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Antibody-targeted horseradish peroxidase associated with indole-3-acetic acid induces apoptosis in vitro in hematological malignancies

DALMAZZO, Leandro F. F.; SANTANA-LEMOS, Barbara A.; JACOMO, Rafael H.; GARCIA, Aglair B.; REGO, Eduardo M.; FONSECA, Luiz M. da; FALCAO, Roberto P.
Fonte: PERGAMON-ELSEVIER SCIENCE LTD Publicador: PERGAMON-ELSEVIER SCIENCE LTD
Tipo: Artigo de Revista Científica
ENG
Relevância na Pesquisa
68.42436%
Indole-3-acetic acid (IAA), when oxidized by horseradish peroxidase (HRP), is transformed into cytotoxic molecules capable of inducing cell injury. The aim of this study was to test if, by targeting hematopoietic tumors with HRP-conjugated antibodies in association with IAA treatment, there is induction of apoptosis. We used two lineages of hematologic tumors: NB4, derived from acute promyelocytic leukemia (APL) and Granta-519 from mantle cell lymphoma (MCL). We also tested cells from 12 patients with acute myeloid leukemia (AML) and from 10 patients with chronic lymphocytic leukemia (CLL). HRP targeting was performed with anti-CD33 or anti-CD19 antibodies (depending on the origin of the cell), followed by incubation with goat anti-mouse antibody conjugated with HRP. Eight experimental groups were analyzed: control, HRP targeted, HRP targeted and incubated with 1, 5 and 10 mM IAA, and cells not HRP targeted but incubated with 1, 5 and 10 mM IAA. Apoptosis was analyzed by flow cytometry using annexin V-FITC and propidium iodide labeling. Results showed that apoptosis was dependent on the dose of IAA utilized, the duration of exposure to the prodrug and the origin of the neoplasia. Targeting HRP with antibodies was efficient in activating IAA and inducing apoptosis. (C) 2010 Elsevier Ltd. All rights reserved.; Fundacao de Amparo a Pesquisa do Estado de Sao Paulo`s (FAPESP)[05/58519-5]; Fundacao de Amparo a Pesquisa do Estado de Sao Paulo`s (FAPESP)[05/04296-5]; Fundacao de Amparo a Pesquisa do Estado de Sao Paulo`s (FAPESP)[98/14247-6]; Fundacao de Amparo a Pesquisa do Estado de Sao Paulo`s (FAPESP)[57877-3/2008]; CNPq[573754/2009]

Enzyme activity of horseradish peroxidase immobilized in chitosan matrices in alternated layers

SCHMIDT, Thais F.; CASELI, Luciano; SANTOS JR., David S. dos; OLIVEIRA JUNIOR, Osvaldo Novais de
Fonte: ELSEVIER SCIENCE BV Publicador: ELSEVIER SCIENCE BV
Tipo: Artigo de Revista Científica
ENG
Relevância na Pesquisa
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The successful immobilization of enzymes such as horseradish peroxidase (HRP) in solid films is essential for applications in sensors and for fundamental studies aimed at identifying possible biotechnological devices. In this study we show that HRP can be immobilized in alternated layers with chitosan as the template material. The activity of HRP in HRP/chitosan films was preserved for several weeks, and could be detected optically upon monitoring the reaction with pyrogallol. The morphology of the film displayed stripes that disappeared after reaction with pyrogallol. Though the activity in the HRP/chitosan film was lower than in a homogeneous solution or in an LB film investigated earlier, the response was linear for a considerable period of time, which may be advantageous for sensing hydrogen peroxide. (C) 2009 Elsevier B.V. All rights reserved.; FAPESP; Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); CNPq; Rede Biomat; Rede Biomat

Enhanced activity of horseradish peroxidase in Langmuir-Blodgett films of phospholipids

