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New iron nitrosyl complexes containing strong electron donating carbaporphyrins
S36
Abstracts / Nitric Oxide 27 (2012) S2–S50
P-70
Abstract Withdrawn http://dx.doi.org/10.1016/j.niox.2012.04.128
P-71 New iron nitrosyl complexes containing strong electron donating carbaporphyrins Chen-Hsiung Hung a, Wei-Min Ching b a Institute of Chemistry, Academia Sinica, Taipei, Taiwan, b Department of Chemistry, National Taiwan Normal University, Taipei, Taiwan A serious of new iron nitrosyl complexes containing carbaporphyrins as the macrocyclic ligand has been synthesized. Two methods have been developed to introduce the axial nitric oxide, either through nitrite reduction or through direct reaction with nitric oxide gas. In the case of nitrite reduction, the formation of the reduced {Fe (NO)}7 complex or the oxidized {Fe (NO)}6 can be controlled through the stoichiometry of the nitrite anion used as the reactant. Both oxidized and reduced carbaporphyrin iron nitrosyl complexes exhibit a 3N1C coordination environment for iron metal center to bind with the carbaporphyrin with which containing an inverted pyrrole ring to give an inner carbon atom in the coordination sphere. The linear and bent bond angles for {Fe (NO)}6 and {Fe (NO)}7, respectively, as well as the NO stretching frequencies in IR spectroscopy resemble regular iron porphyrin nitrosyl complexes. On the other hand, unique chemistry has been observed using the inner carbon methylated carbaporphyrin as the ligand. It is found that the iron oxidation and the nitric oxide coordination synchronized a C–H activation on the inner methyl group to give an unusual {Fe (NO)}6 iron nitrosyl complex. The conformation confirmed by the single crystal X-ray structure suggests a non-planar porphyrinic core with a strong pi-donating from the inner alkene to the iron center. The strong backbonding also resulted a small Fe–N–O angle at 148 degree. The unusual spectroscopies, electronic structures, and reactions of these carbaporphyrin complexes will be discussed. http://dx.doi.org/10.1016/j.niox.2012.04.129
P-72 Formation of nitrated tryptophan residues as a novel posttranslational modification in the physiological state Fumiyuki Yamakura, Hiroaki Kawasaki, Ayako Shigenaga, Munehiro Uda, Takeshi Baba, Kenji Takamori Juntendo University, Inzai, Chiba, Japan Protein nitration is a post-translational modification that is induced by reactive nitrogen species (RNS) such as peroxynitrite (ONOO) and nitrogen dioxide (NO2). Nitration of tyrosine residues in proteins has been studied extensively and found in many diseases under oxidative stress. We have found a novel type of protein nitration, formation of 6-nitrotryptophan (6-NO2Trp) residues, developed its specific antibody, and constructed a method to detect 6-NO2Trp-containing proteins by proteomic analysis using LC–ESI–MS–MS (1). In this study, we applied this method for PC12 cell (naïve PC12 cell) and NGF-treated PC12 cell (differentiated-PC12 cell) to detect 6-NO2Trp-containing proteins
in physiological state. We found several peptides from five ribosomal proteins (RP) (60S RP L7, 60S acidic RP P1, 40s RP S2, 40S RP S6, and 40S RP S19), 14-3-3 protein epsilon, nucleoside diphosphate kinase B, and proteasome subunit alpha type1 as nitrotryptophan containing peptides. We successfully determined the positions of nitrated tryptophan residues in the amino acid sequences of these proteins. Among them, the tryptophan nitration was observed only in the differentiated-PC12 cells for 40S RP S19 protein and 14-3-3 protein. The nitrotryptophan positive signals of 40S RP S19 and 14-3-3 protein were suppressed by treatment of PC12 cells with NOS inhibitor, L-NAME. The tryptophan nitration was not observed by LC–MS–MS analysis of these proteins. Finally, we also found several tryptophan-nitrated proteins in brain of adult rats by using the same method. Reference [1] H. Kawasaki et al., Free Radic. Biol. Med. 50 (2011) 419–427. http://dx.doi.org/10.1016/j.niox.2012.04.130
