Calil Felipe Antunes, Lima Juliana Maria, de Oliveira Arthur Henrique Cavalcante, Mariotini-Moura Christiane, Fietto Juliana Lopes Rangel, Cardoso Carmen Lucia
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.
Departamento de Bioquímica e Biologia Molecular, Universidade Federal de Viçosa, 36570-000 Viçosa, MG, Brazil; Instituto Nacional de Biotecnologia Estrutural e Química Medicinal em Doenças Infecciosas (INBEQMeDI), São Carlos, SP, Brazil.
J Anal Methods Chem. 2016;2016:9846731. doi: 10.1155/2016/9846731. Epub 2016 Dec 14.
The use of IMERs (Immobilized Enzyme Reactors) as a stationary phase coupled to high performance chromatographic systems is an interesting approach in the screening of new ligands. In addition, IMERs offer many advantages over techniques that employ enzymes in solution. The enzyme nucleoside triphosphate diphosphohydrolase (NTPDase-1) from acts as a pathogen infection facilitator, so it is a good target in the search for inhibitors. In this paper, immobilization of NTPDase-1 afforded ICERs (Immobilized Capillary Enzyme Reactors). A liquid chromatography method was developed and validated to monitor the ICER activity. The conditions for the application of these bioreactors were investigated, and excellent results were obtained. The enzyme was successfully immobilized, as attested by the catalytic activity detected in the NTPDase-1-ICER chromatographic system. Kinetic studies on the substrate ATP gave of 0.317 ± 0.044 mmol·L, which still presented high affinity compared to in solution. Besides that, the ICER was stable for 32 days, enough time to investigate samples of possible inhibitors, including especially the compound Suramin, that inhibited 51% the enzyme activity at 100 mol·L, which is in accordance with the data for the enzyme in solution.
将固定化酶反应器（IMERs）用作与高效色谱系统联用的固定相，是筛选新配体的一种有趣方法。此外，与在溶液中使用酶的技术相比，IMERs具有许多优势。来自 的核苷三磷酸二磷酸水解酶（NTPDase-1）作为病原体感染促进因子，因此是寻找抑制剂的良好靶点。在本文中，NTPDase-1的固定化得到了固定化毛细管酶反应器（ICERs）。开发并验证了一种液相色谱方法来监测ICER活性。研究了这些生物反应器的应用条件，并获得了优异的结果。通过在NTPDase-1-ICER色谱系统中检测到的催化活性证明酶已成功固定化。对底物ATP的动力学研究得出 的值为0.317±0.044 mmol·L，与溶液中的情况相比，仍表现出高亲和力。除此之外，ICER在32天内保持稳定，这有足够的时间来研究可能的抑制剂样品，特别是化合物苏拉明，其在100 μmol·L时抑制了51%的酶活性，这与溶液中该酶的数据一致。