SCHMIDT, Thais F.; CASELI, Luciano; VIITALA, Tapani; OLIVEIRA JUNIOR, Osvaldo Novais de
Fonte: ELSEVIER SCIENCE BV Publicador: ELSEVIER SCIENCE BV
Tipo: Artigo de Revista Científica
ENG
Relevância na Pesquisa
68.42436%
The immobilization of enzymes in nanostructured films has potential applications, e.g. in biosensing, for which the activity may not only be preserved, but also enhanced if optimized conditions are identified. Optimization is not straightforward because several requirements must be fulfilled, including a suitable matrix and film-forming technique. In this study, we show that horseradish peroxidase (HRP) has its activity enhanced when immobilized in Langmuir-Blodgett (LB) films, in conjunction with dipalmitoylphosphaticlylglycerol (DPPG). Incorporation of HRP into a DPPG monolayer at the air-water interface was demonstrated with compression isotherms, and Polarization-Modulation Infrared Reflection Absorption Spectroscopy (PM-IRRAS). From the PM-IRRAS data, we inferred that HRP was not denatured when adsorbed on a pre-formed, low pressure DPPG monolayer. A change in orientation was induced by the phospholipid matrix, with the amide C=O and NH groups from HRP being oriented perpendicular to the surface, parallel to the DPPG acyl chains, i.e. the alpha-helix was inserted into the monolayer. The mixed DPPG-HRP monolayer could be transferred onto solid supports, to which HRP activity was ca. 23% higher than in solution. The control of molecular architecture and choice of a suitable phospholipid matrix allowed HRP-containing LB films to be used in sensing peroxide. (c) 2008 Elsevier B.V. All rights reserved.; FAPESP; Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); CNPq; Rede Biomat; Rede Biomat; Ministério da Ciência...

Interaction of horseradish peroxidase with Langmuir monolayers of phospholipids

SCHMIDT, Thais F.; CASELI, Luciano; NOBRE, Thatyane M.; ZANIQUELLI, Maria E. D.; OLIVEIRA JUNIOR, Osvaldo Novais de
Fonte: ELSEVIER SCIENCE BV Publicador: ELSEVIER SCIENCE BV
Tipo: Artigo de Revista Científica
ENG
Relevância na Pesquisa
68.42436%
The method employed to incorporate guest molecules onto phospholipid Langmuir monolayers plays an important role in the interaction between the monolayer and the guest molecules. In this paper, we show that for the interaction between horseradish peroxidase (HRP) and a monolayer of dipalmitoylphosphatidylglycerol (DPPG) does depend on the method of HRP incorporation. The surface pressure isotherms of the mixed DPPG/HRP monolayers, for instance, were less expanded when the two materials were co-spread than in the case where HRP was injected into the subphase. Therefore, the method for incorporation affected not only the penetration of HRP but also the changes in molecular packing caused to the DPPG monolayer. With experiments with the monolayer on a pendant drop, we observed that the incorporation of HRP affects the dynamic elasticity of the DPPG monolayer, on a way that varies with the surface pressure. At low pressures, HRP causes the monolayer to be more rigid, while the converse is true for surface pressures above 8 mN/m. Taken all the results together, we conclude that HRP is more efficiently incorporated if injected into the subphase on which a DPPG monolayer had been spread and that the interaction between HRP and DPPG is maintained even at high surface pressures. This is promising for the possible transfer of mixed films onto solid substrates and for applications in biosensors and drug delivery systems. (c) 2008 Elsevier B.V. All rights reserved.

