RESUMEN
In this work we used affinity capillary electrophoresis (ACE) to investigate the extent of interaction between a pool of drugs and wild-type transthyretin. After qualitative preliminary screening, attention was focused on the most promising molecules, flufenamic acid and flurbiprofen, which underwent a further stage of investigation, the determination of the binding constants, and, when possible, the assessment of the number of binding sites by ACE, frontal analysis (FA) capillary electrophoresis (CE) and parallel ultrafiltration (UF) experiments. Furthermore, our data demonstrate that FA CE is a suitable technique for identifying fibril ligands. This represents a novel CE application of pharmaceutical interest.
Asunto(s)
Antiinflamatorios no Esteroideos/sangre , Ácido Flufenámico/sangre , Flurbiprofeno/sangre , Prealbúmina/metabolismo , Amiloidosis/sangre , Amiloidosis/tratamiento farmacológico , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/uso terapéutico , Sitios de Unión , Electroforesis Capilar/métodos , Ácido Flufenámico/química , Ácido Flufenámico/uso terapéutico , Flurbiprofeno/química , Flurbiprofeno/uso terapéutico , Humanos , Cinética , Prealbúmina/química , Unión Proteica , Análisis de Regresión , Relación Estructura-Actividad , Ultrafiltración/métodosRESUMEN
Recently we described the use of riboflavin binding protein extracted from quail egg-white, as a new HPLC chiral stationary phase. In this study we show the further results obtained with the use of high-performance affinity chromatography to provide a better understanding of the chiral recognition mechanism for the observed enantioselectivity and to gain a deeper knowledge into the binding site that has been recently characterised by X-ray crystallography for chicken egg-white. High-performance affinity chromatography provides information on the potential protein structural changes occurring upon its immobilisation and enables competitive binding studies as well as the assessment of binding constants through frontal analysis experiments.