A família Tiol Peroxidases (TPxs - Peroxirredoxinas e Glutationa peroxidases) purificadas definitivamente reduzem peróxidos rapidamente (peroxinitrito, ONOOH/ONOO; peróxido de hidrogênio, H2O2), mas nenhuma evidência direta desta atividade foi demonstrada em células vivas. Isto é particularmente importante pois o ciclo catalítico da atividade peróxido redutase de TPxs depende de sucessivas reações de trocas de tióis que podem limitar a velocidade de redução do peróxido. Neste trabalho, esta questão foi investigada em Saccharomyces cerevisiae (Sc) por meio de cinética de competição com um indicador fluorescente que é específico para ONOO (ácido borônico de cumarina; CBA), com a expectativa de que quanto maior a atividade peroxinitrito redutase, menor a oxidação do indicador. Também foi investigado o papel de duas peroxirredoxinas (Prxs) específicas na remoção deste peróxido. O estudo mostrou que a oxidação do indicador CBA dependente de ONOO foi sempre significativamente maior em células de Saccharomyces cerevisiae deficientes em TPxs (cepa 8) relativo a cepa nativa (WT). Além disso, a transfecção do gene que codifica a Prx mais abundante em Saccharomyces cerevisiae (Tsa1) na cepa 8 diminui parcialmente a oxidação de CBA. Além disso, a oxidação de CBA foi maior na cepa deficiente apenas da peroxirredoxina Tsa1 (a mais abundante da família) relativo à cepa WT, mostrando a relevância desta isoforma especificamente. De forma adversa, a oxidação de CBA na cepa deficiente da peroxirredoxina Tsa2 foi semelhante à cepa WT. Também, foi constatado que o processo de remoção de ONOO é catalítico (e não estequiométrico) para crescentes fluxos de peroxinitrito em todas as cepas e condições utilizadas no estudo. Finalmente, o estudo sugere que células possuem sistemas catalíticos peroxinitrito redutase redundantes, já que a própria cepa 8 apresenta e pode modular esta atividade. Estes resultados confirmam a expectativa da relevância de TPxs na remoção de ONOO e por extensão de outros peróxidos biologicamente relevantes e são a primeira evidência direta e em tempo real da atividade peroxinitrito redutase de TPxs em células. / The purified Thiol Peroxidases family (TPxs - Peroxiredoxins and Glutathione peroxidases) rapidly reduces peroxides (peroxynitrite, ONOOH/ONOO-, hydrogen peroxide, H2O2), but no direct evidence of this activity has been demonstrated in living cells. This is particularly important since the catalytic cycle of the TPxs peroxide reductase activity depends on successive thiol exchange reactions, which may limit the rate of peroxide reduction. In this work, this question was investigated in Saccharomyces cerevisiae (Sc) by competition kinetics using a fluorescent indicator that is specific for ONOO- (coumarin boronic acid; CBA). It is expected that the higher the peroxynitrite reductase activity, the lower the oxidation of the indicator. The role of two specific peroxiredoxins (Prxs) in the removal of this peroxide has also been investigated. The study showed that the oxidation of ONOO- dependent CBA indicator was always significantly higher in TPxs-deficient Saccharomyces cerevisiae cells (strain 8) compared to the native strain (WT). In addition, the transfection of the gene encoding the most abundant Prx into Saccharomyces cerevisiae (Tsa1) in the 8 strain partially diminishes CBA oxidation. Besides that, CBA oxidation was greater in the deficient strain only of the peroxiredoxin Tsa1 (the most abundant in the family) compared to the WT strain, showing the relevance of this isoform specifically. On the other hand, CBA oxidation in the deficient strain of the Tsa2 peroxiredoxin was similar to the WT strain. Also, it was found that the ONOO- removal process is catalytic (and not stoichiometric) for increasing peroxynitrite fluxes in all strains and conditions used in the study. Finally, the study suggests that cells have redundant peroxynitrite reductase catalytic systems, since the 8 strain itself presents and can modulate this activity. These results confirm the expectation of the relevance of TPxs in the removal of ONOO- and by extension of other biologically relevant peroxides and are the first direct and real-time evidence of peroxynitrite reductase activity of TPxs in cells.

