Label-free offline versus online activity methods for nucleoside diphosphate kinase b using high performance liquid chromatography. 2016

Juliana Maria Lima, and Plínio Salmazo Vieira, and Arthur Henrique Cavalcante de Oliveira, and Carmen Lúcia Cardoso
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.

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.

UI MeSH Term Description Entries
D007700 Kinetics The rate dynamics in chemical or physical systems.
D007891 Leishmania A genus of flagellate protozoa comprising several species that are pathogenic for humans. Organisms of this genus have an amastigote and a promastigote stage in their life cycles. As a result of enzymatic studies this single genus has been divided into two subgenera: Leishmania leishmania and Leishmania viannia. Species within the Leishmania leishmania subgenus include: L. aethiopica, L. arabica, L. donovani, L. enrietti, L. gerbilli, L. hertigi, L. infantum, L. major, L. mexicana, and L. tropica. The following species are those that compose the Leishmania viannia subgenus: L. braziliensis, L. guyanensis, L. lainsoni, L. naiffi, and L. shawi. Leishmania (Leishmania),Leishmania (Viannia),Leishmania leishmania,Leishmania viannia,Leishmania leishmanias,Leishmania viannias,Leishmanias,Leishmanias (Leishmania),Leishmanias (Viannia),leishmanias, Leishmania,viannias, Leishmania
D008024 Ligands A molecule that binds to another molecule, used especially to refer to a small molecule that binds specifically to a larger molecule, e.g., an antigen binding to an antibody, a hormone or neurotransmitter binding to a receptor, or a substrate or allosteric effector binding to an enzyme. Ligands are also molecules that donate or accept a pair of electrons to form a coordinate covalent bond with the central metal atom of a coordination complex. (From Dorland, 27th ed) Ligand
D002851 Chromatography, High Pressure Liquid Liquid chromatographic techniques which feature high inlet pressures, high sensitivity, and high speed. Chromatography, High Performance Liquid,Chromatography, High Speed Liquid,Chromatography, Liquid, High Pressure,HPLC,High Performance Liquid Chromatography,High-Performance Liquid Chromatography,UPLC,Ultra Performance Liquid Chromatography,Chromatography, High-Performance Liquid,High-Performance Liquid Chromatographies,Liquid Chromatography, High-Performance
D004800 Enzymes, Immobilized Enzymes which are immobilized on or in a variety of water-soluble or water-insoluble matrices with little or no loss of their catalytic activity. Since they can be reused continuously, immobilized enzymes have found wide application in the industrial, medical and research fields. Immobilized Enzymes,Enzyme, Immobilized,Immobilized Enzyme
D012822 Silicon Dioxide Transparent, tasteless crystals found in nature as agate, amethyst, chalcedony, cristobalite, flint, sand, QUARTZ, and tridymite. The compound is insoluble in water or acids except hydrofluoric acid. Silica,Aerosil,Aerosil 380,Cristobalite,Quso G-32,Quso G32,Tridymite,380, Aerosil,Dioxide, Silicon,G32, Quso,Quso G 32
D054778 NM23 Nucleoside Diphosphate Kinases A family of nucleotide diphosphate kinases that play a role in a variety of cellular signaling pathways that effect CELL DIFFERENTIATION; CELL PROLIFERATION; and APOPTOSIS. They are considered multifunctional proteins that interact with a variety of cellular proteins and have functions that are unrelated to their enzyme activity. Nucleoside Diphosphate Kinase A,DR-NM23 Nucleoside Diphosphate Kinase,Expressed in Non-Metastatic Cells 2 Protein,Granzyme A-activated DNase,NDP Kinase A,NM23-H2 Nucleoside Diphosphate Kinase,NM23-H3 Nucleoside Diphosphate Kinase,NM23A Nucleoside Diphosphate Kinases,NM23B Nucleoside Diphosphate Kinase,NME1 Nucleoside Diphosphate Kinase,Nm23-H1 Nucleoside Diphosphate Kinase,Non-Metastatic Cells 1 Protein,Nucleoside Diphosphate Kinase 3,Nucleoside Diphosphate Kinase B,Nucleoside Diphosphate Kinase C,Tumor Metastatic Process-Associated Protein,DR NM23 Nucleoside Diphosphate Kinase,Expressed in Non Metastatic Cells 2 Protein,Granzyme A activated DNase,NM23 H2 Nucleoside Diphosphate Kinase,NM23 H3 Nucleoside Diphosphate Kinase,Nm23 H1 Nucleoside Diphosphate Kinase,Non Metastatic Cells 1 Protein,Tumor Metastatic Process Associated Protein

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