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

被引：21

作者：

de Moraes, Marcela Cristina ^{[1
]}

Temporini, Caterina ^{[2
]}

Calleri, Enrica ^{[2
]}

Bruni, Giovanna ^{[3
]}

Ducati, Rodrigo Gay ^{[4
]}

Santos, Diogenes Santiago ^{[4
]}

Cardoso, Carmen Lucia ^{[5
]}

Cass, Quezia Bezerra ^{[1
]}

Massolini, Gabriella ^{[2
]}

机构：

[1] Univ Fed Sao Carlos, Dept Quim, BR-13565905 Sao Paulo, Brazil

[2] Univ Pavia, Dept Drug Sci, I-27100 Pavia, Italy

[3] Univ Pavia, Dept Chem, I-27100 Pavia, Italy

[4] Pontificia Univ Catolica Rio Grande do Sul PUCRS, Ctr Pesquisas Biol Mol & Func, Inst Nacl Ciencia & Tecnol TB, Porto Alegre, RS, Brazil

[5] Univ Sao Paulo, Fac Filosofia Ciencias & Letras Ribeirao Preto, BR-14040901 Sao Paulo, Brazil

来源：

JOURNAL OF CHROMATOGRAPHY A | 2014年 / 1338卷

基金：

巴西圣保罗研究基金会;

关键词：

HPAC; FAC-MS; Immobilized human purine nucleoside; phosphorylase; Ligand affinity determination; Monolithic supports; PROTEIN-COUPLED RECEPTOR; TYROSINE KINASE RECEPTOR; ENZYME REACTOR; SCHISTOSOMA-MANSONI; STATIONARY PHASES; MS DETECTION; DISCOVERY; BINDING; IDENTIFICATION; INHIBITORS;

D O I：

10.1016/j.chroma.2014.02.057

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

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. x 5 cm) and a methacrylate-based monolithic epoxy polymeric capillary column (0.25 mm I.D. x 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. K-d determination was carried out with the based fused silica HsPNP-IMER and the results are herein discussed. (C) 2014 Elsevier B.V. All rights reserved.

引用

页码：77 / 84

页数：8

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