Horseradish peroxidase compound I as a tool to investigate reactive protein-cysteine residues: from quantification to kinetics

TOLEDO JR., Jose Carlos; AUDI, Renata; OGUSUCU, Renata; MONTEIRO, Gisele; SOARES NETTO, Luis Eduardo; AUGUSTO, Ohara
Fonte: ELSEVIER SCIENCE INC Publicador: ELSEVIER SCIENCE INC
Tipo: Artigo de Revista Científica
ENG
Relevância na Pesquisa
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Proteins containing reactive cysteine residues (protein-Cys) are receiving increased attention as mediators of hydrogen peroxide signaling. These proteins are mainly identified by mining the thiol proteomes of oxidized protein-Cys in cells and tissues. However, it is difficult to determine if oxidation occurs through a direct reaction with hydrogen peroxide or by thiol-disulfide exchange reactions. Kinetic studies with purified proteins provide invaluable information about the reactivity of protein-Cys residues with hydrogen peroxide. Previously, we showed that the characteristic UV-Vis spectrum of horseradish peroxidase compound I, produced from the oxidation of horseradish peroxidase by hydrogen peroxide, is a simple, reliable, and useful tool to determine the second-order rate constant of the reaction of reactive protein-Cys with hydrogen peroxide and peroxynitrite. Here, the method is fully described and extended to quantify reactive protein-Cys residues and micromolar concentrations of hydrogen peroxide. Members of the peroxiredoxin family were selected for the demonstration and validation of this methodology. In particular, we determined the pK(a) of the peroxidatic thiol of rPrx6 (5.2) and the second-order rate constant of its reactions with hydrogen peroxide ((3.4 +/- 0.2) x 10(7) M(-1) s(-1)) and peroxynitrite ((3.7 +/- 0.4) x 10(5) M(-1) s(-1)) at pH 7.4 and 25 degrees C. (C) 2011 Elsevier Inc. All rights reserved.; Fundacao de Amparo A Pesquisa do Estado de Sao Paulo (FAPESP); Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP); Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Estudo da adsorção de horseradish peroxidase (HRP) sobre superfícies planas e de sua atividade catalítica; Study on the adsorption of horseradish peroxidase (HRP) onto flat surfaces and its catalytic activity

Naves, Alliny Ferreira
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 10/07/2008 PT
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Este trabalho está dividido em duas partes: (i) estudo da adsorção da enzima horseradish peroxidase (HRP) sobre substratos planos (lâminas de Si/SiO2, filmes ASP/Si/SiO2, filmes CABads/Si/SiO2, filmes CABspin/Si/SiO2 e filmes CMCABspin/Si/SiO2) seguida de testes da atividade enzimática da HRP imobilizada sobre estes substratos e (ii) obtenção de oligômeros divinílicos mediada pela HRP. Na primeira parte, a imobilização da HRP sobre substratos planos foi estudada através de elipsometria, microscopia de força atômica (AFM) e medidas de ângulo de contato. As isotermas de adsorção da HRP sobre Si/SiO2, APS/Si/SiO2 e CABads/Si/SiO2 apresentaram um aumento contínuo da quantidade de material adsorvido ΓHRP em função da concentração da solução de enzima até atingir um patamar de adsorção no qual verificou-se a formação de uma monocamada de HRP adsorvida. Nestes casos, o processo de adsorção da HRP pode ser descrito pelo modelo de adsorção aleatória (RSA). Para filmes HRP/CABspin/Si/SiO2 e HRP/CMCABspin/Si/SiO2 observou-se adsorção cooperativa de moléculas de HRP e formação de multicamadas. A adsorção da HRP sobre Si/SiO2, filmes APS, CABads e CMCABspin é um processo irreversível. Ao contrário...

Estudo da interação da peroxidase de raiz forte em interfaces nanoestruturadas; Study of horseradish peroxidase interaction in nanostructured interfaces

