用于前沿亲和色谱研究中固定化人嘌呤核苷磷酸化酶的毛细管色谱载体的评价。
Evaluation of capillary chromatographic supports for immobilized human purine nucleoside phosphorylase in frontal affinity chromatography studies.
作者信息
de Moraes Marcela Cristina, Temporini Caterina, Calleri Enrica, Bruni Giovanna, Ducati Rodrigo Gay, Santos Diógenes Santiago, Cardoso Carmen Lucia, Cass Quezia Bezerra, Massolini Gabriella
机构信息
Departamento de Química, Universidade Federal de São Carlos, Cx Postal 676, São Carlos 13565-905, São Paulo, Brazil.
Department of Drug Sciences, University of Pavia, 27100 Pavia, Italy.
出版信息
J Chromatogr A. 2014 Apr 18;1338:77-84. doi: 10.1016/j.chroma.2014.02.057. Epub 2014 Feb 28.
The aim of this work was to optimize the preparation of a capillary human purine nucleoside phosphorylase (HsPNP) immobilized enzyme reactor (IMER) for characterization and affinity screening studies of new inhibitors by frontal affinity chromatography coupled to mass spectrometry (FAC-MS). For this purpose two monolithic supports, a Chromolith Speed Rod (0.1mm I.D.×5cm) and a methacrylate-based monolithic epoxy polymeric capillary column (0.25mm I.D.×5cm) with epoxy reactive groups were considered and compared to an IMER previously developed using an open fused silica capillary. Each HsPNP-IMER was characterized in terms of catalytic activity using Inosine as standard substrate. Furthermore, they were also explored for affinity ranking experiments. Kd determination was carried out with the based fused silica HsPNP-IMER and the results are herein discussed.
这项工作的目的是优化毛细管人嘌呤核苷磷酸化酶（HsPNP）固定化酶反应器（IMER）的制备，以通过与质谱联用的前沿亲和色谱法（FAC-MS）对新型抑制剂进行表征和亲和筛选研究。为此，考虑了两种整体式载体，一种是内径0.1mm×5cm的Chromolith Speed Rod和一种带有环氧反应基团的基于甲基丙烯酸酯的整体式环氧聚合物毛细管柱（内径0.25mm×5cm），并将它们与先前使用开放熔融石英毛细管开发的IMER进行比较。每个HsPNP-IMER均以肌苷作为标准底物，根据催化活性进行表征。此外，还对它们进行了亲和排序实验。使用基于熔融石英的HsPNP-IMER进行了Kd测定，本文讨论了结果。
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