Os oxidantes derivados do peroxinitrito e das peroxidases, como mieloperoxidase (MPO), e os danos que ocasionam em proteínas vêm sendo muito estudados pela sua relevância em processos inflamatórios. Neste trabalho, as proteínas RNase e lisozima foram empregadas como alvos de oxidação e nitração mediadas por peroxinitrito e MPO/H<SUB.2O2/NO2-. Experimentos de EPR indicaram que as oxidações envolvem a formação de radicais protéicos sendo que os principais foram caracterizados como RNase-tirosila e lisozima-tirosila exposto e não exposto ao solvente, respectivamente. Estimativas do rendimento de radicais protéicos e produtos nitrados nos pHs 5,4, 6,4 e 7,4 mostrou que o peroxinitrito e o sistema MPO/H<SUB.2O2/NO2- são oxidantes mais efetivos nos pHs 7,4 e 5,4, respectivamente. Na condição ótima para cada oxidante foram identificados produtos de oxidação/nitração de resíduos de Tyr e Trp por HPLC-UV/MS-ESI. Para localização dos resíduos modificados nas estruturas das proteínas tratadas, elas foram digeridas com tripsina e os peptídeos resultantes submetidos a análise por HPLC/MS-MALDI-ToF. Desses resultados pode-se concluir que a RNase foi nitrada preferencialmente nos fragmentos contendo o(s)resíduo(s) Tyr115 > Tyr92/97 > Tyr73/76 por peroxinitrito e em praticamente todos os resíduos de tirosina por MPOH<SUB.2O2/NO2-. No caso da lisozima, o peroxinitrito oxidou principalmente o fragmento contendo os resíduos Trp62/63 que se mostrou nitrado e oxidado a dímero e quinurenina. Já o sistema MPO/H<SUB.2O2/NO2- nitrou o fragmento contendo os resíduos Tyr23/28 e nitrou e oxidou a dímeros e quinurenina o fragmento contendo os resíduos Trp62/63. As relações entre a acessibilidade dos resíduos específicos nas estruturas terciárias e a formação de produtos de oxidação/nitração são discutidas. Também, a possível importância da oxidação de resíduos de triptofano em agregação de proteínas é enfatizada. Paralelamente, examinou-se os efeitos do nitróxido tempol sobre a nitração da RNase mediada por MPO ou HRP/H<SUB.2O2/NO2- em condições de máxima nitração. De fato, as interações de tempol com peroxidases eram pouco conhecidas apesar da eficiência do nitróxido em reduzir a injúria e os níveis de 3-nitrotirosina em proteínas de tecidos de animais submetidos a condições inflamatórias. Foram determinadas as constantes de velocidade da reação do tempol com os intermediários oxidantes da MPO e HRP e também, o consumo de reagentes e a formação de produtos. A simulação dos resultados experimentais indicou que o tempol inibe a nitração da RNase mediada por peroxidases principalmente pela sua capacidade de reagir rapidamente com o &#8226;NO2 com formação de nitrito e cátion oxamônio que, por sua vez, recicla para tempol reagindo com H2O2 para produzir O2. / The oxidants derived from peroxynitrite and peroxidase enzymes, such as myeloperoxidase (MPO), and the lesions they promote in proteins are being extensively investigated because of their relevance in inflammatory processes. Here, the proteins RNase and lysozyme were employed as targets of oxidations/nitrations mediated by peroxynitrite and MPO/H<SUB.2O2/NO2-. EPR experiments showed that the oxidations produced protein radicals of which the prominent ones were characterized as RNase-tyrosyl and lysozyme-tyrosil solvent-exposed and non-exposed, respectively. Estimates of protein radical and nitrated product yields at pH 5.4, 6.4 and 7.4 indicated that peroxynitrite and MPO/H<SUB.2O2/NO2- were more effective oxidants at pH 7.4 and 5.4, respectively. At the best condition for each oxidant, the oxidation/nitration products of Tyr and Trp residues were identified by HPLC-UV/ESI-MS analysis. The site of oxidation in the protein structures were identified by HPLC/MALDI-ToF-MS analysis of tryptic digests after oxidative treatment. From these results, it was concluded that RNase was nitrated mainly in Tyr115 > Tyr92/97 > Tyr62/63 by peroxynitrite and in all Tyr by MPO/H<SUB.2O2/NO2-. In the case of lysozyme, peroxynitrite modified mainly Trp62/63 that resulted nitrated and oxidized to a dimer and kynurenine. The MPO/H<SUB.2O2/NO2- system promoted the nitration of Tyr23/Trp28 and nitration and oxidation to dimer and kynurenine of Trp62/63. The relationships between residue accessibility in the structure of the proteins and their oxidation/nitration are discussed. The possible importance of Trp oxidation in protein aggregation is emphasized. In parallel, the effects of the nitroxide tempol upon RNase nitration mediated by MPO or HRP/H<SUB.2O2/NO2- was examined. Indeed, the interactions of tempol with peroxidases have been little investigated although the nitroxide is very efficient in reducing injury and 3-nitrotyrosine protein levels in tissues of animals submitted to inflammatory conditions. The second order rate constants of tempol reactions with the ferryl oxidants of MPO and HRP were determined. The consumption of reactants and formation of products were also determined. Computer simulation of the results indicated that tempol inhibits RNAse nitration mediated by peroxidases mainly because of its capability to rapidly react with &#8226;NO2 with formation of nitrite and the oxammonium cation, which, in turn, recycles back to tempol, by reacting with H2O2 to produce O2.

