TY - JOUR
T1 - Evaluation of capillary chromatographic supports for immobilized human purine nucleoside phosphorylase in frontal affinity chromatography studies
AU - De Moraes, Marcela Cristina
AU - Temporini, Caterina
AU - Calleri, Enrica
AU - Bruni, Giovanna
AU - Ducati, Rodrigo Gay
AU - Santos, Diógenes Santiago
AU - Cardoso, Carmen Lucia
AU - Cass, Quezia Bezerra
AU - Massolini, Gabriella
N1 - Funding Information:
This work was funded by grants from the São Paulo State Research Foundation (FAPESP, process number 2008/04051-0 ) and the Brazilian Agency for Support and Evaluation of Graduate Education (CAPES, process number 6595/10-3 ). The authors also thank the National Council for Scientific and Technological Development (CNPq) for financial support, and Dr. Corana from Centro Grandi Strumenti, University of Pavia (Italy), for suggestions and advices on MS instrumentation.
PY - 2014/4/18
Y1 - 2014/4/18
N2 - 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.
AB - 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.
KW - FAC-MS
KW - HPAC
KW - Immobilized human purine nucleoside phosphorylase
KW - Ligand affinity determination
KW - Monolithic supports
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U2 - 10.1016/j.chroma.2014.02.057
DO - 10.1016/j.chroma.2014.02.057
M3 - Article
C2 - 24630982
AN - SCOPUS:84897102427
SN - 0021-9673
VL - 1338
SP - 77
EP - 84
JO - Journal of Chromatography A
JF - Journal of Chromatography A
ER -