TY - JOUR
T1 - Capillary bioreactors based on human purine nucleoside phosphorylase
T2 - A new approach for ligands identification and characterization
AU - de Moraes, Marcela Cristina
AU - Ducati, Rodrigo Gay
AU - Donato, Augusto José
AU - Basso, Luiz Augusto
AU - Santos, Diógenes Santiago
AU - Cardoso, Carmen Lucia
AU - Cass, Quezia Bezerra
PY - 2012/4/6
Y1 - 2012/4/6
N2 - 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 K M value for the immobilized enzyme was about twofold higher than that measured for the enzyme in solution (255±29.2μM and 133±14.9μM, respectively). A new fourth-generation immucillin derivative (DI4G; IC 50=40.6±0.36nM), 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.
AB - 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 K M value for the immobilized enzyme was about twofold higher than that measured for the enzyme in solution (255±29.2μM and 133±14.9μM, respectively). A new fourth-generation immucillin derivative (DI4G; IC 50=40.6±0.36nM), 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.
KW - Enzyme immobilization
KW - Immucillin analogues
KW - Ligands screening
KW - Purine nucleoside phosphorylase
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U2 - 10.1016/j.chroma.2011.10.056
DO - 10.1016/j.chroma.2011.10.056
M3 - Article
C2 - 22099222
AN - SCOPUS:84858071823
SN - 0021-9673
VL - 1232
SP - 110
EP - 115
JO - Journal of Chromatography A
JF - Journal of Chromatography A
ER -