2,2-diphenyl-1-picrylhydrazyl radical solubilized by β-cyclodextrin in water for evaluation of...

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antioxidant role at later stages of oxidation. 343 Distribution and Classification of Cys-Based Proteins of the Ohr/OsmC Family Diogo de Abreu Meireles 1 , Renato Mateus Domingos 1 , Robson Francisco de Souza 2 , and Luis Eduardo Soares Netto 1 1 Instituto de Biociências, Universidade de São Paulo, Brazil, 2 Instituto de Ciências Biomédicas, Universidade de São Paulo, Brazil Ohr (Organic Hydroperoxide Resistance Protein) and OsmC (Osmotically inducible protein C) are two Cys-based lypoil- dependent peroxidases that display preference for organic hydroperoxides as substrates and do not share homology to peroxiredoxins. Ohr/OsmC family of proteins can be subdivided into three subgroups (named Ohr, OsmC and YhfA) and the peroxidase activity is probably dependent on the presence of two Cys residues and one conserved Arg residue. YhfA proteins lack this conserved Arg residue and probably the peroxidase activity. Initially, we studied the distribution of Ohr, OsmC and YhfA genes among distinct phyla in an attempt to find an evolutionary pattern. For the first time, through an in deep bioinformatic analysis of a large set of sequences (14166 genes), we found Ohr/OsmC members outside of Eubacteria Domain. Furthermore, the analysis revealed that a more complex classification was required than three sub-groups (Ohr, OsmC and YhfA) initially proposed (Shin et al., 2004). In parallel, through enzymatic and functional complementation assays, we showed that YhfA protein from Escherichia coli did not present thiol dependent peroxidase activity towards distinct substrates. Accordingly, the pK a value of its reactive Cys residue, determined by monobromobimane alkylation method, was 8.27 ±0.11, which is high when compared with Ohr and OsmC proteins. Finally, expression of genes from distinct phyla (including gram positive and negative bacteria and eukaryotes) in an E.coli strain sensitive to organic peroxide DKS&¨ UHVXOWHG LQ FRPSOHPHQWDWLRQ UHVWRUDWLRQ ZLOG-type phenotype) only for ohr or osmC genes. In contrast, YhfA and other less characterized proteins did not rescue wild-type phenotype, suggesting that they are not Cys-based, thiol- dependent peroxidases. These hypotheses will be tested by assaying recombinant proteins. Keywords: Hydroperoxide; Cys-based peroxidase; Acknowledgements: Fapesp, CEPID/INCT Redoxoma, CNPq. 344 2,2-Diphenyl-1-Picrylhydrazyl Radical Solubilized by ȕ-Cyclodextrin in Water for Evaluation of Radical- Scavenging Activity of Antioxidants in Aqueous Systems Ikuo Nakanishi 1 , Kohei Imai 1,2 , Kiyoshi Fukuhara 2 , Toshihiko Ozawa 1,3 , and Ken-ichiro Matsumoto 1 1 National Institute of Radiological Sciences, Japan, 2 Showa University, Japan, 3 Showa Pharmaceutical University, Japan 2,2-Diphenyl-1-picrylhydrazyl radical (DPPH ) is a relatively stable radical and frequently used as a model of reactive oxygen species to evaluate the radical-scavenging activity of antioxidants. However, the poor solubility of DPPH to water has precluded its XVH LQ DTXHRXV V\VWHPV ,Q WKLV VWXG\ ZH XVHG ȕ-cyclodextrin (CD) to solubilize DPPH in water. Ball milling of the mixture containing DPPH and ȕ-CD for 1 h, followed by an addition of water and filtration with a membrane filter gave a purple solution. This solution showed an absorption band at 525 nm, which is diagnostic of DPPH . When ascorbic acid was added to the solution, the band at 525 nm disappeared immediately. This indicates that ascorbic acid efficiently scavenge DPPH in water. Thus, DPPH solubilized by ȕ-CD in water may be a powerful tool to evaluate antioxidative activity of antioxidants in aqueous systems. 345 Mechanisms and Biological Consequences of Damage to Human Coronary Artery Endothelial (HCAEC) Cells by Peroxynitrous Acid Priyashiel Parikh 1,2 , Clare Hawkins 1,2 , and Michael Davies 1,2 1 The Heart Research Institute, Australia, 2 The University of Sydney, Australia Atherosclerosis is a major cause of cardiovascular disease and is responsible for ~35% of deaths in developed and developing nations. Atherosclerosis is characterized by chronic inflammation and lipid accumulation in arterial wall macrophages. Considerable data indicate that activated macrophages generate superoxide radicals and nitric oxide, and hence the powerful oxidant peroxynitrous acid (ONOOH) within human atherosclerotic lesions. We hypothesized that ONOOH would induce dysfunction of human coronary artery endothelial cells via thiol depletion and protein nitration, and this would affect membrane proteins to a greater extent than cytosolic proteins. Intact cells or lysate were treated with ONOOH (0- ȝ0 IRU PLQV DW qC. under these conditions no significant loss of viability was detected with < ȝ0 2122+ XVLQJ WKH 077 DVVD\ )UDFWLRQDO centrifugation was used to examine the nature and extents of ONOOH-mediated damage to cytosolic and membrane proteins as assessed by Cys oxidation and 3-nitrotyrosine (3NT) formation. Significant thiol loss was detected for both cytosolic and membrane proteins when intact cells were treated with > 500 ONOOH. In contrast treatment of cell lysates induced thiol loss on cytosolic and membranic SURWHLQV DW ȝ0 2122+ 7KH SUHVHQFH of CO2 (from 25 mM NaHCO3), with which ONOOH reacts with to form the alternative oxidant ONOOCO2 - , did not alter the pattern or extent of thiol loss. 3NT was generated in a dose-dependent manner on multiple cytosolic and membranic proteins as detected by Western blotting, and silver staining of SDS-PAGE gels. WB and MS peptide mass mapping indicate modification of specific proteins including actin and GAPDH. Overall these results indicate that ONOOH induces significant thiol loss and 3NT formation on cytosolic and membranic proteins, with the latter being particularly susceptible targets. 346 Reaction of Phenols with Nitrous Acid and Nitrogen Dioxide: A Kinetic and Spectroelectrochemistry Study Jael Lucila Reyes 1 1 Pontificia Universidad Católica, Chile The reactions mediated by nitrous acid (HNO2) and nitrogen S142 SFRBM 2014 doi: 10.1016/j.freeradbiomed.2014.10.117 doi: 10.1016/j.freeradbiomed.2014.10.118 doi: 10.1016/j.freeradbiomed.2014.10.120 doi: 10.1016/j.freeradbiomed.2014.10.119

