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

Author(s)

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]

Affiliation

^[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

• Document type: Journal article
• Source: JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES; v. 1008, p. 98-107, JAN 1 2016.
• Web of Science Citations: 3

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: 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)

• FAPESP's process: 13/01710-1 - Enzyme ligand: new models of screening
• Grantee: Quezia Bezerra Cass
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 12/10749-6 - Development of capillary columns enzymatic to application of screening of selective inhibitors for the enzyme NTPDase- 2 of Leishmania infantun
• Grantee: Luana Magalhães
• Support Opportunities: Scholarships in Brazil - Doctorate