Capillary bioreactors based on human purine nucleoside phosphorylase: A new approach for ligands identification and characterization

被引:25
作者
de Moraes, Marcela Cristina [1 ]
Ducati, Rodrigo Gay [2 ]
Donato, Augusto Jose [3 ]
Basso, Luiz Augusto [2 ]
Santos, Diogenes Santiago [2 ]
Cardoso, Carmen Lucia [4 ]
Cass, Quezia Bezerra [1 ]
机构
[1] Univ Fed Sao Carlos, Dept Quim, BR-13565905 Sao Paulo, Brazil
[2] Pontificia Univ Catal Rio Grande do Sul PUCRS, Ctr Pesquisas Biol Mol & Func, Inst Nacl Ciencia & Tecnol TB, Porto Alegre, RS, Brazil
[3] Pontificia Univ Catolica Rio Grande do Sul, Fac Quim, Porto Alegre, RS, Brazil
[4] Univ Sao Paulo, Fac Filosofia Ciencias & Letras Ribeirao Preto, BR-14040901 Sao Paulo, Brazil
关键词
Purine nucleoside phosphorylase; Enzyme immobilization; Ligands screening; Immucillin analogues; TRANSITION-STATE ANALOGS; PERFORMANCE LIQUID-CHROMATOGRAPHY; IMMOBILIZED ENZYME REACTOR; MYCOBACTERIUM-TUBERCULOSIS; GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE; TRYPANOSOMA-CRUZI; IMMUCILLIN-H; INHIBITOR; PHOSPHATE; SUBSTRATE;
D O I
10.1016/j.chroma.2011.10.056
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
The enzyme purine nucleoside phosphorylase (PNP) is a target for the discovery of new lead compounds employed on the treatment severe T-cell mediated disorders. Within this context, the development of new, direct, and reliable methods for ligands screening is an important task. This paper describes the preparation of fused silica capillaries human PNP (HsPNP) immobilized enzyme reactor (IMER). The activity of the obtained IMER is monitored on line in a multidimensional liquid chromatography system, by the quantification of the product formed throughout the enzymatic reaction. The Km value for the immobilized enzyme was about twofold higher than that measured for the enzyme in solution (255 +/- 29.2 mu M and 133 +/- 114.9 mu M, respectively). A new fourth-generation immucillin derivative (DI4G: IC50 = 40.6 +/- 0.36 nM), previously identified and characterized in HsPNP free enzyme assays, was used to validate the IMER as a screening method for HsPNP ligands. The validated method was also used for mechanistic studies with this inhibitor. This new approach is a valuable tool to PNP ligand screening, since it directly measures the hypoxanthine released by inosine phosphorolysis, thus furnishing more reliable results than those one used in a coupled enzymatic spectrophotometric assay. (C) 2011 Elsevier B.V. All rights reserved.
引用
收藏
页码:110 / 115
页数:6
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