Publications by authors named "Fernanda M Prado"

25 Publications

  • Page 1 of 1

l-Tryptophan Interactions with the Horseradish Peroxidase-Catalyzed Generation of Triplet Acetone.

Photochem Photobiol 2021 Mar 11;97(2):327-334. Epub 2021 Feb 11.

Departamento de Química Fundamental, Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brazil.

Triplet carbonyls generated by chemiexcitation are involved in typical photobiochemical processes in the absence of light. Due to their biradical nature, ultraweak light emission and long lifetime, electronically excited triplet species display typical radical reactions such as isomerization, fragmentation, cycloaddition and hydrogen abstraction. In this paper, we report chemical reactions in a set of amino acid residues induced by the isobutanal/horseradish peroxidase (IBAL/HRP) system, a well-known source of excited triplet acetone (Ac ). Accordingly, quenching of Ac by tryptophan (Trp) unveiled parallel enzyme damage and inactivation, likely explained by scavenging of IBAL tertiary radical reaction intermediate and Ac -derived 2-hydroxy-i-propyl radical. Quenching constants were calculated from Stern-Volmer plots, and the structure of radical adducts was revealed by mass spectrometry. As expected, a concurrent Schiff-type adduct was found to be one of the reaction by-products. These findings draw attention to potential structural and functional changes in enzymes involved in the electronic chemiexcitation of their products.
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http://dx.doi.org/10.1111/php.13363DOI Listing
March 2021

Singlet oxygen generation by the reaction of acrolein with peroxynitrite via a 2-hydroxyvinyl radical intermediate.

Free Radic Biol Med 2020 05 4;152:83-90. Epub 2020 Mar 4.

Departamento de Química Fundamental, Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brazil; Departamento Ciências Exatas e da Terra, Instituto de Ciências Ambientais, Químicas e Farmacêuticas, Universidade Federal de São Paulo, Diadema, SP, Brazil. Electronic address:

Acrolein (2-propenal) is an environmental pollutant, food contaminant, and endogenous toxic by-product formed in the thermal decomposition and peroxidation of lipids, proteins, and carbohydrates. Like other α,β-unsaturated aldehydes, acrolein undergoes Michael addition of nucleophiles such as basic amino acids residues of proteins and nucleobases, triggering aging associated disorders. Here, we show that acrolein is also a potential target of the potent biological oxidant, nitrosating and nitrating agent peroxynitrite. In vitro studies revealed the occurrence of 1,4-addition of peroxynitrite (k = 6 × 10 M s, pH 7.2, 25 °C) to acrolein in air-equilibrated phosphate buffer. This is attested by acrolein concentration-dependent oxygen uptake, peroxynitrite consumption, and generation of formaldehyde and glyoxal as final products. These products are predicted to be originated from the Russell termination of OOCH=CH(OH) radical which also includes molecular oxygen at the singlet delta state (OΔ). Accordingly, EPR spin trapping studies with the 2,6-nitrosobenzene-4-sulfonate ion (DBNBS) revealed a 6-line spectrum attributable to the 2-hydroxyvinyl radical adduct. Singlet oxygen was identified by its characteristic monomolecular IR emission at 1,270 nm in deuterated buffer, which was expectedly quenched upon addition of water and sodium azide. These data represent the first report on singlet oxygen creation from a vinylperoxyl radical, previously reported for alkyl- and formylperoxyl radicals, and may contribute to better understand the adverse acrolein behavior in vivo.
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http://dx.doi.org/10.1016/j.freeradbiomed.2020.03.003DOI Listing
May 2020

Generation of Singlet Molecular Oxygen by Lipid Hydroperoxides and Nitronium Ion.

Photochem Photobiol 2020 05 4;96(3):560-569. Epub 2020 May 4.

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil.

Singlet molecular oxygen is a reactive species involved in biological oxidative processes. The major cellular targets of singlet molecular oxygen are unsaturated fatty acids in the membrane, as well as nucleic acids and proteins. The aim of this study was to investigate whether lipids and commercial hydroperoxides generate singlet molecular oxygen, in presence of nitronium and activated nitronium ion. For this purpose, monomol light emitted in the near-infrared region (λ = 1270 nm) was used to monitor singlet molecular oxygen decay in different solvents, with different hydroperoxides and in the presence of azide. Direct measurements of the singlet molecular oxygen spectrum at 1270 nm recorded during the reaction between lipids and commercial hydroperoxides and nitronium ions unequivocally demonstrated the formation of this excited species.
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http://dx.doi.org/10.1111/php.13236DOI Listing
May 2020

, an Endophyte That Establishes a Nutrient-Transfer Symbiosis With Banana Plants and Protects Against the Black Sigatoka Pathogen.

Front Microbiol 2019 7;10:804. Epub 2019 May 7.

Department of Chemistry, Universidad Autónoma de Guadalajara, Zapopan, Mexico.

