The relationship between A beta-associated free radical generation and A beta fibril formation revealed by negative stain electron microscopy and thioflavine-T fluorometric assay

In the present study, we investigated whether or not the A beta peptide its elf spontaneously generates free radicals using electron spin resonance (ES R) spectroscopy while also observing the A beta fibril formation by negativ e stain electron microscopy. The present results demonstrated a four-line s pectrum in the presence of A beta (1-40) with N-tert-butyl-alpha -phenylnit rone (PBN) but not in the presence of PBN alone in phosphate-buffered salin e. Negative stain electron microscopy has shown that A beta peptides after 96 h of incubation showed more amyloid-like fibrils than those after 72 h o f incubation while the four-line spectrum obtained by ESR spectroscopy atta ined a maximum intensity after 72 h of incubation and thereafter its intens ity immediately decreased during the 4-day incubation period. These results were also supported by a thioflavine-T (Th-T) fluorometric assay. In concl usion, the present results suggest that A beta -associated free radical gen eration is correlated with A beta fibril formation while its generation is only observed transiently during the process of A beta fibril formation. (C ) 2001 Elsevier Science Ireland Ltd. All rights reserved.