Mechanism of A beta(1-40) fibril-induced fluorescence of (trans,trans)-1-bromo-2,5-bis(4-hydroxystyryl)benzene (K114)

K114, (trans,trans)-1-bromo-2,5-bis(4-hydroxystyryl)benzene, is a fluorescent Congo Red analogue that binds tightly to amyloid fibrils, but not the monomeric proteins, with a concomitant enhancement in fluorescence. The mechanism for the low aqueous fluorescence and the subsequent enhancement by A beta(1-40) fibrils was investigated by fluorescence spectroscopy and binding analysis. K114's unusually low buffer fluorescence is due to self-quenching in sedimentable aggregates or micelles which upon interacting with amyloid fibrils undergo an enhancement in fluorescence intensity and shifts in the excitation and emission spectra. These spectral changes are suggestive of a stabilization of the phenolate anion, perhaps by hydrogen bonding, rather than an increase in the microenvironment dielectric constant or dye immobilization. 1,4-Bis(4-aminophenylethenyl)-2-methoxybenzene, which lacks the phenol moiety, and X-34, which contains a stabilized phenol (pK approximately 13.4), do not display the phenolate anion fluorescence in the presence of fibrils. The apparent affinity of K114 for fibril binding is 20-30 nM with a stoichiometry of 2.2 mol of K114/mol of A beta(1-40) monomer. Competition studies indicate that K114 and Congo Red share a site, but K114 does not bind to sites on A beta(1-40) fibrils for neutral benzothiazole (BTA-1), cationic thioflavin T, or the hydrophobic (S)-naproxen and (R)-ibuprofen molecules. Comparison of benzothiazole binding stoichiometry which has been suggested to reflect disease-relevant amyloid structures to that of Congo Red analogues which reflect total fibril content may be useful in defining biologically pertinent conformational forms of amyloid.