Immobilization of NTPDase-1 from Trypanosoma cruzi and Development of an Online Label-Free Assay
The use of IMERs (Immobilized Enzyme Reactors) as a stationary phase coupled to high performance chromatographic systems is an interesting approach in the screening of new ligands. In addition, IMERs offer many advantages over techniques that employ enzymes in solution. The enzyme nucleoside triphos...
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| Format: | Article |
| Language: | English |
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Wiley
2016-01-01
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| Series: | Journal of Analytical Methods in Chemistry |
| Online Access: | http://dx.doi.org/10.1155/2016/9846731 |
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| author | Felipe Antunes Calil Juliana Maria Lima Arthur Henrique Cavalcante de Oliveira Christiane Mariotini-Moura Juliana Lopes Rangel Fietto Carmen Lucia Cardoso |
| author_facet | Felipe Antunes Calil Juliana Maria Lima Arthur Henrique Cavalcante de Oliveira Christiane Mariotini-Moura Juliana Lopes Rangel Fietto Carmen Lucia Cardoso |
| author_sort | Felipe Antunes Calil |
| collection | DOAJ |
| description | The use of IMERs (Immobilized Enzyme Reactors) as a stationary phase coupled to high performance chromatographic systems is an interesting approach in the screening of new ligands. In addition, IMERs offer many advantages over techniques that employ enzymes in solution. The enzyme nucleoside triphosphate diphosphohydrolase (NTPDase-1) from Trypanosoma cruzi acts as a pathogen infection facilitator, so it is a good target in the search for inhibitors. In this paper, immobilization of NTPDase-1 afforded ICERs (Immobilized Capillary Enzyme Reactors). A liquid chromatography method was developed and validated to monitor the ICER activity. The conditions for the application of these bioreactors were investigated, and excellent results were obtained. The enzyme was successfully immobilized, as attested by the catalytic activity detected in the TcNTPDase-1-ICER chromatographic system. Kinetic studies on the substrate ATP gave KM of 0.317 ± 0.044 mmol·L−1, which still presented high affinity compared to in solution. Besides that, the ICER was stable for 32 days, enough time to investigate samples of possible inhibitors, including especially the compound Suramin, that inhibited 51% the enzyme activity at 100 µmol·L−1, which is in accordance with the data for the enzyme in solution. |
| format | Article |
| id | doaj-art-761b796d68a94525bbb80b466d1d3204 |
| institution | Kabale University |
| issn | 2090-8865 2090-8873 |
| language | English |
| publishDate | 2016-01-01 |
| publisher | Wiley |
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| series | Journal of Analytical Methods in Chemistry |
| spelling | doaj-art-761b796d68a94525bbb80b466d1d32042025-02-03T01:32:46ZengWileyJournal of Analytical Methods in Chemistry2090-88652090-88732016-01-01201610.1155/2016/98467319846731Immobilization of NTPDase-1 from Trypanosoma cruzi and Development of an Online Label-Free AssayFelipe Antunes Calil0Juliana Maria Lima1Arthur Henrique Cavalcante de Oliveira2Christiane Mariotini-Moura3Juliana Lopes Rangel Fietto4Carmen Lucia Cardoso5Departamento de Química, Grupo de Cromatografia de Bioafinidade e Produtos Naturais, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, 14040-901 Ribeirão Preto, SP, BrazilDepartamento de Química, Grupo de Cromatografia de Bioafinidade e Produtos Naturais, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, 14040-901 Ribeirão Preto, SP, BrazilDepartamento de Química, Grupo de Cromatografia de Bioafinidade e Produtos Naturais, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, 14040-901 Ribeirão Preto, SP, BrazilDepartamento de Bioquímica e Biologia Molecular, Universidade Federal de Viçosa, 36570-000 Viçosa, MG, BrazilDepartamento de Bioquímica e Biologia Molecular, Universidade Federal de Viçosa, 36570-000 Viçosa, MG, BrazilDepartamento de Química, Grupo de Cromatografia de Bioafinidade e Produtos Naturais, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, 14040-901 Ribeirão Preto, SP, BrazilThe use of IMERs (Immobilized Enzyme Reactors) as a stationary phase coupled to high performance chromatographic systems is an interesting approach in the screening of new ligands. In addition, IMERs offer many advantages over techniques that employ enzymes in solution. The enzyme nucleoside triphosphate diphosphohydrolase (NTPDase-1) from Trypanosoma cruzi acts as a pathogen infection facilitator, so it is a good target in the search for inhibitors. In this paper, immobilization of NTPDase-1 afforded ICERs (Immobilized Capillary Enzyme Reactors). A liquid chromatography method was developed and validated to monitor the ICER activity. The conditions for the application of these bioreactors were investigated, and excellent results were obtained. The enzyme was successfully immobilized, as attested by the catalytic activity detected in the TcNTPDase-1-ICER chromatographic system. Kinetic studies on the substrate ATP gave KM of 0.317 ± 0.044 mmol·L−1, which still presented high affinity compared to in solution. Besides that, the ICER was stable for 32 days, enough time to investigate samples of possible inhibitors, including especially the compound Suramin, that inhibited 51% the enzyme activity at 100 µmol·L−1, which is in accordance with the data for the enzyme in solution.http://dx.doi.org/10.1155/2016/9846731 |
| spellingShingle | Felipe Antunes Calil Juliana Maria Lima Arthur Henrique Cavalcante de Oliveira Christiane Mariotini-Moura Juliana Lopes Rangel Fietto Carmen Lucia Cardoso Immobilization of NTPDase-1 from Trypanosoma cruzi and Development of an Online Label-Free Assay Journal of Analytical Methods in Chemistry |
| title | Immobilization of NTPDase-1 from Trypanosoma cruzi and Development of an Online Label-Free Assay |
| title_full | Immobilization of NTPDase-1 from Trypanosoma cruzi and Development of an Online Label-Free Assay |
| title_fullStr | Immobilization of NTPDase-1 from Trypanosoma cruzi and Development of an Online Label-Free Assay |
| title_full_unstemmed | Immobilization of NTPDase-1 from Trypanosoma cruzi and Development of an Online Label-Free Assay |
| title_short | Immobilization of NTPDase-1 from Trypanosoma cruzi and Development of an Online Label-Free Assay |
| title_sort | immobilization of ntpdase 1 from trypanosoma cruzi and development of an online label free assay |
| url | http://dx.doi.org/10.1155/2016/9846731 |
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