Label-free offline versus online activity methods for nucleoside diphosphate kinase b using high performance liquid chromatography

Author(s)

Lima, Juliana Maria ; Vieira, Plinio Salmazo ; Cavalcante de Oliveira, Arthur Henrique ; Cardoso, Carmen Lucia

• Document type: Journal article
• Source: ANALYST; v. 141, n. 15, p. 4733-4741, 2016.
• Web of Science Citations: 5

Abstract

Nucleoside diphosphate kinase from Leishmania spp. (LmNDKb) has recently been described as a potential drug target to treat leishmaniasis disease. Therefore, screening of LmNDKb ligands requires methodologies that mimic the conditions under which LmNDKb acts in biological systems. Here, we compare two label-free methodologies that could help screen LmNDKb ligands and measure NDKb activity: an offline LC-UV assay for soluble LmNDKb and an online two-dimensional LC-UV system based on LmNDKb immobilised on a silica capillary. The target enzyme was immobilised on the silica capillary via Schiff base formation (to give LmNDKb-ICER-Schiff) or affinity attachment (to give LmNDKb-ICER-His). Several aspects of the ICERs resulting from these procedures were compared, namely kinetic parameters, stability, and procedure steps. Both the LmNDKb immobilisation routes minimised the conformational changes and preserved the substrate binding sites. However, considering the number of steps involved in the immobilisation procedure, the cost of reagents, and the stability of the immobilised enzyme, immobilisation via Schiff base formation proved to be the optimal procedure. (AU)

• FAPESP's process: 14/06907-0 - Immobilization nucleoside diphosphate kinase (NDK) enzyme Leishmania major: the study of conditions for the application in the screening of ligands
• Grantee: Juliana Maria de Lima
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• 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: 11/20569-2 - Role of amino acids of the active site and interface in oligomeric in the structure and function of the nucleoside diphosphate kinase of Leishmania major
• Grantee: Arthur Henrique Cavalcante de Oliveira
• Support Opportunities: Regular Research Grants