Author/Authors :
de Moraes، نويسنده , , Marcela Cristina and Temporini، نويسنده , , Caterina and Calleri، نويسنده , , Enrica and Bruni، نويسنده , , Giovanna and Ducati، نويسنده , , Rodrigo Gay and Santos، نويسنده , , Diَgenes Santiago and Cardoso، نويسنده , , Carmen Lucia and Cass، نويسنده , , Quezia Bezerra and Massolini، نويسنده , , Gabriella، نويسنده ,
Abstract :
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. × 5 cm) and a methacrylate-based monolithic epoxy polymeric capillary column (0.25 mm I.D. × 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. Kd determination was carried out with the based fused silica HsPNP-IMER and the results are herein discussed.
Keywords :
HPAC , FAC-MS , Immobilized human purine nucleoside phosphorylase , Ligand affinity determination , Monolithic supports
