Label-free assay based on immobilized capillary enzyme reactor of Leishmania infantum nucleoside triphosphate diphosphohydrolase (LicNTPDase-2-ICER-LC/UV)

Autor(es)

Magalhaes, Luana ^[1] ; Cavalcante de Oliveira, Arthur Henrique ^[1] ; Vasconcellos, Raphael de Souza ^{[2, 3]} ; Mariotini-Moura, Christiane ^{[2, 3]} ; Firmino, Rafaela de Cassia ^[2] ; Rangel Fietto, Juliana Lopes ^{[2, 3]} ; Cardoso, Carmen Lucia ^[1]

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo, Dept Quim, Fac Filosofia Ciencias & Letras Ribeirao Preto, BR-14040901 Ribeirao Preto, SP - Brazil

^[2] Univ Fed Vicosa, Dept Bioquim & Biol Mol, BR-36570000 Vicosa, MG - Brazil

^[3] INBEQMeDI, Sao Carlos, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES; v. 1008, p. 98-107, JAN 1 2016.
• Citações Web of Science: 3

Resumo

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: K-M 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. (C) 2015 Elsevier B.V. All rights reserved. (AU)

• Processo FAPESP: 13/01710-1 - Ligantes enzimáticos: novos modelos de triagem
• Beneficiário: Quezia Bezerra Cass
• Modalidade de apoio: Auxílio à Pesquisa - Temático
• Processo FAPESP: 12/10749-6 - Desenvolvimento e aplicação de colunas capilares enzimáticas na triagem de inibidores seletivos para a enzima NTPDase 2 de Leishmania
• Beneficiário: Luana Magalhães
• Modalidade de apoio: Bolsas no Brasil - Doutorado