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

Autor(es)

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

• Tipo de documento: Artigo Científico
• Fonte: ANALYST; v. 141, n. 15, p. 4733-4741, 2016.
• Citações Web of Science: 5

Resumo

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)

• Processo FAPESP: 14/06907-0 - Imobilização da enzima nucleosídeo difosfato quinase (NDK) de Leishmania major: do estudo de condições à aplicação na triagem de ligantes
• Beneficiário: Juliana Maria de Lima
• Modalidade de apoio: Bolsas no Brasil - Doutorado Direto
• Processo FAPESP: 13/01710-1 - Ligantes enzimáticos: novos modelos de triagem
• Beneficiário: Quezia Bezerra Cass
• Modalidade de apoio: Auxílio à Pesquisa - Temático
• Processo FAPESP: 11/20569-2 - Papel de aminoácidos do sítio ativo e da interface oligomérica na estrutura e função da nucleosídeo difosfato kinase de Leishmania major
• Beneficiário: Arthur Henrique Cavalcante de Oliveira
• Modalidade de apoio: Auxílio à Pesquisa - Regular