Label-free offline versus online activity methods for nucleoside diphosphate kinase b using high performance liquid chromatography.
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.
Departamento de Química, Grupo de Cromatografia de Bioafinidade e Produtos Naturais, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, 14040-901, Ribeirão Preto-SP, Brazil. ccardoso@ffclrp.usp.br.
Juliana Maria
JM
Lima
Plínio
P
Salmazo Vieira
Arthur Henrique
AH
Cavalcante de Oliveira
Carmen Lúcia
CL
Cardoso
D002851:Chromatography, High Pressure Liquid; D004800:Enzymes, Immobilized; D007700:Kinetics; D007891:Leishmania / Q000201:enzymology*; D008024:Ligands; D054778:NM23 Nucleoside Diphosphate Kinases / Q000032:analysis*; D012822:Silicon Dioxide
D004800:Enzymes, Immobilized; D008024:Ligands; D054778:NM23 Nucleoside Diphosphate Kinases; D012822:Silicon Dioxide