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Page 1: 2,2-Diphenyl-1-Picrylhydrazyl Radical Solubilized by β-Cyclodextrin in Water for Evaluation of Radical-Scavenging Activity of Antioxidants in Aqueous Systems

antioxidant role at later stages of oxidation.

343 Distribution and Classification of Cys-Based Proteins of the Ohr/OsmC Family Diogo de Abreu Meireles1, Renato Mateus Domingos1, Robson Francisco de Souza2, and Luis Eduardo Soares Netto1 1Instituto de Biociências, Universidade de São Paulo, Brazil, 2Instituto de Ciências Biomédicas, Universidade de São Paulo, Brazil Ohr (Organic Hydroperoxide Resistance Protein) and OsmC (Osmotically inducible protein C) are two Cys-based lypoil-dependent peroxidases that display preference for organic hydroperoxides as substrates and do not share homology to peroxiredoxins. Ohr/OsmC family of proteins can be subdivided into three subgroups (named Ohr, OsmC and YhfA) and the peroxidase activity is probably dependent on the presence of two Cys residues and one conserved Arg residue. YhfA proteins lack this conserved Arg residue and probably the peroxidase activity. Initially, we studied the distribution of Ohr, OsmC and YhfA genes among distinct phyla in an attempt to find an evolutionary pattern. For the first time, through an in deep bioinformatic analysis of a large set of sequences (14166 genes), we found Ohr/OsmC members outside of Eubacteria Domain. Furthermore, the analysis revealed that a more complex classification was required than three sub-groups (Ohr, OsmC and YhfA) initially proposed (Shin et al., 2004). In parallel, through enzymatic and functional complementation assays, we showed that YhfA protein from Escherichia coli did not present thiol dependent peroxidase activity towards distinct substrates. Accordingly, the pKa value of its reactive Cys residue, determined by monobromobimane alkylation method, was 8.27 ±0.11, which is high when compared with Ohr and OsmC proteins. Finally, expression of genes from distinct phyla (including gram positive and negative bacteria and eukaryotes) in an E.coli strain sensitive to organic peroxide

-type phenotype) only for ohr or osmC genes. In contrast, YhfA and other less characterized proteins did not rescue wild-type phenotype, suggesting that they are not Cys-based, thiol-dependent peroxidases. These hypotheses will be tested by assaying recombinant proteins. Keywords: Hydroperoxide; Cys-based peroxidase; Acknowledgements: Fapesp, CEPID/INCT Redoxoma, CNPq.

