Asunto(s)
Aldehído Oxidorreductasas/aislamiento & purificación , Hígado/enzimología , Aldehído Oxidasa , Animales , Cromatografía de Afinidad , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Cromatografía Liquida , Femenino , Proteínas/análisis , Conejos , Espectrofotometría UltravioletaRESUMEN
Highly purified rabbit liver aldehyde oxidase was prepared in high yield using affinity chromatography on Benzamidine Sepharose 6B. Rabbit liver was homogenised, heat treated and ammonium sulphate was added to the supernatant to give a crude preparation of the enzyme. Aliquots of the crude preparation were chromatographed on a Benzamidine Sepharose 6B column at pH 9 and the aldehyde oxidase was eluted by a benzamidine containing buffer. This single affinity step resulted in a 38-fold increase in purity over the crude preparation with an 84% recovery of enzyme activity. Further purification on a Mono Q ion-exchange column gave an additional 1.7-fold increase in specific activity to yield a highly purified preparation of the enzyme. The new method described is considerably simpler and faster than ones hitherto employed and gives a much better yield of the highly purified enzyme.