Label-free offline versus online activity methods for nucleoside diphosphate kinase b using high performance liquid chromatography
文献信息
发布日期
2016-05-27
DOI
10.1039/C6AN00655H
影响因子
4.616
作者
Juliana Maria Lima, Plínio Salmazo Vieira, Arthur Henrique Cavalcante de Oliveira, Carmen Lúcia Cardoso
查看原文
摘要
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.
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来源期刊
Analyst
CiteScore:
7.8
自引率:
5.6%
年发文量:
653
Analyst publishes analytical and bioanalytical research that reports premier fundamental discoveries and inventions, and the applications of those discoveries, unconfined by traditional discipline barriers.
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