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.
Analyst. 2016 Aug 7;141(15):4733-41. doi: 10.1039/c6an00655h. Epub 2016 Jun 7.
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.
来自利什曼原虫属（LmNDKb）的核苷二磷酸激酶最近被描述为一种治疗利什曼病的潜在药物靶点。因此，筛选 LmNDKb 配体需要模拟 LmNDKb 在生物系统中作用条件的方法。在这里，我们比较了两种无标记方法，这些方法可用于筛选 LmNDKb 配体并测量 NDKb 活性：用于可溶性 LmNDKb 的离线 LC-UV 测定法和基于固定在硅胶毛细管上的 LmNDKb 的在线二维 LC-UV 系统。靶酶通过席夫碱形成（得到 LmNDKb-ICER-Schiff）或亲和附着（得到 LmNDKb-ICER-His）固定在硅胶毛细管上。比较了这两种方法得到的 ICER 的几个方面，即动力学参数、稳定性和步骤。两种 LmNDKb 固定化方法都最小化了构象变化并保留了底物结合位点。然而，考虑到固定化过程中涉及的步骤数量、试剂成本和固定化酶的稳定性，通过席夫碱形成进行固定化被证明是最佳的方法。
