniumethylcarboxyl)phenyl]benzene trications. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) showed formation of three-dimensional perforated coral-shaped amorphous materials with the organic cations surrounding polyoxometalate anions. N2 sorption analysis showed that the hybrid materials have a BET surface area of approximately 30-50 m2 g(-1) and an average pore diameter of 36 A leading to the classification of these materials as mesoporous materials with moderate surface areas. These hybrid materials behaved as very effective and selective heterogeneous catalysts for the epoxidation of allylic alcohols and oxidation of secondary alcohols to ketones with hydrogen peroxide as oxidant. The activity and selectivity of the heterogeneous catalysts based on the hybrid materials was similar to those of homogeneous catalysts based on the same [ZnWZn1(H2O)2(ZnW9O34)2]12- polyoxometalate.

IN VIVO ASSESSMENT OF FREE RADICAL ACTIVITY DURING SHOCK WAVE LITHOTRIPSY USING A MICRODIALYSIS SYSTEM: THE RENOPROTECTIVE ACTION OF ALLOPURINOL

Shock wave lithotripsy is believed to cause renal damage directly through cellular injury from high energy shock waves and indirectly through vascular injury and resultant ischemia, which gives rise to oxygen free radical compounds. The transient and volatile nature of free radicals and derived products makes their detection difficult. Moreover, certain medications may provide a protective effect against shock wave lithotripsy induced renal parenchymal injury. We introduced an innovative microdialysis system for in vivo sampling of interstitial fluids that can be analyzed for free radical mediated lipid peroxidation products after shock wave lithotripsy treatment in the swine model. In addition, this system was used to test the antioxidant or renoprotective action of allopurinol. Ten juvenile swine were assigned to a nonmedicated control group that underwent shock wave lithotripsy or to a group that was premedicated with allopurinol before shock wave lithotripsy. Each group of animals underwent shock wave lithotripsy to the lower pole of the right kidney and received a total of 10,000 shock waves. Dialysate fluid was collected at 1,000-shock wave increments via probes surgically implanted into the lower pole of the right and left kidneys before lithotripsy. Samples were immediately preserved in liquid nitrogen and subsequently analyzed for the presence and concentration of conjugated diene levels, a measure of lipid peroxidation. Five additional juvenile swine were assigned to a sham treated group that did not undergo shock wave lithotripsy. Dialysate fluid was collected from the lower pole of the right and left kidneys to establish baseline or pre-lithotripsy levels of conjugated dienes. After shock wave lithotripsy conjugated diene levels increased almost 100-fold over that in the right kidneys of the nonmedicated control group. The difference was statistically significant compared to levels in the contralateral untreated kidneys (p <0.01). Right kidneys in the group premedicated with allopurinol did not demonstrate an increase in conjugated diene levels during shock wave lithotripsy. The results of this study confirm shock wave lithotripsy induced free radical activity as well the antioxidant and protective nature of allopurinol. The newly described microdialysis system enables real-time sampling of interstitial fluids during shock wave lithotripsy. It represents a unique method for assessing free radical formation and evaluating the protective effects of additional antioxidant medications.

Hematoporphyrin interacts with myoglobin and alters its functions.

The binding parameters of hematoporphyrin, a photosensitizing drug used in photodynamic therapy, interacting with myoglobin, an oxygen storage protein, have been studied spectrofluorometrically and spectrophotometrically. Two concentration ranges of hematoporphyrin, representing significantly monomeric and aggregated (dimeric) states have been used. The binding affinity constant (K) decreases and the possible number of binding sites (p) increases as the porphyrin changes from significantly monomeric state to predominantly dimeric state. Titration of the protein with hematoporphyrin in a spectrophotometric study (differential spectroscopy) exhibits an isosbestic point indicating a ground state complex formation. The interaction leads to a conformational change of the protein as observed in a circular dichroism study. The hematoporphyrin-myoglobin interaction causes oxygen release from the protein and it varies with the stoichiometric ratio of the porphyrin:protein. Hematoporphyrin also increases the myoglobin-catalysed hydrogen peroxide-mediated oxidation of o-dianisidine and NADH. These findings on the effects of hematoporphrin-myoglobin interaction should be given due consideration in therapeutic uses of the porphyrin and its derivatives.

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.