Banana ( spp.) is an important crop worldwide, but black Sigatoka disease caused by the fungus threatens fruit production. In this work, we examined the potential of the endophytes of banana plants and , as antagonists of and support plant growth in nutrient limited soils by N-transfer. The two bacterial isolates were identified by MALDI-TOF mass spectrometry and corroborated by 16S rRNA sequence analysis. Both bacteria were positive for beneficial traits such as N-fixation, indole acetic acid production, phosphate solubilization, negative for 1-aminocyclopropane 1-carboxylic acid deaminase and were antagonistic to . To measure the effects on plant growth, the two plant bacteria and an strain (as non-endophyte), were inoculated weekly for 60 days as active cells (AC) and heat-killed cells (HKC) into plant microcosms without nutrients and compared to a water only treatment, and a mineral nutrients solution (MMN) treatment. Bacterial treatments increased growth parameters and prevented accelerated senescence, which was observed for water and mineral nutrients solution (MMN) treatments used as controls. Plants died after the first 20 days of being irrigated with water; irrigation with MMN enabled plants to develop some new leaves, but plants lost weight (-30%) during the same period. Plants treated with bacteria showed good growth, but AC treated plants had significantly greater biomass than the HKC. After 60 days, plants inoculated with AC showed intracellular bacteria within root cells, suggesting that a stable symbiosis was established. To evaluate the transference of organic N from bacteria into the plants, the 3 bacteria were grown with NHCl or NaNO as the nitrogen source. The N transferred from bacteria to plant tissues was measured by pheophytin isotopomer abundance. The relative abundance of the isotopomers 872.57, 873.57, 874.57, 875.57, 876.57 unequivocally demonstrated that plants acquired N atoms directly from bacterial cells, using them as a source of N, to support plant growth in restricted nutrient soils. might be a new alternative to promote growth and health of banana crops.
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http://dx.doi.org/10.3389/fmicb.2019.00804DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6513882PMC
May 2019

Singlet molecular oxygen regulates vascular tone and blood pressure in inflammation.

Nature 2019 02 13;566(7745):548-552. Epub 2019 Feb 13.

Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales, Australia.

Singlet molecular oxygen (O) has well-established roles in photosynthetic plants, bacteria and fungi, but not in mammals. Chemically generated O oxidizes the amino acid tryptophan to precursors of a key metabolite called N-formylkynurenine, whereas enzymatic oxidation of tryptophan to N-formylkynurenine is catalysed by a family of dioxygenases, including indoleamine 2,3-dioxygenase 1. Under inflammatory conditions, this haem-containing enzyme is expressed in arterial endothelial cells, where it contributes to the regulation of blood pressure. However, whether indoleamine 2,3-dioxygenase 1 forms O and whether this contributes to blood pressure control have remained unknown. Here we show that arterial indoleamine 2,3-dioxygenase 1 regulates blood pressure via formation of O. We observed that in the presence of hydrogen peroxide, the enzyme generates O and that this is associated with the stereoselective oxidation of L-tryptophan to a tricyclic hydroperoxide via a previously unrecognized oxidative activation of the dioxygenase activity. The tryptophan-derived hydroperoxide acts in vivo as a signalling molecule, inducing arterial relaxation and decreasing blood pressure; this activity is dependent on Cys42 of protein kinase G1α. Our findings demonstrate a pathophysiological role for O in mammals through formation of an amino acid-derived hydroperoxide that regulates vascular tone and blood pressure under inflammatory conditions.
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http://dx.doi.org/10.1038/s41586-019-0947-3DOI Listing
February 2019

Caloric restriction protects livers from ischemia/reperfusion damage by preventing Ca-induced mitochondrial permeability transition.

Free Radic Biol Med 2017 09 19;110:219-227. Epub 2017 Jun 19.

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brazil. Electronic address:

Caloric restriction (CR) promotes lifespan extension and protects against many pathological conditions, including ischemia/reperfusion injury to the brain, heart and kidney. In the liver, ischemia/reperfusion damage is related to excessive mitochondrial Ca accumulation, leading to the mitochondrial permeability transition. Indeed, liver mitochondria isolated from animals maintained on CR for 4 months were protected against permeability transition and capable of taking up Ca at faster rates and in larger quantities. These changes were not related to modifications in mitochondrial respiratory activity, but rather to a higher proportion of ATP relative to ADP in CR liver mitochondria. Accordingly, both depletion of mitochondrial adenine nucleotides and loading mitochondria with exogenous ATP abolished the differences between CR and ad libitum (AL) fed groups. The prevention against permeability transition promoted by CR strongly protected against in vivo liver damage induced by ischemia/reperfusion. Overall, our results show that CR strongly protects the liver against ischemia/reperfusion and uncover a mechanism for this protection, through a yet undescribed diet-induced change in liver mitochondrial Ca handling related to elevated intramitochondrial ATP.
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http://dx.doi.org/10.1016/j.freeradbiomed.2017.06.013DOI Listing
September 2017

Chemical Characterization of Urate Hydroperoxide, A Pro-oxidant Intermediate Generated by Urate Oxidation in Inflammatory and Photoinduced Processes.

Chem Res Toxicol 2015 Aug 5;28(8):1556-66. Epub 2015 Aug 5.

§Centre for Free Radical Research, Department of Pathology, University of Otago, Christchurch, 8140 Christchurch, New Zealand.

Urate hydroperoxide is a strong oxidant generated by the combination of urate free radical and superoxide. The formation of urate hydroperoxide as an intermediate in urate oxidation is potentially responsible for the pro-oxidant effects of urate in inflammatory disorders, protein degradation, and food decomposition. To understand the molecular mechanisms that sustain the harmful effects of urate in inflammatory and oxidative stress related conditions, we report a detailed structural characterization and reactivity of urate hydroperoxide toward biomolecules. Urate hydroperoxide was synthesized by photo-oxidation and by a myeloperoxidase/hydrogen peroxide/superoxide system. Multiple reaction monitoring (MRM) and MS(3) ion fragmentation revealed that urate hydroperoxide from both sources has the same chemical structure. Urate hydroperoxide has a maximum absorption at 308 nm, ε308nm = 6.54 ± 0.38 × 10(3) M(-1) cm(-1). This peroxide decays spontaneously with a rate constant of k = 2.80 ± 0.18 × 10(-4) s(-1) and a half-life of 41 min at 22 °C. Urate hydroperoxide undergoes electrochemical reduction at potential values less negative than -0.5 V (versus Ag/AgCl). When incubated with taurine, histidine, tryptophan, lysine, methionine, cysteine, or glutathione, urate hydroperoxide reacted only with methionine, cysteine, and glutathione. The oxidation of these molecules occurred by a two-electron mechanism, generating the alcohol, hydroxyisourate. No adduct between cysteine or glutathione and urate hydroperoxide was detected. The second-order rate constant for the oxidation of glutathione by urate hydroperoxide was 13.7 ± 0.8 M(-1) s(-1). In conclusion, the oxidation of sulfur-containing biomolecules by urate hydroperoxide is likely to be a mechanism by which the pro-oxidant and damaging effects of urate are mediated in inflammatory and photo-oxidizing processes.
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http://dx.doi.org/10.1021/acs.chemrestox.5b00132DOI Listing
August 2015

Nitrogen acquisition in Agave tequilana from degradation of endophytic bacteria.