Thesis (doctoral)--Umeå Universitet, 1987. / Extra t.p. with thesis statement inserted. Includes bibliographical references.

Thesis (doctoral)--Umeå Universitet, 1987. / Extra t.p. with thesis statement inserted. Includes bibliographical references.

No description available.

Being the most abundant carbon-containing terrestrial biopolymer, lignocellulose serves as one of the best candidate feedstocks for biofuel production. The current cost-ineffective method for lignocellulose pretreatment is one of the major barriers that hinder the development of biofuel production. This leads to an exploration in the potential application of lignocellulolytic enzymes in this biorefinery process. Taking advantage of the strong activity of ligninolytic enzymes in L. edodes, we aimed at cloning and heterologously expressing these enzymes. The present project applied a yeast expression system, Pichia pastoris, as a laboratory-scale platform for heterologous expression of one of our target ligninolytic enzymes, manganese peroxidase (MnP). We successfully cloned and expressed recombinant MnP. Its enzymatic activity was the highest when grown in the presence of hemoglobin. Our long-term goal is to establish a platform for the large-scale production of recombinant lignocellulotyic enzymes at low-cost, which would strengthen their application in biofuel production. / The shitake mushroom, Lentinula edodes, is one of the most commonly consumed edible mushrooms in Asian countries. It is a saprophyte that naturally colonizes dead wood. As a member of wood-decaying white rot basidiomycete, L. edodes is able to depolymerize lignin and hydrolyze wood polysaccharides. However, the enzymatic mechanism for its lignocellulolytic system is poorly understood. Examination on the L. edodes genome and transcriptome revealed a unique lignocellulolytic system. L. edodes has a diverse enzymatic arsenal for lignin degradation. The enzymes include laccase, manganese peroxidase, cellobiose dehydrogenase and various lignin degrading auxiliary enzymes. When compared to another white rot fungus Phanerochaete chrysosporium, L. edodes possesses more hemicellulase- and pectinase-coding genes, and fewer genes encoding cellulases, suggesting that it preferentially attacks non-cellulosic polysaccharides. The transcription analysis on genes related to antioxidative mechanisms also offers insights to the oxidative stress encountered by mycelium during the free radical-mediated lignin degradation. / Kwok, Sze Wai. / Adviser: Hoi-Shan Kwan. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 141-160). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Includes bibliographical references.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Includes bibliographical references.

Spectroscopic characterization of ferric soybean peroxidase with peroxides were studied to determine the ligand coordination and to characterize the structure of the heme active site and its intermediates (ferryl species). The lifetime, chemical reactivity and distinctive colors of the ferryl species (FeIV) formed during the oxidation of peroxidase (FeIII) by peroxides enabled structure, dynamics and reaction mechanisms to be studied. Resonance Raman spectroscopy was used as a means of characterizing the structure of the soybean peroxidase and its intermediates. Excitation in the Soret absorption band at 406.7nm with 2-5mW laser power was used for this study. Resonance Raman spectra in the 200 to 1700 cm-1 region were obtained for the soybean peroxidase. However, the focus of this study was on the vibrational region of the resonance Raman spectra from 900 to 500cm-1 where the FeIV=O stretching frequencies for heme compound II intermediates are expected. Several pH and pD (deuterium substitution) samples of the soybean peroxidase were analyzed using resonance Raman spectroscopy. The vibrational stretching frequencies of the ferryl peroxidases varied with varying pH/pD were observed within the 773–787cm-1 range. From the deuterium experiment, accompanied with changes in the vibrational frequencies of the iron-ligand, a 3cm-1 upshift and intense resonant enhancement of the peaks, we observed the ferryl nature of compound II intermediate for soybean peroxidase. Badger’s rule was used to estimate the bond distances that existed within Fe-O which offers additional insight into the structure of the ferryl species. The estimated bond distance for the soybean peroxidase was significantly less than Fe-O bond distances proposed by X-ray crystallographers for other peroxidases in the same family. Comparing the vibrational frequencies of the ferryl intermediates in soybean peroxidase to that in heme proteins portrayed the effect the protein environment has on the vibrational frequencies.

