Ferritin Radicals Generated by Iron Uptake and Peroxynitrite Treatment

GALLEANO MONICA; ALCALDE MYRIAM; PUNTARULO SUSANA; AUGUSTO OHARA

Buenos Aire

Congreso; 12th Biennial Meeting International Society for Free Radical Research; 2004

International Society for Free Radical Research

Ferritin-radicals produced during aerobic iron (II) uptake by ferritin was studied by electronic paramagnetic resonance (EPR) in the presence of the trapping agents N-tert-butyl-a-phenyl nitrone (PBN) or 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The spectrum in the presence of PBN was a superposition of two types of signals, an anisotropic signal from a slowly tumbling adduct (2aNzz = 68.1 G) and an isotropic signal from a rapidly tumbling adduct (aN=15.9 G and aH=3.75 G). In the presence of DMPO, the spectrum obtained showed the dominant triplet pattern expected for a slowly tumbling nitroxide (aHb=22.3±1.1 G). In order to identify the detected radicals, chemical modifications were carried out on the native protein. Blocking of thiol groups did not affect significantly the signal whereas blockage of tyrosine residues decreased signal intensity in approximately 50%. This signal was also obtained upon ferritin treatment with peroxynitrite, a strong biological oxidant. In this case, a significant decrease in tryptophan-ferritin fluorescence was also observed. Taken together, these results suggest that iron uptake by ferritin produces, at least, two protein radicals. Most likely, an initially produced ferritin-tyrosyl radical oxidizes another residue to produce a secondary radical tentatively identified as a ferritin-tryptophanyl radical. Supported by University of Buenos Aires, ANPYCT and CONICET.