Sci Rep 2014 Nov 6;4:6938. Epub 2014 Nov 6.

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, 05508-000, São Paulo, SP, Brazil.

Plants form symbiotic associations with endophytic bacteria within tissues of leaves, stems, and roots. It is unclear whether or how plants obtain nitrogen from these endophytic bacteria. Here we present evidence showing nitrogen flow from endophytic bacteria to plants in a process that appears to involve oxidative degradation of bacteria. In our experiments we employed Agave tequilana and its seed-transmitted endophyte Bacillus tequilensis to elucidate organic nitrogen transfer from (15)N-labeled bacteria to plants. Bacillus tequilensis cells grown in a minimal medium with (15)NH4Cl as the nitrogen source were watered onto plants growing in sand. We traced incorporation of (15)N into tryptophan, deoxynucleosides and pheophytin derived from chlorophyll a. Probes for hydrogen peroxide show its presence during degradation of bacteria in plant tissues, supporting involvement of reactive oxygen in the degradation process. In another experiment to assess nitrogen absorbed as a result of endophytic colonization of plants we demonstrated that endophytic bacteria potentially transfer more nitrogen to plants and stimulate greater biomass in plants than heat-killed bacteria that do not colonize plants but instead degrade in the soil. Findings presented here support the hypothesis that some plants under nutrient limitation may degrade and obtain nitrogen from endophytic microbes.
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http://dx.doi.org/10.1038/srep06938DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4221784PMC
November 2014

Excited singlet molecular O₂(¹Δg) is generated enzymatically from excited carbonyls in the dark.

Sci Rep 2014 Aug 4;4:5938. Epub 2014 Aug 4.

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, CEP 05513-970, CP 26077, São Paulo, SP, Brazil.

In mammalian tissues, ultraweak chemiluminescence arising from biomolecule oxidation has been attributed to the radiative deactivation of singlet molecular oxygen [O2 ((1)Δg)] and electronically excited triplet carbonyl products involving dioxetane intermediates. Herein, we describe evidence of the generation of O2 ((1)Δg) in aqueous solution via energy transfer from excited triplet acetone. This involves thermolysis of 3,3,4,4-tetramethyl-1,2-dioxetane, a chemical source, and horseradish peroxidase-catalyzed oxidation of 2-methylpropanal, as an enzymatic source. Both sources of excited carbonyls showed characteristic light emission at 1,270 nm, directly indicative of the monomolecular decay of O2 ((1)Δg). Indirect analysis of O2 ((1)Δg) by electron paramagnetic resonance using the chemical trap 2,2,6,6-tetramethylpiperidine showed the formation of 2,2,6,6-tetramethylpiperidine-1-oxyl. Using [(18)O]-labeled triplet, ground state molecular oxygen [(18)O2 ((3)Σg(-))], chemical trapping of (18)O2 ((1)Δg) with disodium salt of anthracene-9,10-diyldiethane-2,1-diyl disulfate yielding the corresponding double-[(18)O]-labeled 9,10-endoperoxide, was detected through mass spectrometry. This corroborates formation of O2 ((1)Δg). Altogether, photoemission and chemical trapping studies clearly demonstrate that chemically and enzymatically nascent excited carbonyl generates (18)O2 ((1)Δg) by triplet-triplet energy transfer to ground state oxygen O2 ((3)Σg(-)), and supports the long formulated hypothesis of O2 ((1)Δg) involvement in physiological and pathophysiological events that might take place in tissues in the absence of light.
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http://dx.doi.org/10.1038/srep05938DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4120373PMC
August 2014

Singlet molecular oxygen generation by light-activated DHN-melanin of the fungal pathogen Mycosphaerella fijiensis in black Sigatoka disease of bananas.

PLoS One 2014 19;9(3):e91616. Epub 2014 Mar 19.

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brazil.

In pathogenic fungi, melanin contributes to virulence, allowing tissue invasion and inactivation of the plant defence system, but has never been implicated as a factor for host cell death, or as a light-activated phytotoxin. Our research shows that melanin synthesized by the fungal banana pathogen Mycosphaerella fijiensis acts as a virulence factor through the photogeneration of singlet molecular oxygen O2 (1Δg). Using analytical tools, including elemental analysis, ultraviolet/infrared absorption spectrophometry and MALDI-TOF mass spectrometry analysis, we characterized both pigment content in mycelia and secreted to the culture media as 1,8-dihydroxynaphthalene (DHN)-melanin type compound. This is sole melanin-type in M. fijiensis. Isolated melanins irradiated with a Nd:YAG laser at 532 nm produced monomol light emission at 1270 nm, confirming generation of O2 (1Δg), a highly reactive oxygen specie (ROS) that causes cellular death by reacting with all cellular macromolecules. Intermediary polyketides accumulated in culture media by using tricyclazole and pyroquilon (two inhibitors of DHN-melanin synthesis) were identified by ESI-HPLC-MS/MS. Additionally, irradiation at 532 nm of that mixture of compounds and whole melanized mycelium also generated O2 (1Δg). A pigmented-strain generated more O2 (1Δg) than a strain with low melanin content. Banana leaves of cultivar Cavendish, naturally infected with different stages of black Sigatoka disease, were collected from field. Direct staining of the naturally infected leaf tissues showed the presence of melanin that was positively correlated to the disease stage. We also found hydrogen peroxide (H2O2) but we cannot distinguish the source. Our results suggest that O2 (1Δg) photogenerated by DHN-melanin may be involved in the destructive effects of Mycosphaerella fijiensis on banana leaf tissues. Further studies are needed to fully evaluate contributions of melanin-mediated ROS to microbial pathogenesis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0091616PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3960117PMC
December 2014

