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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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Autores principales: Calil, Felipe Antunes, Lima, Juliana Maria, de Oliveira, Arthur Henrique Cavalcante, Mariotini-Moura, Christiane, Fietto, Juliana Lopes Rangel, Cardoso, Carmen Lucia
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Hindawi Publishing Corporation 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5192316/
https://www.ncbi.nlm.nih.gov/pubmed/28070446
http://dx.doi.org/10.1155/2016/9846731
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author Calil, Felipe Antunes
Lima, Juliana Maria
de Oliveira, Arthur Henrique Cavalcante
Mariotini-Moura, Christiane
Fietto, Juliana Lopes Rangel
Cardoso, Carmen Lucia
author_facet Calil, Felipe Antunes
Lima, Juliana Maria
de Oliveira, Arthur Henrique Cavalcante
Mariotini-Moura, Christiane
Fietto, Juliana Lopes Rangel
Cardoso, Carmen Lucia
author_sort Calil, Felipe Antunes
collection PubMed
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 K (M) 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.
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spelling pubmed-51923162017-01-09 Immobilization of NTPDase-1 from Trypanosoma cruzi and Development of an Online Label-Free Assay Calil, Felipe Antunes Lima, Juliana Maria de Oliveira, Arthur Henrique Cavalcante Mariotini-Moura, Christiane Fietto, Juliana Lopes Rangel Cardoso, Carmen Lucia J Anal Methods Chem Research Article 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 K (M) 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. Hindawi Publishing Corporation 2016 2016-12-14 /pmc/articles/PMC5192316/ /pubmed/28070446 http://dx.doi.org/10.1155/2016/9846731 Text en Copyright © 2016 Felipe Antunes Calil et al. https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Calil, Felipe Antunes
Lima, Juliana Maria
de Oliveira, Arthur Henrique Cavalcante
Mariotini-Moura, Christiane
Fietto, Juliana Lopes Rangel
Cardoso, Carmen Lucia
Immobilization of NTPDase-1 from Trypanosoma cruzi and Development of an Online Label-Free Assay
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
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5192316/
https://www.ncbi.nlm.nih.gov/pubmed/28070446
http://dx.doi.org/10.1155/2016/9846731
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