Use este identificador para citar ou linkar para este item: https://locus.ufv.br//handle/123456789/21772
Tipo: Artigo
Título: Label-free assay based on immobilized capillary enzyme reactor of Leishmania infantum nucleoside triphosphate diphosphohydrolase (LicNTPDase-2-ICER-LC/UV)
Autor(es): Vasconcellos, Raphael de Souza
Magalhães, Luana
Oliveira, Arthur Henrique Cavalcante de
Mariotini-Moura, Christiane
Firmino, Rafaela de Cássia
Fietto, Juliana Lopes Rangel
Cardoso, Carmen Lúcia
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 1.8 mmol L^−1 for the ADP and ATP substrates, respectively. Suramin (1 mmol L^−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.
Palavras-chave: Nucleoside triphosphate diphosphohydrolase
NTPDase-2
Leishmania infantum
Enzyme
Immobilization
Multidimensional enzymatic assay
Editor: Journal of Chromatography B
Tipo de Acesso: Elsevier B.V.
URI: https://doi.org/10.1016/j.jchromb.2015.11.028
http://www.locus.ufv.br/handle/123456789/21772
Data do documento: 1-Jan-2016
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