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

Marcela Cristina De Moraes, Caterina Temporini, Enrica Calleri, Giovanna Bruni, Rodrigo Gay Ducati, Diógenes Santiago Santos, Carmen Lucia Cardoso, Quezia Bezerra Cass, Gabriella Massolini

Research output: Contribution to journalArticle

14 Scopus citations

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 (0.1mm 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.

Original languageEnglish (US)
Pages (from-to)77-84
Number of pages8
JournalJournal of Chromatography A
Volume1338
DOIs
StatePublished - Apr 18 2014

Keywords

  • FAC-MS
  • HPAC
  • Immobilized human purine nucleoside phosphorylase
  • Ligand affinity determination
  • Monolithic supports

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Organic Chemistry

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    De Moraes, M. C., Temporini, C., Calleri, E., Bruni, G., Ducati, R. G., Santos, D. S., Cardoso, C. L., Cass, Q. B., & Massolini, G. (2014). Evaluation of capillary chromatographic supports for immobilized human purine nucleoside phosphorylase in frontal affinity chromatography studies. Journal of Chromatography A, 1338, 77-84. https://doi.org/10.1016/j.chroma.2014.02.057