Schmidt, Thaís Fernandes
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 01/08/2008 PT
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Neste projeto estudou-se a interação da enzima peroxidase de raiz forte (HRP) em interfaces nanoestruturadas e sua possível aplicação em biossensores de peróxido de hidrogênio. Foram utilizadas as técnicas de Langmuir, Langmuir-Blodgett (LB) e automontagem por adsorção física para formar filmes nanoestruturados. A interação da enzima com espécies em interfaces foi investigada com materiais que serviram de matrizes de adsorção, ou seja, a quitosana (Ch) e o fosfolipídio 1,2-dipalmitoil-sn-glicero-3-[fosfatidil-rac-(1-glicerol)] (sal de sódio) (DPPG). Os filmes de Langmuir foram caracterizados com medidas de pressão e potencial de superfície, espectroscopia no infravermelho, e tensão superficial dinâmica. Para os filmes LB e automontados, empregaram-se espectroscopias de fluorescência, ultravioleta-visível e infravermelho e microgravimetria por cristal de quartzo. A peroxidase de raiz forte apresentou forte interação com DPPG, confirmada em filmes de Langmuir por medidas de pressão de superfície, elasticidade dinâmica e de espectroscopia de reflexão e absorção no infravermelho, com modulação por polarização (PM-IRRAS). A massa de peroxidase transferida em filmes Langmuir-Blodgett (LB) mistos com DPPG foi de aproximadamente 200 ng...

Antibody-targeted horseradish peroxidase associated with indole-3-acetic acid induces apoptosis in vitro in hematological malignancies

Dalmazzo, Leandro F. F.; Santana-Lemos, Barbara A.; Jacomo, Rafael H.; Garcia, Aglair B.; Rego, Eduardo M.; da Fonseca, Luiz M.; Falcao, Roberto P.
Fonte: Pergamon-Elsevier B.V. Ltd Publicador: Pergamon-Elsevier B.V. Ltd
Tipo: Artigo de Revista Científica Formato: 657-662
ENG
Relevância na Pesquisa
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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); Processo FAPESP: 05/58519-5; Processo FAPESP: 05/04296-5; Processo FAPESP: 98/14247-6; Processo FAPESP: 57877-3/2008; Indole-3-acetic acid (IAA), when oxidized by horseradish peroxidase (HRP), is transformed into cytotoxic molecules capable of inducing cell injury. The aim of this study was to test if, by targeting hematopoietic tumors with HRP-conjugated antibodies in association with IAA treatment, there is induction of apoptosis. We used two lineages of hematologic tumors: NB4, derived from acute promyelocytic leukemia (APL) and Granta-519 from mantle cell lymphoma (MCL). We also tested cells from 12 patients with acute myeloid leukemia (AML) and from 10 patients with chronic lymphocytic leukemia (CLL). HRP targeting was performed with anti-CD33 or anti-CD19 antibodies (depending on the origin of the cell), followed by incubation with goat anti-mouse antibody conjugated with HRP. Eight experimental groups were analyzed: control, HRP targeted, HRP targeted and incubated with 1, 5 and 10 mM IAA, and cells not HRP targeted but incubated with 1, 5 and 10 mM IAA. Apoptosis was analyzed by flow cytometry using annexin V-FITC and propidium iodide labeling. Results showed that apoptosis was dependent on the dose of IAA utilized...

The effect of pH on horseradish peroxidase-catalyzed oxidation of melatonin: production of N-1-acetyl-N-2-formyl-5-methoxykynuramine versus radical-mediated degradation

Ximenes, Valdecir F.; Fernandes, Joao Roberto; Bueno, Vania B.; Catalani, Luiz H.; de Oliveira, Georgino H.; Machado, Rosangela G. P.
Fonte: Blackwell Publishing Publicador: Blackwell Publishing
Tipo: Artigo de Revista Científica Formato: 291-296
ENG
Relevância na Pesquisa
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There is a g-rowing body of evidence that melatonin and its oxidation product, N-1-acetyl-N-2-formyl-5-methoxykynuramine (AFMK), have anti-inflammatory properties. From a nutritional point of view, the discovery of melatonin in plant tissues emphasizes the importance of its relationship with plant peroxidases. Here we found that the pH of the reaction mixture has a profound influence in the reaction rate and products distribution when melatonin is oxidized by the plant enzyme horseradish peroxidase. At pH 5.5. 1 mm of melatonin was almost completely oxidized within 2 min, whereas only about 3% was consumed at pH 7.4. However, the relative yield of AFMK was higher in physiological pH. Radical-mediated oxidation products, including 2-hydroxymelatonin a dimer of, 2-hydroxymelatonin and O-demethylated dimer of melatonin account for the fast consumption of melatonin at pH 5.5. The higher production of AFMK at pH 7.4 was explained by the involvement of compound III of peroxidases as evidenced by spectral studies. on the other hand, the fast oxidative degradation at pH 5.5 was explained by the classic peroxidase cycle.