344 2,2-Diphenyl-1-Picrylhydrazyl Radical Solubilized by

-Cyclodextrin in Water for Evaluation of Radical-Scavenging Activity of Antioxidants in Aqueous Systems Ikuo Nakanishi1, Kohei Imai1,2, Kiyoshi Fukuhara2, Toshihiko Ozawa1,3, and Ken-ichiro Matsumoto1 1National Institute of Radiological Sciences, Japan, 2Showa University, Japan, 3Showa Pharmaceutical University, Japan 2,2-Diphenyl-1-picrylhydrazyl radical (DPPH ) is a relatively stable radical and frequently used as a model of reactive oxygen species to evaluate the radical-scavenging activity of antioxidants. However, the poor solubility of DPPH to water has precluded its

-cyclodextrin (CD) to solubilize DPPH in water. Ball milling of the mixture containing DPPH and -CD for 1 h, followed by an addition of water and filtration with a membrane filter gave a purple solution. This solution showed an absorption band at 525 nm, which is diagnostic of DPPH . When ascorbic acid was added to the solution, the band at 525 nm disappeared immediately. This indicates that ascorbic acid efficiently scavenge DPPH in water. Thus, DPPH solubilized by -CD in water may be a powerful tool to evaluate antioxidative activity of antioxidants in aqueous systems.

345 Mechanisms and Biological Consequences of Damage to Human Coronary Artery Endothelial (HCAEC) Cells by Peroxynitrous Acid Priyashiel Parikh1,2, Clare Hawkins1,2, and Michael Davies1,2 1The Heart Research Institute, Australia, 2The University of Sydney, Australia Atherosclerosis is a major cause of cardiovascular disease and is responsible for ~35% of deaths in developed and developing nations. Atherosclerosis is characterized by chronic inflammation and lipid accumulation in arterial wall macrophages. Considerable data indicate that activated macrophages generate superoxide radicals and nitric oxide, and hence the powerful oxidant peroxynitrous acid (ONOOH) within human atherosclerotic lesions. We hypothesized that ONOOH would induce dysfunction of human coronary artery endothelial cells via thiol depletion and protein nitration, and this would affect membrane proteins to a greater extent than cytosolic proteins. Intact cells or lysate were treated with ONOOH (0- C. under these conditions no significant loss of viability was detected with <

centrifugation was used to examine the nature and extents of ONOOH-mediated damage to cytosolic and membrane proteins as assessed by Cys oxidation and 3-nitrotyrosine (3NT) formation. Significant thiol loss was detected for both cytosolic and membrane proteins when intact cells were treated with > 500 ONOOH. In contrast treatment of cell lysates induced thiol loss on cytosolic and membranic

of CO2 (from 25 mM NaHCO3), with which ONOOH reacts with to form the alternative oxidant ONOOCO2

-, did not alter the pattern or extent of thiol loss. 3NT was generated in a dose-dependent manner on multiple cytosolic and membranic proteins as detected by Western blotting, and silver staining of SDS-PAGE gels. WB and MS peptide mass mapping indicate modification of specific proteins including actin and GAPDH. Overall these results indicate that ONOOH induces significant thiol loss and 3NT formation on cytosolic and membranic proteins, with the latter being particularly susceptible targets.

346 Reaction of Phenols with Nitrous Acid and Nitrogen Dioxide: A Kinetic and Spectroelectrochemistry Study Jael Lucila Reyes1 1Pontificia Universidad Católica, Chile The reactions mediated by nitrous acid (HNO2) and nitrogen

S142 SFRBM 2014

doi: 10.1016/j.freeradbiomed.2014.10.117

doi: 10.1016/j.freeradbiomed.2014.10.118

doi: 10.1016/j.freeradbiomed.2014.10.120

doi: 10.1016/j.freeradbiomed.2014.10.119