title: Label-free assay based on immobilized capillary enzyme reactor of Leishmania infantum nucleoside triphosphate diphosphohydrolase (LicNTPDase-2-ICER-LC/UV).
abstract: Nucleoside triphosphate diphosphohydrolase (NTPDase) is an enzyme belonging to the apyrase family that participates in the hydrolysis of the nucleosides di- and triphosphate to the corresponding nucleoside monophosphate. This enzyme underlies the virulence of parasites such as Leishmania. Recently, an NTPDase from Leishmania infantum (LicNTPDase-2) was cloned and expressed and has been considered as a new drug target for the treatment of leishmaniasis. With the intent of developing label-free online screening methodologies, LicNTPDase-2 was covalently immobilized onto a fused silica capillary tube in the present study to create an immobilized capillary enzyme reactor (ICER) based on LicNTPDase-2 (LicNTPDase-2-ICER). To perform the activity assays, a multidimensional chromatographic method was developed employing the LicNTPDase-2-ICER in the first dimension, and an analytical Ascentis C8 column was used in the second dimension to provide analytical separation of the substrates and products. The validated LicNTPDase-2-ICER method provided the following kinetic parameters of the immobilized enzyme: KM of 2.2 and 1.8mmolL(-1) for the ADP and ATP substrates, respectively. Suramin (1mmolL(-1)) was also shown to inhibit 32.9% of the enzymatic activity. The developed method is applicable to kinetic studies and enables the recognition of the ligands. Furthermore, a comparison of the values of LicNTPDase-2-ICER with those obtained with an LC method using free enzyme in solution showed that LicNTPDase-2-ICER-LC/UV was an accurate and reproducible method that enabled automated measurements for the rapid screening of ligands.
authors.affiliation: Departamento de Química-Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto-Universidade de São Paulo, 14040-901 Ribeirão Preto, SP, Brazil.
authors.forename: Luana
authors.initials: L
authors.lastname: Magalhães
authors.affiliation: Departamento de Química-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: AHC
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, São Paulo, Brazil.
authors.forename: Raphael
authors.initials: R
authors.lastname: de Souza Vasconcellos
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, São Paulo, Brazil.
authors.forename: Christiane
authors.initials: C
authors.lastname: Mariotini-Moura
authors.affiliation: Departamento de Bioquímica e Biologia Molecular-Universidade Federal de Viçosa, 36570-000 Viçosa, MG, Brazil.
authors.forename: Rafaela
authors.initials: R
authors.lastname: de Cássia Firmino
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, São Paulo, Brazil.
authors.forename: Juliana Lopes Rangel
authors.initials: JLR
authors.lastname: Fietto
authors.affiliation: Departamento de Química-Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto-Universidade de São Paulo, 14040-901 Ribeirão Preto, SP, Brazil. Electronic address: ccardoso@ffclrp.usp.br.
authors.forename: Carmen Lúcia
authors.initials: CL
authors.lastname: Cardoso
mesh_terms: D000818:Animals; D019149:Bioreactors; D018314:Leishmania infantum / Q000201:enzymology*; D008855:Microscopy, Electron, Scanning; D043583:Nucleoside-Triphosphatase / Q000378:metabolism*
chemical_list: D043583:Nucleoside-Triphosphatase
keywords: Enzyme Immobilization; Leishmania infantum; Multidimensional enzymatic assay; NTPDase-2; Nucleoside triphosphate diphosphohydrolase