The promoter of filamentation (POF1) protein from Saccharomyces cerevisiae is an ATPase involved in the protein quality control process.

BMC Microbiol 2011 Dec 28;11:268. Epub 2011 Dec 28.

Departamento de Tecnologia Bioquímico-Farmacêutica, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo - USP, São Paulo-SP, Brazil.

Background: The gene YCL047C, which has been renamed promoter of filamentation gene (POF1), has recently been described as a cell component involved in yeast filamentous growth. The objective of this work is to understand the molecular and biological function of this gene.

Results: Here, we report that the protein encoded by the POF1 gene, Pof1p, is an ATPase that may be part of the Saccharomyces cerevisiae protein quality control pathway. According to the results, Δpof1 cells showed increased sensitivity to hydrogen peroxide, tert-butyl hydroperoxide, heat shock and protein unfolding agents, such as dithiothreitol and tunicamycin. Besides, the overexpression of POF1 suppressed the sensitivity of Δpct1, a strain that lacks a gene that encodes a phosphocholine cytidylyltransferase, to heat shock. In vitro analysis showed, however, that the purified Pof1p enzyme had no cytidylyltransferase activity but does have ATPase activity, with catalytic efficiency comparable to other ATPases involved in endoplasmic reticulum-associated degradation of proteins (ERAD). Supporting these findings, co-immunoprecipitation experiments showed a physical interaction between Pof1p and Ubc7p (an ubiquitin conjugating enzyme) in vivo.

Conclusions: Taken together, the results strongly suggest that the biological function of Pof1p is related to the regulation of protein degradation.
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http://dx.doi.org/10.1186/1471-2180-11-268DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3282682PMC
December 2011

Singlet molecular oxygen trapping by the fluorescent probe diethyl-3,3'-(9,10-anthracenediyl)bisacrylate synthesized by the Heck reaction.

Photochem Photobiol Sci 2011 Oct 7;10(10):1546-55. Epub 2011 Jul 7.

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, CP CP26077, CEP 05513-970, São Paulo, SP, Brazil.

Singlet molecular oxygen O(2)((1)Δ(g)) is a potent oxidant that can react with different biomolecules, including DNA, lipids and proteins. Many polycyclic aromatic hydrocarbons have been studied as O(2)((1)Δ(g)) chemical traps. Nevertheless, a suitable modification in the polycyclic aromatic ring must be made to increase the yield of O(2)((1)Δ(g)) chemical trapping. With this goal, an anthracene derivative, diethyl-3,3'-(9,10-anthracenediyl)bisacrylate (DADB), was obtained from the reaction of 9,10-dibromoanthracene and ethyl acrylate through the Heck coupling reaction. The coupling of ethyl acrylate with the anthracene ring produced a new lipophilic, esterified, fluorescent probe reactive toward O(2)((1)Δ(g)). This compound reacts with O(2)((1)Δ(g)) at a rate of k(r) = 1.69 × 10(6) M(-1) s(-1) forming a stable endoperoxide (DADBO(2)), which was characterized by UV-Vis, fluorescence, HPLC/MS and (1)H and (13)C NMR techniques. The photophysical, photochemical and thermostability features of DADB were also evaluated. Furthermore, this compound has the potential for great application in biological systems because it is easily synthetized in large amount and generates specific endoperoxide (DADBO(2)), which can be easily detected by HPLC tandem mass spectrometry (HPLC/MS/MS).
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http://dx.doi.org/10.1039/c1pp05120bDOI Listing
October 2011

Cholesterol hydroperoxides generate singlet molecular oxygen [O(2) ((1)Δ(g))]: near-IR emission, (18)O-labeled hydroperoxides, and mass spectrometry.

Chem Res Toxicol 2011 Jun 5;24(6):887-95. Epub 2011 May 5.

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, CEP 05513-970, CP26077, São Paulo, SP, Brazil.

In mammalian membranes, cholesterol is concentrated in lipid rafts. The generation of cholesterol hydroperoxides (ChOOHs) and their decomposition products induces various types of cell damage. The decomposition of some organic hydroperoxides into peroxyl radicals is known to be a potential source of singlet molecular oxygen [O(2) ((1)Δ(g))] in biological systems. We report herein on evidence of the generation of O(2) ((1)Δ(g)) from ChOOH isomers in solution or in liposomes containing ChOOHs, which involves a cyclic mechanism from a linear tetraoxide intermediate originally proposed by Russell. Characteristic light emission at 1270 nm, corresponding to O(2) ((1)Δ(g)) monomolecular decay, was observed for each ChOOH isomer or in liposomes containing ChOOHs. Moreover, the presence of O(2) ((1)Δ(g)) was unequivocally demonstrated using the direct spectral characterization of near-infrared light emission. Using (18)O-labeled cholesterol hydroperoxide (Ch(18)O(18)OH), we observed the formation of (18)O-labeled O(2) ((1)Δ(g)) [(18)O(2) ((1)Δ(g))] by the chemical trapping of (18)O(2) ((1)Δ(g)) with 9,10-diphenylanthracene (DPA) and detected the corresponding (18)O-labeled DPA endoperoxide (DPA(18)O(18)O) and the (18)O-labeled products of the Russell mechanism using high-performance liquid chromatography coupled to tandem mass spectrometry. Photoemission properties and chemical trapping clearly demonstrate that the decomposition of Ch(18)O(18)OH generates (18)O(2) ((1)Δ(g)), which is consistent with the Russell mechanism and points to the involvement of O(2) ((1)Δ(g)) in cholesterol hydroperoxide-mediated cytotoxicity.
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http://dx.doi.org/10.1021/tx200079dDOI Listing
June 2011

