Peroxynitrite effect on ferritin

GALLEANO MONICA; ALCALDE MYRIAM; PUNTARULO SUSANA

La Paz, Baja California Sur

Workshop; Second Workshop on Comparative Aspects of Oxidative Stress in Biological Systems; 2005

CIBNOR

              The aim of this work was to study the in vitro effect of peroxynitrite on ferritin structure and the possible functional alterations. Purified horse spleen apoferritin was exposed to a peroxynitrite donor linsidomine (SIN-1; 0-500 mM) by incubation in 100 mM phosphate buffer, pH 7.4, 25 mM bicarbonate at 37°C for 0-60 min. Fluorescence associated to tryptophan (lexc=285 nm; lem=345 nm) decreased significantly during incubation in a dose dependent manner (22±4 and 35±2% after 15 min of exposure to 30 and 100 mM SIN-1, respectively). The dityrosine content was not affected by SIN-1 exposure. Cysteine content (assessed by electronic paramagnetic resonance ?EPR-, using the spin label 4-maleimido-TEMPO) showed a significant decrease (45±5%) after 15 min of exposure to 100 mM SIN-1. Ferroxidase activity, studied spectrophotometrically, was 18±2 and 9.2±0.9 mM Fe(II)/min.mM ferritin for control and ferritin exposed during 30 min to 100 mM SIN-1, respectively. Protein-derived radicals production was monitored by low-temperature EPR following the kinetic of the reaction over the initial 30 min of exposure to 100 mM SIN-1 and, at least, one radical species was detected. The concentration of this radical increased as a function of time exposure and its characteristics were compatible with a tryptophan-centered radical. These results suggest that an increased peroxynitrite steady-state concentration could lead to amino acids modifications in ferritin with possible alterations in its functionality. Further studies will be necessary to determine the patho-physiological relevance of this phenomena. Supported by CONICET, ANPCyT (11187) and University of Buenos Aires (B017).