Evaluation of capillary chromatographic supports for immobilized human purine nucleoside phosphorylase in frontal affinity chromatography studies

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 (0.1 mm I.D. x 5 cm) and a methacrylate-based monolithic epoxy polymeric capillary column (0.25 mm I.D. x 5 cm) 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. K-d determination was carried out with the based fused silica HsPNP-IMER and the results are herein discussed. (C) 2014 Elsevier B.V. All rights reserved.