Oxidation of acetylacetone catalyzed by horseradish peroxidase in the absence of hydrogen peroxide

Rodrigues, Ana Paula; da Fonseca, Luiz Marcos; de Faria Oliveira, Olga M.; Brunetti, Iguatemy Lourenço; Ximenes, Valdecir Farias
Fonte: Elsevier B.V. Publicador: Elsevier B.V.
Tipo: Artigo de Revista Científica Formato: 1755-1761
ENG
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Horseradish peroxidase (HRP) is a plant enzyme widely used in biotechnology, including antibody-directed enzyme prodrug therapy (ADEPT). Here, we showed that HRP is able to catalyze the autoxidation of acetylacetone in the absence of hydrogen peroxide. This autoxidation led to generation of methylglyoxal and reactive oxygen species. The production of superoxide anion was evidenced by the effect of superoxide dismutase and by the generation of oxyperoxidase during the enzyme turnover. The HRP has a high specificity for acetylacetone, since the similar beta-dicarbonyls dimedon and acetoacetate were not oxidized. As this enzyme prodrug combination was highly cytotoxic for neutrophils and only requires the presence of a non-human peroxidase and acetylacetone, it might immediately be applied to research on the ADEPT techniques. The acetylacetone could be a starting point for the design of new drugs applied in HRP-related ADEPT techniques. (c) 2006 Elsevier B.V. All rights reserved.

Horseradish peroxidase-catalyzed oxidation of rifampicin: Reaction rate enhancement by co-oxidation with anti-inflammatory drugs

Dos Santos, Fernanda de Jesus Notário; Ximenes, Valdecir Farias; Da Fonseca, Luiz Marcos; De Faria Oliveira, Olga Maria Mascarenhas; Brunetti, Iguatemy Lourenço
Fonte: Universidade Estadual Paulista Publicador: Universidade Estadual Paulista
Tipo: Artigo de Revista Científica Formato: 1822-1826
ENG
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The tuberculostatic drug rifampicin has been described as a scavenger of reactive species. Additionally, the recent demonstration that oral therapy with a complex of rifampicin and horseradish peroxidase (HRP) was more effective than rifampicin alone, in an animal model of experimental leprosy, suggested the importance of redox reactions involving rifampicin and their relevance to the mechanism of action. Hence, we studied the oxidation of rifampicin catalyzed by HRP, since this enzyme may represent the prototype of peroxidation-mediated reactions. We found that the antibiotic is efficiently oxidized and that rifampicin-quinone is the product, in a reaction dependent on both HRP and hydrogen peroxide. The steady-state kinetic constants Km app (101±23 mmol/l), Vmax app (0.78±0.09 μmol/l·s-1) and kcat (5.1±0.6 s-1) were measured (n=4). The reaction rate was increased by the addition of co-substrates such as tetramethylbenzidine, salicylic acid, 5-aminosalicylic acid and paracetamol. This effect was explained by invoking an electron-transfer mechanism by which these drugs acted as mediators of rifampicin oxidation. We suggested that this drug interaction might be important at the inflammatory site. © 2005 Pharmaceutical Society of Japan.

