RESUMEN
Protein 4.1 is a multifunctional structural protein occupying a strategic position in the erythrocyte membrane. It is present in the erythrocyte membrane skeleton and in many nonerythroid cells. This report describes a novel method for purifying this protein based on its selective interaction with inositol hexaphosphate dimagnesium tetrapotassium salt. This interaction was discovered in the course of chromatography of high-salt extract of inside-out membrane vesicles on Procion orange MX-2R-Sepharose. The new procedure is simple and selective and produces protein 4.1 with better yield than that obtained with a previously published procedure. The purified protein 4.1 has the same immunoreactivity and the same alpha-chymotryptic digest profile as protein 4.1 purified by published methods and is fully functional in enhancing the interaction between F-actin and spectrin dimers.
Asunto(s)
Cromatografía de Afinidad/métodos , Proteínas del Citoesqueleto , Membrana Eritrocítica/química , Proteínas de la Membrana/aislamiento & purificación , Neuropéptidos , Ácido Fítico/metabolismo , Precipitación Química , Quimotripsina/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/aislamiento & purificación , Humanos , Concentración de Iones de Hidrógeno , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Fragmentos de Péptidos/aislamiento & purificación , Sefarosa , Solubilidad , TriazinasRESUMEN
Readily available elements were used to build an automatic apparatus dedicated to the preparation of erythrocyte ghosts. The apparatus is designed around a low-cost re-usable hollow-fiber filtration cartridge (marketed for therapeutic plasmapheresis). The apparatus is controlled by a simple programmer (based on a diode matrix and low cost timers and liquid level sensors): once the apparatus is loaded with whole red blood cells, washing of cells, as well as hemolysis and washing of ghosts, is performed by the machine in about 4.5 h without any operator intervention. Automatic filter cleaning takes a further 110 min.