P-73 Abstract Withdrawn http://dx.doi.org/10.1016/j.niox.2012.04.131
P-74 Quantification of a novel sulfhydrylated cGMP in mammalian cells and tissues by liquid chromatography tandem mass spectrometry Hideshi Ihara a, Tomoaki Ida b, Shingo Kasamatsu a, Kouhei Kunieda a, Yoshikazu Ikeda c, Tomohiro Sawa b,d, Takaaki Akaike b a Department of Biological Science, Graduate School of Science, Osaka Prefecture University, Osaka, Japan, b Department of Microbiology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan, c MEGMILK SNOW BRAND Co., Ltd., Saitama, Japan, d PRESTO, Japan Science and Technology Agency (JST), Saitama, Japan 8-Nitroguanosine 30 ,50 -cyclic monophosphate (8-nitro-cGMP) is formed endogenously under conditions of excess production of NO coupled with oxidative stress. Recently, we found that the electrophilic nitro moiety underwent nucleophilic substitution with hydrogen sulfide anion (HS) to yield the novel product 8-SH-cGMP. Endogenous 8-SH-cGMP levels are yet to be identified, however. In this study, we precisely quantified endogenous formation of 8-SHcGMP in various tissues and cells by means of liquid chromatographyelectrospray ionization (ESI)-tandem mass spectrometry (LC–ESI–MS/ MS). Mouse tissue homogenates as well as lysates of various mammalian cultured cells were subjected to LC–ESI–MS/MS analysis with a Thermo TSQ vantage triplequadrupole mass spectrometer equipped with a reverse-phase HPLC column. For precise quantification, the stable isotope dilution method was employed: recovery efficiency of the analyte was corrected based on the recovery of the stable isotopelabeled derivative (8-SH-[13C10]cGMP) spiked into the extracts. This method warrants highly sensitive and specific identification of 8SH-cGMP: detection limit, >1 pmol/ml. According to the LC–MS/MS analyses with stable isotope dilution method, we could quantify the formation of 8-SH-cGMP in mouse tissues and various cells in culture. The highest concentration of 8-SH-cGMP determined in cells was over 10 lM. This sulfhydryl derivative of cGMP is also shown to have a
Abstracts / Nitric Oxide 27 (2012) S2–S50
P-70
Abstract Withdrawn http://dx.doi.org/10.1016/j.niox.2012.04.128
P-71 New iron nitrosyl complexes containing strong electron donating carbaporphyrins Chen-Hsiung Hung a, Wei-Min Ching b a Institute of Chemistry, Academia Sinica, Taipei, Taiwan, b Department of Chemistry, National Taiwan Normal University, Taipei, Taiwan A serious of new iron nitrosyl complexes containing carbaporphyrins as the macrocyclic ligand has been synthesized. Two methods have been developed to introduce the axial nitric oxide, either through nitrite reduction or through direct reaction with nitric oxide gas. In the case of nitrite reduction, the formation of the reduced {Fe (NO)}7 complex or the oxidized {Fe (NO)}6 can be controlled through the stoichiometry of the nitrite anion used as the reactant. Both oxidized and reduced carbaporphyrin iron nitrosyl complexes exhibit a 3N1C coordination environment for iron metal center to bind with the carbaporphyrin with which containing an inverted pyrrole ring to give an inner carbon atom in the coordination sphere. The linear and bent bond angles for {Fe (NO)}6 and {Fe (NO)}7, respectively, as well as the NO stretching frequencies in IR spectroscopy resemble regular iron porphyrin nitrosyl complexes. On the other hand, unique chemistry has been observed using the inner carbon methylated carbaporphyrin as the ligand. It is found that the iron oxidation and the nitric oxide coordination synchronized a C–H activation on the inner methyl group to give an unusual {Fe (NO)}6 iron nitrosyl complex. The conformation confirmed by the single crystal X-ray structure suggests a non-planar porphyrinic core with a strong pi-donating from the inner alkene to the iron center. The strong backbonding also resulted a small Fe–N–O angle at 148 degree. The unusual spectroscopies, electronic structures, and reactions of these carbaporphyrin complexes will be discussed. http://dx.doi.org/10.1016/j.niox.2012.04.129