The effect of pH on horseradish peroxidase-catalyzed oxidation of melatonin: Production of N1-acetyl-N2-formyl-5- methoxykynuramine versus radical-mediated degradation

Ximenes, Valdecir F.; Fernandes, João Roberto; Bueno, Vânia B.; Catalani, Luiz H.; Oliveira, Georgino H. de; Machado, Rosângela G. P.
Fonte: Universidade Estadual Paulista Publicador: Universidade Estadual Paulista
Tipo: Artigo de Revista Científica Formato: 291-296
ENG
Relevância na Pesquisa
68.92309%
There is a growing body of evidence that melatonin and its oxidation product, N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), have anti-inflammatory properties. From a nutritional point of view, the discovery of melatonin in plant tissues emphasizes the importance of its relationship with plant peroxidases. Here we found that the pH of the reaction mixture has a profound influence in the reaction rate and products distribution when melatonin is oxidized by the plant enzyme horseradish peroxidase. At pH 5.5, 1 mm of melatonin was almost completely oxidized within 2 min, whereas only about 3% was consumed at pH 7.4. However, the relative yield of AFMK was higher in physiological pH. Radical-mediated oxidation products, including 2-hydroxymelatonin, a dimer of 2-hydroxymelatonin and O-demethylated dimer of melatonin account for the fast consumption of melatonin at pH 5.5. The higher production of AFMK at pH 7.4 was explained by the involvement of compound III of peroxidases as evidenced by spectral studies. On the other hand, the fast oxidative degradation at pH 5.5 was explained by the classic peroxidase cycle. © 2007 The Authors.

Inactivation and reactivation kinetics of horseradish peroxidase in phosphate buffer and buffer – dimethylformamide solutions

Machado, Maria F.; Saraiva, Jorge
Fonte: Elsevier Publicador: Elsevier
Tipo: Artigo de Revista Científica
ENG
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68.42436%
The influence of the regeneration of horseradish peroxidase activity at 4 °C on both the thermal inactivation and the reactivation kinetics of the enzyme was studied in phosphate buffer and a mixture of this buffer with 10% (v/v) N,N-dimethylformamide, at temperatures ranging from 70 to 85 °C. A series-type model was fitted to the experimental data that exhibited typical biphasic patterns, which became less pronounced as enzyme activity was regenerated. The kinetic parameters were found to be, in general, significantly different in both inactivation media. The magnitude of the activation energies for both the reactions of formation and inactivation of the intermediate enzyme indicated that conformational changes might play a major role in both processes when the solvent is added. A logarithmic model, although lacking a theoretical background, was able to predict the enzyme activity regeneration upon storage of the inactivated samples at 4 °C. There was evidence that the rate of reactivation may be dependent on the amount of intermediate formed in the first reaction or remaining after inactivation, as suggested by both the inactivation temperatures and extent of heating time relationships verified in this work.

Thermal stability and activity regain of horseradish peroxidase in aqueous mixtures of imidazolium-based ionic liquids

Machado, Maria Fátima; Saraiva, Jorge Manuel
Fonte: Springer Verlag Publicador: Springer Verlag
Tipo: Artigo de Revista Científica
ENG
Relevância na Pesquisa
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Thermal deactivation kinetics of horseradish peroxidase (HRP) were studied from 45 to 90 degrees C in phosphate buffer and 5-25% (v,w/v) 1-butyl-3-methylimidazolium tetrafluoroborate [BMIM][BF4] and 1-butyl-3-methylimidazolium chloride [BMIM][Cl]. HRP activity at 25 degrees C was not affected by the presence of ionic liquids up to 20% (v,w/v). Increasing the ionic liquids concentration up to 25% (v,w/v) changed the biphasic character of deactivation kinetics to an apparent single first-order step. The presence of 5-10% (v/v) [BMIM][BF4] significantly improved HRP thermal stability with lower activation energies for the deactivation second phase (83-87 kJ mol(-1)). After deactivation, enhanced activity regain of the enzyme, up to 70-80% of the initial activity, was found in 25% (v/v) [BMIM][BF4] and 10% (w/v) [BMIM][Cl] and correlated to prevalence of the deactivation first phase.