Highly sensitive fluorescent method for the detection of cholesterol aldehydes formed by ozone and singlet molecular oxygen.

Anal Chem 2010 Aug;82(16):6775-81

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, CP26077, CEP 05513-970, São Paulo, SP, Brazil.

Cholesterol oxidation gives rise to a mixture of oxidized products. Different types of products are generated according to the reactive species being involved. Recently, attention has been focused on two cholesterol aldehydes, 3beta-hydroxy-5beta-hydroxy-B-norcholestane-6beta-carboxyaldehyde (1a) and 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al (1b). These aldehydes can be generated by ozone-, as well as by singlet molecular oxygen-mediated cholesterol oxidation. It has been suggested that 1b is preferentially formed by ozone and 1a is preferentially formed by singlet molecular oxygen. In this study we describe the use of 1-pyrenebutyric hydrazine (PBH) as a fluorescent probe for the detection of cholesterol aldehydes. The formation of the fluorescent adduct between 1a with PBH was confirmed by HPLC-MS/MS. The fluorescence spectra of PBH did not change upon binding to the aldehyde. Moreover, the derivatization was also effective in the absence of an acidified medium, which is critical to avoid the formation of cholesterol aldehydes through Hock cleavage of 5alpha-hydroperoxycholesterol. In conclusion, PBH can be used as an efficient fluorescent probe for the detection/quantification of cholesterol aldehydes in biological samples. Its analysis by HPLC coupled to a fluorescent detector provides a sensitive and specific way to quantify cholesterol aldehydes in the low femtomol range.
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http://dx.doi.org/10.1021/ac1006427DOI Listing
August 2010

Detection and characterization of cholesterol-oxidized products using HPLC coupled to dopant assisted atmospheric pressure photoionization tandem mass spectrometry.

Anal Chem 2010 Sep;82(17):7293-301

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Brazil.

Oxidation of cholesterol (Ch) by a variety of reactive oxygen species gives rise mainly to hydroperoxides and aldehydes. Despite the growing interest in Ch-oxidized products, the detection and characterization of these products is still a matter of concern. In this work, the main Ch-oxidized products, namely, 3beta-hydroxycholest-5-ene-7alpha-hydroperoxide (7alpha-OOH), 3beta-5alpha-cholest-6-ene-5-hydroperoxide (5alpha-OOH), 3beta-hydroxycholest-4-ene-6alpha-hydroperoxide (6alpha-OOH), 3beta-hydroxycholest-4-ene-6beta-hydroperoxide (6beta-OOH), and 3beta-hydroxy-5beta-hydroxy-B-norcholestane-6beta-carboxaldehyde (ChAld), were detected in the same analysis using high-performance liquid chromatography (HPLC) coupled to dopant assisted atmospheric pressure photoionization tandem mass spectrometry. The use of selected reaction monitoring mode (SRM) allowed a sensitive detection of each oxidized product, while the enhanced product ion mode (EPI) helped to improve the confidence of the analyses. Isotopic labeling experiments enabled one to elucidate mechanistic features during fragmentation processes. The characteristic fragmentation pattern of Ch-oxidized products is the consecutive loss of H(2)O molecules, yielding cationic fragments at m/z 401, 383, and 365. Homolytic scissions of the peroxide bond are also seen. With (18)O-labeling approach, it was possible to establish a fragmentation order for each isomer. The SRM transitions ratio along with EPI and (18)O-labeled experiments give detailed information about differences for water elimination, allowing a proper discrimination between the isomers. This is of special interest considering the emerging role of Ch-oxidized products in the development of diseases.
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http://dx.doi.org/10.1021/ac1011987DOI Listing
September 2010

Thymine hydroperoxide as a potential source of singlet molecular oxygen in DNA.

Free Radic Biol Med 2009 Aug 6;47(4):401-9. Epub 2009 May 6.

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, CEP 05513-970, CP 26077, São Paulo, SP, Brazil.

The decomposition of organic hydroperoxides into peroxyl radicals is a potential source of singlet molecular oxygen [O2 (1Deltag)] in biological systems. This study shows that 5-(hydroperoxymethyl)uracil (5-HPMU), a thymine hydroperoxide within DNA, reacts with metal ions or HOCl, generating O2 (1Deltag). Spectroscopic evidence for generation of O2 (1Deltag) was obtained by measuring (i) the bimolecular decay, (ii) the monomolecular decay, and (iii) the observation of D2O enhancement of O2 (1Deltag) production and the quenching effect of NaN3. Moreover, the presence of O2 (1Deltag) was unequivocally demonstrated by the direct characterization of the near-infrared light emission. For the sake of comparison, O2 (1Deltag) derived from the H2O2/HOCl system and from the thermolysis of the N,N'-di(2,3-dihydroxypropyl)-1,4-naphthalenedipropanamide endoperoxide was also monitored. More evidence of O2 (1Deltag) generation was obtained by chemical trapping of O2 (1Deltag) with anthracene-9,10-divinylsulfonate (AVS) and detection of the specific AVS endoperoxide by HPLC/MS/MS. The detection by HPLC/MS of 5-(hydroxymethyl)uracil and 5-formyluracil, two thymine oxidation products generated from the reaction of 5-HPMU and Ce4+ ions, supports the Russell mechanism. These photoemission properties and chemical trapping clearly demonstrate that the decomposition of 5-HPMU generates O2 (1Deltag) by the Russell mechanism and point to the involvement of O2 (1Deltag) in thymidine hydroperoxide cytotoxicity.
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http://dx.doi.org/10.1016/j.freeradbiomed.2009.05.001DOI Listing
August 2009

Oxidation and nitration of ribonuclease and lysozyme by peroxynitrite and myeloperoxidase.

