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

  • J Chromatogr B Analyt Technol Biomed Life Sci. 2016 Jan 1:1008:98-107. doi: 10.1016/j.jchromb.2015.11.028.
Luana Magalhães  1 ,  Arthur Henrique Cavalcante de Oliveira  1 ,  Raphael de Souza Vasconcellos  2 ,  Christiane Mariotini-Moura  2 ,  Rafaela de Cássia Firmino  3 ,  Juliana Lopes Rangel Fietto  2 ,  Carmen Lúcia Cardoso  4
Affiliations
  • 1. Departamento de Química-Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto-Universidade de São Paulo, 14040-901 Ribeirão Preto, SP, Brazil.
  • 2. 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.
  • 3. Departamento de Bioquímica e Biologia Molecular-Universidade Federal de Viçosa, 36570-000 Viçosa, MG, Brazil.
  • 4. Departamento de Química-Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto-Universidade de São Paulo, 14040-901 Ribeirão Preto, SP, Brazil. Electronic address: [email protected].
PMID: 26638034 DOI: 10.1016/j.jchromb.2015.11.028
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.

Keywords
Enzyme Immobilization; Leishmania infantum; Multidimensional enzymatic assay; NTPDase-2; Nucleoside triphosphate diphosphohydrolase.