RESUMO
A simple and fast spectrophotometric methodology able to quantify superoxide released by NADPH oxidase from differentiated promyelocytic leukaemia (HL-60) cells using pyrogallol red is described.The latter is based on the known stoichiometry of the reaction between superoxide and pyrogallol red and the inability of pyrogallol red to react with hydrogen peroxide. In addition, we developed a 96-wells microplate-based method able to determine NADPH oxidase activity. Using this method, we determined pharmacological properties of the NADPH oxidase inhibitors VAS2870 and diphenyleneiodonium and the obtained IC50 values were in good agreement with previous reported data. NOX2 is highly expressed in differentiated promyelocytic leukaemia cells, whereas other isoforms are not detected or expressed at low amounts. Likewise, this methodology may be a useful assay for NOX2 inhibitor screening. NADPH oxidases are involved in several physiological and pathological processes, rendering its pharmacological modulation an attractive research target. In this context, this simple assay can be used for NADPH oxidase inhibitor screening as well as aiding in the study of different biological conditions that involve NADPH oxidase activity.
Assuntos
NADPH Oxidases/metabolismo , Pirogalol/análogos & derivados , Superóxidos/metabolismo , Benzoxazóis/farmacologia , Células HL-60 , Humanos , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/metabolismo , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/química , Oniocompostos/farmacologia , Pirogalol/química , Pirogalol/metabolismo , Superóxidos/química , Triazóis/farmacologiaRESUMO
Polyphenol oxidase (PPO) is an enzyme widely distributed in the plant kingdom that has been detected in most fruits and vegetables. PPO was extracted and purified from Manila mango (Mangifera indica), and its biochemical properties were studied. PPO was purified 216-fold by hydrophobic interaction and ion exchange chromatography. PPO was purified to homogeneity, and the estimated PPO molecular weight (MW) by SDS-PAGE was ≈31.5 kDa. However, a MW of 65 kDa was determined by gel filtration, indicating a dimeric structure for the native PPO. The isolated PPO showed the highest affinity to pyrogallol (Km = 2.77 mM) followed by 4-methylcatechol (Km = 3.14 mM) and catechol (Km = 15.14 mM). The optimum pH for activity was 6.0. PPO was stable in the temperature range of 20-70 °C. PPO activity was completely inhibited by tropolone, ascorbic acid, sodium metabisulfite, and kojic acid at 0.1 mM.
Assuntos
Catecol Oxidase/isolamento & purificação , Catecol Oxidase/metabolismo , Mangifera/enzimologia , Catecol Oxidase/antagonistas & inibidores , Catecóis/metabolismo , Cromatografia em Gel , Cromatografia por Troca Iônica , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Peso Molecular , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , Pirogalol/metabolismo , Especificidade por Substrato , TemperaturaRESUMO
INTRODUCTION: The phytochemistry of species of the genus Piper has been studied extensively, including Piper solmsianum. However, no studies have addressed the phytochemistry of the sap content of Piper species. OBJECTIVE: To evaluate the transferring of secondary compounds from the saps of P. solmsianum to the honeydew of Edessa meditabunda. METHODOLOGY: The honeydew of E. meditabunda and saps of P. solmsianum were analysed by GC-MS, (1) H-NMR and LC-MS. RESULTS: The lignan (-)-grandisin and the phenylpropanoid (E)-isoelemicin were detected in both saps of P. solmsianum and honeydew of E. meditabunda. CONCLUSION: Analysis of honeydew secreted by the sap-sucking insect E. meditabunda indicated that (-)-grandisin and (E)-isoelemicin are absorbed from the phloem of Piper solmsianum.
Assuntos
Furanos/análise , Heterópteros/química , Lignanas/análise , Piper/química , Piper/metabolismo , Pirogalol/análogos & derivados , Animais , Furanos/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Heterópteros/metabolismo , Lignanas/metabolismo , Floema/química , Floema/metabolismo , Folhas de Planta/química , Folhas de Planta/metabolismo , Pirogalol/análise , Pirogalol/metabolismo , Xilema/química , Xilema/metabolismoRESUMO
The bleaching of the pyrogallol red (PGR) dye mediated by superoxide anion radicals (O(2)(-)) generated from the xanthine/xanthine oxidase system (X/XO) was studied by UV-visible spectrophotometry. The absorption band (at 540 nm) of PGR quickly decreased in the presence of X/XO, implying an efficient reaction of O(2)(-) with PGR. The process was unaffected by catalase (CAT), but completely abolished by superoxide dismutase (SOD). A mechanism of the reaction involving the consumption of one PGR molecule by two O(2)(-) to generate one molecule of H(2)O(2) is proposed. PGR was used as a probe to estimate the rate of O(2)(-) generation in redox cycling reactions of a series of nitro compounds mediated by rat liver microsomes. The consumption of PGR induced by the redox cycling of nitrofurantoin was totally eliminated by the addition of SOD but unaffected by CAT. The initial rate of consumption of PGR mediated by the redox cycling of others nitro derivatives follows the order: furazolidindione > nitrofurantoin > nifurtimox > benznidazole > chloramphenicol. We concluded that PGR can be used as a probe to estimate the release of O(2)(-) from enzymatic systems or from the redox cycling of nitro compounds.
Assuntos
Nitrocompostos/metabolismo , Pirogalol/análogos & derivados , Superóxidos/química , Animais , Citocromos c/metabolismo , Etídio/análogos & derivados , Peróxido de Hidrogênio , Masculino , Microssomos Hepáticos/metabolismo , Oxirredução , Pirogalol/metabolismo , Ratos , Ratos Sprague-Dawley , Xantina/metabolismo , Xantina Oxidase/metabolismoRESUMO
While a long shelf life for fruit products is highly desired, enzymatic browning is the main cause of quality loss in fruits and is therefore a main problem for the food industry. In this study polyphenol oxidase (PPO), the main enzyme responsible for browning was isolated from mamey fruit (Pouteria sapota) and characterized biochemically. Two isoenzymes (PPO 1 and PPO 2) were obtained upon ammonium sulfate precipitation and hydrophobic and ion exchange chromatography; PPO 1 was purified up to 6.6-fold with 0.28% yield, while PPO 2 could not be characterized as enzyme activity was completely lost after 24 h of storage. PPO 1 molecular weight was estimated to be 16.1 and 18 kDa by gel filtration and SDS-PAGE, respectively, indicating that the native state of the PPO 1 is a monomer. The optimum pH for PPO 1 activity was 7. The PPO 1 was determined to be maximum thermally stable up to 35°C. Kinetic constants for PPO 1 were K(m)=44 mM and K(m)=1.3 mM using catechol and pyrogallol as substrate, respectively. The best substrates for PPO 1 were pyrogallol, 4-methylcatechol and catechol, while ascorbic acid and sodium metabisulfite were the most effective inhibitors.