Analysis of the inactivation and reactivation kinetics of horseradish peroxidase in mixtures of phosphate buffer/dimethylformamide

Machado, M. F.; Saraiva, J.
Fonte: Universidade de Aveiro Publicador: Universidade de Aveiro
Tipo: Conferência ou Objeto de Conferência
ENG
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In this work, the kinetics of the thermal inactivation and reactivation of horseradish peroxidase were analysed in buffered solutions of dimethylformamide at severa1 concentrations (up to 40% vlv) and temperatures ranging fiom 70 to 85 "C. The experimental data of the inactivation and reactivation processes showed a biphasic behaviour, which was well described by a series-type model. The significant activity regain at low temperature of the thermal inactivated enzyme had a strong influence on the estimated value for the a parameter of the model, thus reflecting a clear change of the profile of the experimental curves.

Construction of a mutant library of horseradish peroxidase gene by directed evolution and development of an in situ screening method

Mendive,F.M.; Segura,M.M.; Targovnik,H.M.; Cascone,O.; Miranda,M.V.
Fonte: Brazilian Society of Chemical Engineering Publicador: Brazilian Society of Chemical Engineering
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/03/2003 EN
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A process of directed evolution applied to obtain a library of mutants of horseradish peroxidase (HRP) enzyme is described. We have introduced slight variations into the original DNA shuffling protocol. A DNA template was prepared by PCR amplification and digested with Dnase I during 1 hour. Dnase I products were concentrated by precipitation with isopropanol. Gel electrophoresis showed fragments of the desired size range (20-600 pb) without a full-length template remaining in the reaction mixture. A high concentration of fragments was crucial for performing PCR without primers. In this case, a template concentration of 32.5 ng/mu l was appropriate. Amplification of recombinant genes in a standard PCR reaction (template dilution 1:100) produced a smear with a low yield for the full-length sequence. A single product of the correct size was obtained by PCR with nested primers separated from the previously used primers by 40 pb. In our laboratory, native HRP has been functionally expressed in a baculovirus expression vector system. The purpose is to develop the screening of the first generation of random mutants in this system. To facilitate detection of those clones that have high peroxidase activity, we developed a rapid method: after five days postinfection agarose plates with six wells were covered with DAB (3...

Synthesis and characterization of starch-poly(methyl acrylate) graft copolymers using horseradish peroxidase

Su Wang; Qiang Wang; Xuerong Fan; Jin Xu; Ying Zhang; Jiugang Yuan; Heling Jin; Cavaco-Paulo, Artur
Fonte: Elsevier Publicador: Elsevier
Tipo: Artigo de Revista Científica
Publicado em 20/01/2016 ENG
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"Available online 9 October 2015"; Horseradish peroxidase (HRP)-mediated graft polymerization in the presence of hydrogen peroxide (H2O2) and acetylacetone (Acac) has been successfully applied to the synthesis of starch-poly(methyl acrylate) (PMA). The graft copolymer was characterized by fourier transform infrared (FT-IR), elemental analysis, nuclear magnetic resonance (1H NMR and 13C NMR), and differential scanning calorimetry (DSC). FT-IR, elemental analysis and NMR confirmed that methyl acrylate (MA) was grafted onto starch successfully. DSC results showed the graft reaction had changed the crystalline regions of the gelatinized starch. The effects of pH, MA content, HRP dosage, incubation temperature and time on grafting percentage (GP) and grafting efficiency (GE) were also investigated. The GP and GE under optimal conditions reached 30.21% and 45.13%, respectively.