Made available in DSpace on 2014-06-11T19:31:03Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-01-11Bitstream added on 2014-06-13T18:41:31Z : No. of bitstreams: 1 vinuezagalarraga_jc_dr_arafcf.pdf: 807158 bytes, checksum: 514ddc5de134cb9baa9baf27939376ec (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Universidade Estadual Paulista (UNESP) / As peroxidases (EC 1.11.1.7) são hemoproteínas que catalisam processos redox e durante a oxidação são gerados radicais livres, formando produtos poliaromáticos insolúveis em água, facilitando sua remoção do meio aquoso. O objetivo deste trabalho foi extrair as peroxidases da casca de soja e do chuchu, realizar aminação na superfície da estrutura terciária das enzimas e imobilizarem estas enzimas nos suportes alternativos de celulose vegetal (pó de sabugo de milho) e de celulose bacteriana ativada e utilizar os derivados obtidos para descoloração do azul de bromofenol 0.01 mM. A quantidade de proteínas nos extratos foram determinada por Bradford com valores médios de 0,235 mg mL -1 para soja e 0,290 mg mL -1 para o chuchu, a atividade específica de peroxidase foi determinada com ABTS 1 mM em presença de H2O2 100 mM em λ= 430nm, obtendo-se os valores médios de 86,06 μmol min -1 mg -1 e 9,04 μmol min -1 mg -1 respectivamente, em tampão acetato de sódio 100 mM, pH 4,0. Aminação das peroxidases solúveis foram feitas em tampão etilenodiamina pH 4,75 e carbodiimida, 10 e 50 mM, a funcionalização das celuloses e imobilização covalente multipontual foram semelhantes às descritas para agarose (Guisan). 19 As peroxidases aminadas 10 e 50 mM foram imobilizadas covalentemente nos suportes ativados SM-glioxil e CB-glioxil. Para a descoloração do azul de bromofenol 0.01 mM, utilizou-se os derivados estabilizados em presença de H2O2 em λ= 590nm. Os derivados foram reutilizados por cinco reciclos, mantendo-se a propriedade catalítica, sugerindo que estes derivados são uma alternativa de baixo custo, não só para o tratamento de águas residuais como também com potenciais usos em outros processos industriais / The peroxidases (EC 1.11.1.7) are hemoproteins that catalyze redox processes; during the oxidation the generated free radicals are forming poly-aromatic products insoluble in water, facilitating their removal from aqueous medium. The objective of this study was to extract peroxidases from the coat soybean and the chuchu (Sechium edule L), to make aminations on the tertiary structure of the enzymes surface, and immobilize these enzymes in the alternative supports of plant cellulose (powdered corn cob) and active bacterial cellulose, to use the derivatives obtained for the discoloration of the bromophenol blue 0.01 mM. The amount of protein in the extracts were determined by Bradford methods, with average values of 0,235 mg mL -1 for soybean and 0,290 mg mL -1 for chuchu peroxidases, the specific activity of peroxidase was determined with ABTS 1 mM in the presence of H2O2 100 mM in λ= 430nm, obtaining the mean values of 86,06 μmol min -1 mg -1 and 9,04 μmol min -1 mg -1 respectively, in sodium acetate buffer 100mM, pH 4,0. Amination of soluble peroxidases were made in buffer ethylenediamine pH 4,75 and carbodiimide, 10 and 50mM, the functionalization of cellulose and multipoint covalent immobilization were similar to those described for agarose (Guisan). 19 The amino peroxidases 10 and 50 mM were covalently immobilized on activated supports SM-glioxil and CB-glioxil. For discoloration of bromophenol blue 0.01 mM, we used derivatives of stabilized in H2O2 presence in λ= 590nm. The derivatives were reused for five recycling maintaining the catalytic property, suggesting that these products are a low cost alternative, not only for the treatment of wastewater as well as potential uses in other industrial processes