Arch Biochem Biophys 2009 Apr 30;484(2):127-33. Epub 2008 Dec 30.

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Caixa Postal 26077, São Paulo, CEP 05513-970, Brazil.

In spite of the many studies on protein modifications by reactive species, knowledge about the products resulting from the oxidation of protein-aromatic residues, including protein-derived radicals and their stable products, remains limited. Here, we compared the oxidative modifications promoted by peroxynitrite and myeloperoxidase/hydrogen peroxide/nitrite in two model proteins, ribonuclease (6Tyr) and lysozyme (3Tyr/6Trp). The formation of protein-derived radicals and products was higher at pH 5.4 and 7.4 for myeloperoxidase and peroxynitrite, respectively. The main product was 3-nitro-Tyr for both proteins and oxidants. Lysozyme rendered similar yields of nitro-Trp, particularly when oxidized by peroxynitrite. Hydroxylated and dimerized products of Trp and Tyr were also produced, but in lower yields. Localization of the main modified residues indicates that peroxynitrite decomposes to radicals within the proteins behaving less specifically than myeloperoxidase. Nitrogen dioxide is emphasized as an important protein modifier.
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http://dx.doi.org/10.1016/j.abb.2008.12.017DOI Listing
April 2009

Peroxidase catalytic cycle of MCM-41-entrapped microperoxidase-11 as a mechanism for phenol oxidation.

J Nanosci Nanotechnol 2007 Oct;7(10):3643-52

Centro Interdisciplinar de Investigação Bioquímica-CIIB Universidade de Mogi das Cruzes-UMC, Mogi das Cruzes-SPF Brazil.

The encapsulation of microperoxidases (MPs) into molecular sieves with controlled pore size, such as the mesoporous silica MCM-41, represents a nanotechnology strategy to control the catalytic properties of MPs and mimic the enzymatic activity of hemoproteins. In this work, the ferric microperoxidase-11 (MP-11), obtained from trypsin-catalyzed hydrolysis of horse-heart cytochrome c, was entrapped in MCM-41, thus resulting in a catalyst (Fe(III)MP11MCM41) with catalase and monooxygenase properties. The entrapment of MP-11 inside MCM-41 was confirmed by elemental analysis and UV-visible spectrum, with a red shift in the Soret band indicating that the heme group was in a hydrophobic microenvironment. Similarly to catalase, the catalyst Fe(III)MP11MCM41 exhibited specificity for hydrogen peroxide to be converted to a high-valence oxidized intermediate, Compound II. Also mimicking catalase, the cleavage of hydrogen peroxide by MP11MCM41 resulted in O2 production detected by a Clark electrode. Phenol was able to act as reducing agent of MP11MCM41 Compound II leading to the completion of a peroxidase cycle, as confirmed by UV-visible spectrometry and EPR measurements. The analysis of the reaction products by high performance liquid chromatogram coupled to tandem mass spectrometry (HPLC/MS) revealed 2,4-dihydroxyphenol as the product of phenol oxidation by MP11MCM41. Therefore, in addition to catalase activity, the catalyst MP11MCM41 also displayed monooxygenase properties, which was possible because the MP-11 heme iron promotes homolytic cleavage of the hydrogen peroxide generating hydroxyl radicals. With such characteristics, MCM-41-entrapped MP-11 is a promising catalyst for nanobiotechnological devices.
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http://dx.doi.org/10.1166/jnn.2007.853DOI Listing
October 2007

Biological hydroperoxides and singlet molecular oxygen generation.

IUBMB Life 2007 Apr-May;59(4-5):322-31

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo. São Paulo, SP, Brazil.

The decomposition of lipid hydroperoxides (LOOH) into peroxyl radicals is a potential source of singlet molecular oxygen ((1)O(2)) in biological systems. Recently, we have clearly demonstrated the generation of (1)O(2) in the reaction of lipid hydroperoxides with biologically important oxidants such as metal ions, peroxynitrite and hypochlorous acid. The approach used to unequivocally demonstrate the generation of (1)O(2) in these reactions was the use of an isotopic labeled hydroperoxide, the (18)O-labeled linoleic acid hydroperoxide, the detection of labeled compounds by HPLC coupled to tandem mass spectrometry (HPLC-MS/MS) and the direct spectroscopic detection and characterization of (1)O(2) light emission. Using this approach we have observed the formation of (18)O-labeled (1)O(2) by chemical trapping of (1)O(2) with anthracene derivatives and detection of the corresponding labeled endoperoxide by HPLC-MS/MS. The generation of (1)O(2) was also demonstrated by direct spectral characterization of (1)O(2) monomol light emission in the near-infrared region (lambda = 1270 nm). In summary, our studies demonstrated that LOOH can originate (1)O(2). The experimental evidences indicate that (1)O(2) is generated at a yield close to 10% by the Russell mechanism, where a linear tetraoxide intermediate is formed in the combination of two peroxyl radicals. In addition to LOOH, other biological hydroperoxides, including hydroperoxides formed in proteins and nucleic acids, may also participate in reactions leading to the generation (1)O(2). This hypothesis is currently being investigated in our laboratory.
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http://dx.doi.org/10.1080/15216540701242508DOI Listing
December 2007

Novel rhythms of N1-acetyl-N2-formyl-5-methoxykynuramine and its precursor melatonin in water hyacinth: importance for phytoremediation.