Hofmeister specific-ion effects on enzyme activity and buffer pH: Horseradish peroxidase in citrate buffer

Bauduin, Pierre; Nohmie, Fawaz; Touraud, Didier; Neueder, Roland; Kunz, Werner; Ninham, Barry
Fonte: Elsevier Publicador: Elsevier
Tipo: Artigo de Revista Científica
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Salt addition to enzymes in buffer always induce the problem of the respective influences of electrostatic interactions and anion specificity on buffer pH and enzyme kinetics. In the present paper the influence of some sodium salts (Na2SO4, NaCl, NaBr and NaNO3) on the pH of a citrate buffer (c = 0.025 M), and on the catalytic constants of horseradish peroxidase (HRP) is studied. First, the pH changes due to the presence of salts in the buffer are examined; second, catalytic constants, KmABTS, VmaxABTS and V maxABTS/KmABTS, are studied as a function of pH in buffer with and without added salt, at various salt concentrations and different pH values due to the salt additions. With a simple electrostatic model, it is possible to show that the glass electrode yields reasonable pH values even in the presence of fairly high 1 : 1 salt concentrations. For the catalytic efficiency, VmaxABTS/KmABTS, a Hofmeister series is found with opposite deviations from the pure pH effect for salting-in and salting-out ions over a large range of salt concentrations. This usual Hofmeister series is a consequence of three, for the moment inseparable salt concentration and specific ion-induced phenomena: global bulk effects, local active site effects and surface effects.

Hofmeister Effects in Biology: Effect of Choline Addition on the Salt-Induced Super Activity of Horseradish Peroxidase and Its Implication for Salt Resistance of Plants

Pinna, M Cristina; Bauduin, Pierre; Touraud, Didier; Monduzzi, Maura; Ninham, Barry; Kunz, Werner
Fonte: American Chemical Society Publicador: American Chemical Society
Tipo: Artigo de Revista Científica
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The effect of choline addition on the salt-induced super activity of horseradish peroxidase (HRP) is investigated. HRP is presented in the literature as an efficient H2O2 scavenger, and choline is the precursor of glycine betaine, a strong osmoprotectant

Avalia??o da produ??o de esp?cies reativas de oxig?nio e da citotoxicidade in vitro mediada pelo sistema 2,4-pentanodiona/horseradish peroxidase/oxig?nio

Pinheiro, N?thale Rodrigues
Fonte: UFVJM Publicador: UFVJM
Tipo: Dissertação de Mestrado
POR
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O sistema ADEPT (antibody-directed enzyme prodrug therapy) ? uma terapia antitumoral que envolve a ativa??o catal?tica de um pr?-f?rmaco, nas proximidades do s?tio tumoral, por uma enzima conjugada a um anticorpo monoclonal com afinidade para ant?genos espec?ficos das c?lulas tumorais. O sistema composto pela enzima Horseradish peroxidase (HRP) e ?cido indol-3-ac?tico (IAA) tem sido estudado para o emprego na terapia ADEPT, e associado ? indu??o de apoptose de c?lulas tumorais. A 2,4-pentanodiona (PD) tamb?m atua como substrato da HRP sendo oxidada por ela atrav?s de uma rea??o cuja cin?tica ? semelhante ? da cat?lise do IAA e, portanto, pode representar uma alternativa para essa terapia. Este trabalho teve como objetivo realizar uma avalia??o da citotoxicidade mediada pelos produtos provenientes da oxida??o da PD pela HRP frente a diferentes linhagens celulares, utilizando para isso diferentes metodologias que determinam a viabilidade celular como o azul de Trypan, MTT e vermelho neutro assim, como a an?lise microsc?pica das altera??es celulares induzidas por esses sistemas; estabelecer uma compara??o com a citotoxicidade mediada pela oxida??o do IAA catalisada pela mesma enzima; verificar a incid?ncia de morte celular por apoptose mediada pelos sistemas IAA/HRP/O2 e PD/HRP/O2; al?m de verificar a produ??o e os tipos de esp?cies reativas de oxig?nio (ERO) produzidas pelos dois sistemas. Os experimentos permitiram evidenciar que as combina??es PD/HRP/O2 e IAA/HRP/O2 levam a forma??o de ERO...