title: Immobilization of NTPDase-1 fromTrypanosoma cruziand Development of an Online Label-Free Assay.
abstract: 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) fromTrypanosoma cruziacts 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 theTcNTPDase-1-ICER chromatographic system. Kinetic studies on the substrate ATP gaveKMof 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 100µmol·L-1, which is in accordance with the data for the enzyme in solution.
authors.affiliation: Departamento 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, Brazil.
authors.forename: Felipe Antunes
authors.identifier: 0000-0001-7503-4078
authors.initials: FA
authors.lastname: Calil
authors.affiliation: Departamento 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, Brazil.
authors.forename: Juliana Maria
authors.initials: JM
authors.lastname: Lima
authors.affiliation: Departamento 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, Brazil.
authors.forename: Arthur Henrique Cavalcante
authors.initials: AH
authors.lastname: de Oliveira
authors.affiliation: Departamento de Bioquímica e Biologia Molecular, Universidade Federal de Viçosa, 36570-000 Viçosa, MG, Brazil; Instituto Nacional de Biotecnologia Estrutural e Química Medicinal em Doenças Infecciosas (INBEQMeDI), São Carlos, SP, Brazil.
authors.forename: Christiane
authors.identifier: 0000-0003-1101-6895
authors.initials: C
authors.lastname: Mariotini-Moura
authors.affiliation: Departamento de Bioquímica e Biologia Molecular, Universidade Federal de Viçosa, 36570-000 Viçosa, MG, Brazil; Instituto Nacional de Biotecnologia Estrutural e Química Medicinal em Doenças Infecciosas (INBEQMeDI), São Carlos, SP, Brazil.
authors.forename: Juliana Lopes Rangel
authors.identifier: 0000-0001-6122-1710
authors.initials: JL
authors.lastname: Fietto
authors.affiliation: Departamento 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, Brazil.
authors.forename: Carmen Lucia
authors.identifier: 0000-0001-6239-6651
authors.initials: CL
authors.lastname: Cardoso