FASEB J 2007 Jun 21;21(8):1724-9. Epub 2007 Feb 21.

Department of Cellular Structural Biology, The University of Texas Health Science Center, 7703 Floyd Curl Dr., San Antonio, TX 78229, USA.

N1-acetyl-N2-formyl-5-methoxykynuramine (AMFK) is a major metabolite of melatonin in mammals. To investigate whether AFMK exists in plants, an aquatic plant, water hyacinth, was used. To achieve this, LC/MS/MS with a deuterated standard was employed. AFMK was identified in any plant for the first time. Both it and its precursor, melatonin, were rhythmic with peaks during the late light phase. These novel rhythms indicate that these molecules do not serve as the chemical signal of darkness as in animals but may relate to processes of photosynthesis or photoprotection. These possibilities are supported by higher production of melatonin and AFMK in plants grown in sunlight (10,000-15,000 microW/cm2) compared to those grown under artificial light (400-450 microW/cm2). Melatonin and AFMK, as potent free radical scavengers, may assist plants in coping with harsh environmental insults, including soil and water pollutants. High levels of melatonin and AFMK in water hyacinth may explain why this plant more easily tolerates environmental pollutants, including toxic chemicals and heavy metals and is successfully used in phytoremediation. These novel findings could lead to improvements in the phytoremediative capacity of plants by either stimulating endogenous melatonin synthesis or by adding melatonin to water/soil in which they are grown.
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http://dx.doi.org/10.1096/fj.06-7745comDOI Listing
June 2007

Reaction route control by microperoxidase-9/CTAB micelle ratios.

Phys Chem Chem Phys 2006 Apr 14;8(16):1963-73. Epub 2006 Mar 14.

Centro Interdisciplinar de Investigação Bioquímica-CIIB Universidade de Mogi das Cruzes-UMC, Mogi das Cruzes, SP, Brazil.

Microperoxidases (MP) as water-soluble models attract interest to studying the reaction mechanism of peroxidases because these heme peptides are able to form the same enzyme intermediates during the reaction with peroxides. In this work we have demonstrated that the association of Fe(III)MP-9 and Fe(III)MP-11 with CTAB micelles (MP-9/CTAB and MP11/CTAB) provides a microenvironment with an alkaline interface and a hydrophobic core that exhibits peroxidase behavior. This microenvironment shifts positively the redox potential of microperoxidases by approximately 100 mV. tert-Butylhydroperoxide (t-BuOOH) when added to the medium, converted Fe(III)MP-9/CTAB to MP-9/CTAB Compound II, a high valence oxidized intermediate of the heme peptide. Subsequent addition of diphenylacetaldehyde (DPAA) to MP-9/CTAB Compound II regenerated the native form of the enzyme, Fe(III)MP-9/CTAB, what characterizes the occurrence of a peroxidase cycle. Fe(III)MP-9/CTAB regenerated during the peroxidase cycle reacted with residual DPAA in the medium to form Fe(II)MP-9/CTAB, which indicates that both Fe(III)MP-9/CTAB and its oxyferryl form can use aldehydes as reducing agents. According to the determined reduction potential, Fe(III)MP-9 and Fe(III)MP-9/CTAB should be able to oxidize DPAA (reduction potential -630 mV). The reaction of MP-9/CTAB with DPAA produced benzophenone as final product, detected by infrared spectroscopy and mass spectrometry. Interestingly, a significant difference was observed in the benzophenone yield according to the micelle/MP-9 molar ratio.
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http://dx.doi.org/10.1039/b601671eDOI Listing
April 2006

Measurement of melatonin and its metabolites: importance for the evaluation of their biological roles.

Endocrine 2005 Jul;27(2):111-8

Departamento de Bioquímica e Biologia Molecular, Setor de Ciências Biológicas, Universidade Federal do Paraná, Curitiba, PR, Brazil.

Many physiologic changes related to light-dark cycles and antioxidant effects have been related to melatonin (N-acetyl-5-methoxytryptamine) and its metabolites, N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) and N1-acetyl-5-methoxykynuramine (AMK). In this review, we discuss some methodologies, in particular, those employing high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) assays to quantitatively determine melatonin, AFMK, and AMK. These approaches offer a highly specific and an accurate quantification of melatonin and its metabolites. These characteristics are essential to point out correctly the biological effects of these compounds in physiological and pathological conditions.
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http://dx.doi.org/10.1385/endo:27:2:111DOI Listing
July 2005

pH-Dependent interaction of cytochrome c with mitochondrial mimetic membranes: the role of an array of positively charged amino acids.

J Biol Chem 2005 Oct 11;280(41):34709-17. Epub 2005 Jul 11.

Centro Interdisciplinar de Investigação Bioquímica, Universidade de Mogi das Cruzes, Mogi das Cruzes, São Paulo CEP 08780-911, Brazil.

