title: Evaluation of capillary chromatographic supports for immobilized human purine nucleoside phosphorylase in frontal affinity chromatography studies.
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.
authors.affiliation: Departamento de Química, Universidade Federal de São Carlos, Cx Postal 676, São Carlos 13565-905, São Paulo, Brazil.
authors.forename: Marcela Cristina
authors.initials: MC
authors.lastname: de Moraes
authors.affiliation: Department of Drug Sciences, University of Pavia, 27100 Pavia, Italy.
authors.forename: Caterina
authors.initials: C
authors.lastname: Temporini
authors.affiliation: Department of Drug Sciences, University of Pavia, 27100 Pavia, Italy.
authors.forename: Enrica
authors.initials: E
authors.lastname: Calleri
authors.affiliation: Department of Chemistry, University of Pavia, 27100 Pavia, Italy.
authors.forename: Giovanna
authors.initials: G
authors.lastname: Bruni
authors.affiliation: Instituto Nacional de Ciência e Tecnologia em Tuberculose, Centro de Pesquisas em Biologia Molecular e Funcional, Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Porto Alegre, Rio Grande do Sul, Brazil.
authors.forename: Rodrigo Gay
authors.initials: RG
authors.lastname: Ducati
authors.affiliation: Instituto Nacional de Ciência e Tecnologia em Tuberculose, Centro de Pesquisas em Biologia Molecular e Funcional, Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Porto Alegre, Rio Grande do Sul, Brazil.
authors.forename: Diógenes Santiago
authors.initials: DS
authors.lastname: Santos
authors.affiliation: Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto 14040-901, São Paulo, Brazil.
authors.forename: Carmen Lucia
authors.initials: CL
authors.lastname: Cardoso
authors.affiliation: Departamento de Química, Universidade Federal de São Carlos, Cx Postal 676, São Carlos 13565-905, São Paulo, Brazil.
authors.forename: Quezia Bezerra
authors.initials: QB
authors.lastname: Cass
authors.affiliation: Department of Drug Sciences, University of Pavia, 27100 Pavia, Italy. Electronic address: g.massolini@unipv.it.
authors.forename: Gabriella
authors.initials: G
authors.lastname: Massolini
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D004800:Enzymes, Immobilized / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D008855:Microscopy, Electron, Scanning; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry*
chemical_list: D004800:Enzymes, Immobilized; D011683:Purine-Nucleoside Phosphorylase
keywords: FAC-MS; HPAC; Immobilized human purine nucleoside phosphorylase; Ligand affinity determination; Monolithic supports