The relationship between the aggregational state of the amyloid-beta peptides and free radical generation by the peptides

In the present study, we investigated whether or not the amyloid-beta prote in (A beta) peptide itself spontaneously generates free radicals using elec tron spin resonance (ESR) spectroscopy while also monitoring the aggregatio nal state of A beta and A beta -induced cytotoxicity. The present results d emonstrated a four-line spectrum in the presence of both A beta 40 and A be ta 42 with N-terf-butyl-alpha -phenylnitrone (PBN), but not in the presence of PEN alone in phosphate-buffered saline (PBS). The fact that the four-li ne spectrum obtained for the A beta /PBN in PBS was completely abolished in the presence of the iron-chelating agent Desferal demonstrated the observe d four-line spectrum to be iron-dependent. The present study also revealed that either A beta 40 or A beta 42 with PEN in phosphate buffer (PB) did no t produce any definite four-line spectrum. Both a thioflavine-T (Th-T) fluo rometric assay and circular dichroism (CD) spectroscopy showed the amyloid fibril formation of A beta in PBS to be much higher than that of a beta in PB. Moreover, A beta -induced cytotoxicity assays showed A beta incubated i n PBS to be more cytotoxic than that incubated in PB. These results thus su ggest that A beta -associated free radical generation is strongly influence d by the aggregational state of the peptides.