Ensayo frente a PNP de Plasmodium falciparum

El curso de la reacción de fosforolisis se siguió al acoplarla a una reacción de xantinoxidasa, que cataliza la oxidación de hipoxantina a ácido úrico y cuya formación se puede seguir a 293nm (coeficiente de extinción molar para el ácido úrico: 12.9 mM-1

cm-1_{). Para determinar la actividad inhibitoria se incubó una mezcla que contenía}

KH2PO4 (50 mM, pH 7.5), xantinaoxidasa (60mU), PfPNP (0.73mg, 25nM) e inosina

(25 M) en un volumen final de 1 mL a 25 ºC. Para los estudios de inhibición, se llevaron a cabo varios experimentos en donde la concentración de inhibidor era variable (5 concentraciones fueron ensayadas) y la concentración de inosina fija (25 M).

Todos los espectros se registraron a una concentración 50 µM en PBS a partir de una disolución stock 5 mM en DMSO. Todas las medidas se realizaron en celdas de cuarzo de paso óptico 10 mm. La rendija de excitación se fijó a 15 nm y la de emisión a 20 nm, y los espectros fueron registrados en un intervalo de 1 nm y con una velocidad de barrido de 100 nm por minuto.

Una mezcla que contiene 50 mM KH2PO4 pH 7.5, 268 nM (7.83mg) PfPNP y 0.5 mM

de nucleósido se incubó a temperatura ambiente entre 30min y 2 horas, y se registraron los espectros de fluorescencia ( ex = 288nm; em = 300-450nm). Como controles se

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