P-72 Formation of nitrated tryptophan residues as a novel posttranslational modification in the physiological state Fumiyuki Yamakura, Hiroaki Kawasaki, Ayako Shigenaga, Munehiro Uda, Takeshi Baba, Kenji Takamori Juntendo University, Inzai, Chiba, Japan Protein nitration is a post-translational modification that is induced by reactive nitrogen species (RNS) such as peroxynitrite (ONOO) and nitrogen dioxide (NO2). Nitration of tyrosine residues in proteins has been studied extensively and found in many diseases under oxidative stress. We have found a novel type of protein nitration, formation of 6-nitrotryptophan (6-NO2Trp) residues, developed its specific antibody, and constructed a method to detect 6-NO2Trp-containing proteins by proteomic analysis using LC–ESI–MS–MS (1). In this study, we applied this method for PC12 cell (naïve PC12 cell) and NGF-treated PC12 cell (differentiated-PC12 cell) to detect 6-NO2Trp-containing proteins
in physiological state. We found several peptides from five ribosomal proteins (RP) (60S RP L7, 60S acidic RP P1, 40s RP S2, 40S RP S6, and 40S RP S19), 14-3-3 protein epsilon, nucleoside diphosphate kinase B, and proteasome subunit alpha type1 as nitrotryptophan containing peptides. We successfully determined the positions of nitrated tryptophan residues in the amino acid sequences of these proteins. Among them, the tryptophan nitration was observed only in the differentiated-PC12 cells for 40S RP S19 protein and 14-3-3 protein. The nitrotryptophan positive signals of 40S RP S19 and 14-3-3 protein were suppressed by treatment of PC12 cells with NOS inhibitor, L-NAME. The tryptophan nitration was not observed by LC–MS–MS analysis of these proteins. Finally, we also found several tryptophan-nitrated proteins in brain of adult rats by using the same method. Reference [1] H. Kawasaki et al., Free Radic. Biol. Med. 50 (2011) 419–427. http://dx.doi.org/10.1016/j.niox.2012.04.130
P-73 Abstract Withdrawn http://dx.doi.org/10.1016/j.niox.2012.04.131
P-74 Quantification of a novel sulfhydrylated cGMP in mammalian cells and tissues by liquid chromatography tandem mass spectrometry Hideshi Ihara a, Tomoaki Ida b, Shingo Kasamatsu a, Kouhei Kunieda a, Yoshikazu Ikeda c, Tomohiro Sawa b,d, Takaaki Akaike b a Department of Biological Science, Graduate School of Science, Osaka Prefecture University, Osaka, Japan, b Department of Microbiology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan, c MEGMILK SNOW BRAND Co., Ltd., Saitama, Japan, d PRESTO, Japan Science and Technology Agency (JST), Saitama, Japan 8-Nitroguanosine 30 ,50 -cyclic monophosphate (8-nitro-cGMP) is formed endogenously under conditions of excess production of NO coupled with oxidative stress. Recently, we found that the electrophilic nitro moiety underwent nucleophilic substitution with hydrogen sulfide anion (HS) to yield the novel product 8-SH-cGMP. Endogenous 8-SH-cGMP levels are yet to be identified, however. In this study, we precisely quantified endogenous formation of 8-SHcGMP in various tissues and cells by means of liquid chromatographyelectrospray ionization (ESI)-tandem mass spectrometry (LC–ESI–MS/ MS). Mouse tissue homogenates as well as lysates of various mammalian cultured cells were subjected to LC–ESI–MS/MS analysis with a Thermo TSQ vantage triplequadrupole mass spectrometer equipped with a reverse-phase HPLC column. For precise quantification, the stable isotope dilution method was employed: recovery efficiency of the analyte was corrected based on the recovery of the stable isotopelabeled derivative (8-SH-[13C10]cGMP) spiked into the extracts. This method warrants highly sensitive and specific identification of 8SH-cGMP: detection limit, >1 pmol/ml. According to the LC–MS/MS analyses with stable isotope dilution method, we could quantify the formation of 8-SH-cGMP in mouse tissues and various cells in culture. The highest concentration of 8-SH-cGMP determined in cells was over 10 lM. This sulfhydryl derivative of cGMP is also shown to have a