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 G. Ducati, Diógenes Santiago Santos, Carmen Lucia Cardoso, Quezia Bezerra Cass, Gabriella Massolini

Research output: Contribution to journalArticle

10 Citations (Scopus)

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
Externally publishedYes

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Purine-Nucleoside Phosphorylase
Affinity chromatography
Immobilized Enzymes
Affinity Chromatography
Fused silica
Silicon Dioxide
Inosine
Methacrylates
Mass spectrometry
Catalyst activity
Mass Spectrometry
Screening
Substrates
Experiments

Keywords

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

ASJC Scopus subject areas

  • Analytical Chemistry
  • Organic Chemistry
  • Biochemistry

Cite this

Evaluation of capillary chromatographic supports for immobilized human purine nucleoside phosphorylase in frontal affinity chromatography studies. / De Moraes, Marcela Cristina; Temporini, Caterina; Calleri, Enrica; Bruni, Giovanna; Ducati, Rodrigo G.; Santos, Diógenes Santiago; Cardoso, Carmen Lucia; Cass, Quezia Bezerra; Massolini, Gabriella.

In: Journal of Chromatography A, Vol. 1338, 18.04.2014, p. 77-84.

Research output: Contribution to journalArticle

De Moraes, Marcela Cristina ; Temporini, Caterina ; Calleri, Enrica ; Bruni, Giovanna ; Ducati, Rodrigo G. ; Santos, Diógenes Santiago ; Cardoso, Carmen Lucia ; Cass, Quezia Bezerra ; Massolini, Gabriella. / Evaluation of capillary chromatographic supports for immobilized human purine nucleoside phosphorylase in frontal affinity chromatography studies. In: Journal of Chromatography A. 2014 ; Vol. 1338. pp. 77-84.
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AU - Ducati, Rodrigo G.

AU - Santos, Diógenes Santiago

AU - Cardoso, Carmen Lucia

AU - Cass, Quezia Bezerra

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