The interaction of cytochrome c (cyt c) with mitochondrial mimetic vesicles of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, and heart cardiolipin (PCPECL) was investigated over the 7.4-6.2 pH range by means of turbidimetry and photon correlation spectroscopy. In the presence of cyt c, the decrease of pH induced an increase in vesicle turbidity and mean diameter resulting from vesicle fusion as determined by a rapid decrease in the excimer/monomer ratio of 2-(10-(1-pyrene)-decanoyl)-phosphatidylcholine (PyPC). N-acetylated cyt c and protamine, a positively charged protein, increased vesicle turbidity in a pH-independent manner, whereas albumin did not affect PCPECL vesicle turbidity. pH-dependent turbidity kinetics revealed a role for cyt c-ionizable groups with a pK(a)((app)) of approximately 7.0. The carbethoxylation of these groups by diethylpyrocarbonate prevented cyt c-induced vesicle fusion, although cyt c association to vesicles remained unaffected. Matrix-assisted laser desorption ionization time-of-flight analysis revealed that Lys-22, Lys-27, His-33, and Lys-87 cyt c residues were the main targets for carbethoxylation performed at low pH values (<7.5). In fact, these amino acid residues belong to clusters of positively charged amino acids that lower the pK(a). Thus, at low pH, protonation of these invariant and highly conserved amino acid residues produced a second positively charged region opposite to the Lys-72 and Lys-73 region in the cyt c structure. These two opposing sites allowed two vesicles to be brought together by the same cyt c molecule for fusion. Therefore, a novel pH-dependent site associating cyt c to mitochondrial mimetic membranes was established in this study.
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http://dx.doi.org/10.1074/jbc.M412532200DOI Listing
October 2005

Changes in the spin state and reactivity of cytochrome C induced by photochemically generated singlet oxygen and free radicals.

J Biol Chem 2004 Sep 9;279(38):39214-22. Epub 2004 Jul 9.

Centro Interdisciplinar de Investigação Bioquímica CIIB Universidade de Mogi das Cruzes UMC, Mogi das Cruzes, SP, CEP 08780-911, Brazil.

This work compares the effect of photogenerated singlet oxygen (O(2)((1)Delta(g))) (type II mechanism) and free radicals (type I mechanism) on cytochrome c structure and reactivity. Both reactive species were obtained by photoexcitation of methylene blue (MB(+)) in the monomer and dimer forms, respectively. The monomer form is predominant at low dye concentrations (up to 8 microm) or in the presence of an excess of SDS micelles, while dimers are predominant at 0.7 mm SDS. Over a pH range in which cytochrome c is in the native form, O(2) ((1)Delta(g)) and free radicals induced a Soret band blue shift (from 409 to 405 nm), predominantly. EPR measurements revealed that the blue shift of the Soret band was compatible with conversion of the heme iron from its native low spin state to a high spin state with axial symmetry (g approximately 6.0). Soret band bleaching, due to direct attack on the heme group, was only detected under conditions that favored free radical production (MB(+) dimer in SDS micelles) or in the presence of a less structured form of the protein (above pH 9.3). Matrix-assisted laser desorption ionization time-of-flight mass spectrometry of the heme group and the polypeptide chain of cytochrome c with Soret band at 405 nm (cytc405) revealed no alterations in the mass of the cytc405 heme group but oxidative modifications on methionine (Met(65) and Met(80)) and tyrosine (Tyr(74)) residues. Damage of cytc405 tyrosine residue impaired its reduction by diphenylacetaldehyde, but not by beta-mercaptoethanol, which was able to reduce cytc405, generating cytochrome c Fe(II) in the high spin state (spin 2).
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http://dx.doi.org/10.1074/jbc.M402093200DOI Listing
September 2004

Protonation of two adjacent tyrosine residues influences the reduction of cytochrome c by diphenylacetaldehyde: a possible mechanism to select the reducer agent of heme iron.

Free Radic Biol Med 2004 Mar;36(6):802-10

Centro Interdisciplinar de Investigação Bioquímica, Universidade de Mogi das Cruzes, Mogi das Cruzes, São Paulo, Brazil.

We have shown that diphenlacetaldehyde (DPAA) is able to promote mitochondrial DeltaPsi disruption accompanied by damage in mitochondrial DNA, lipids, and proteins [Almeida, A. M.; Bechara, E. J. H.; Vercesi, A. E.; Nantes, I. L. Free Radic. Biol. Med. 27:744-747; 1999]. In this work, DPAA was used as a model of carbonyl reagent for cytochrome c. The results suggest that DPAA is a redox cytochrome c modifier. Conversion of Fe(III) to Fe(II) cytochrome c promoted by DPAA is pH dependent. The second-order rate determined for heme iron reduction (k2) is 698 M(-1) s(-1) and this process occurs with an activation energy of 8.5 +/- 0.8 kcal/mol. Analysis of the pH profile suggests the presence of two ionizable cytochrome c groups (pKa1 = 8.9 and pKa2 = 11.4) related to the electron transfer from DPAA to heme iron. The heats of ionization of the two prototropic groups, pKa1 (DeltaH(ion) = 6.5 kcal/mol, DeltaS(ion) = -29.0 cal/mol.K), and pKa2 (DeltaH(ion) = 5.0 kcal/mol, DeltaS(ion) = -24.0 cal/mol.K), suggest involvement of two tyrosine residues, probably Y67 and Y74, related to DPAA-promoted heme iron reduction. The cytochrome c chemical modification by iodination of tyrosine groups significantly decreased the reduction rate promoted by DPAA, and shifted the pH(opt) value from 10.0 to 9.25. The cytochrome c-promoted DPAA electron abstraction quickly produces the expected enol-derived radical, as indicated by 3,5-dibromo-4-nitrosobenzenesulfonate (DBNBS) spin trapping EPR measurements. This radical reacts with molecular oxygen, producing a peroxyl intermediate radical that, via a putative dioxetane intermediate, promotes formation of benzophenone as the main final product of this reaction, detected by high-performance liquid chromatography coupled with tandem mass spectrometry.
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http://dx.doi.org/10.1016/j.freeradbiomed.2003.12.002DOI Listing
March 2004