A new method for measuring fibrillar amyloid β‐protein using diphenylhexatriene, a fluorescent probe

Abstract

The two current methods for the measurement of fibrillar (f) amyloid beta‐protein (Aβ), namely Thioflavin T (ThT) fluorescence spectroscopy and Congo red (CR) absorbance spectroscopy, are based on the interaction of fAβ with cationic ThT and anionic CR molecules. The presence of cations and colored factors in the reaction often interferes with ThT and CR assays. Recently, we reported that fibrillar Aβ forms a membrane‐like hydrophobic domain (Chauhan et al. 2001; Neuroreport12, 587–590). We report here that diphenylhexatriene (DPH), a neutral fluorescent probe, interacts with fibrillar Aβ 1‐40 and fibrillar Aβ 1‐42, but not with soluble Aβ. As a result, DPH fluorescence increases several fold in the presence of fibrillar Aβ at the excitation and emission wavelength of 360 and 430 nm, respectively. The blank sample (DPH in buffer) had minimal fluorescence, and soluble Aβ had no effect on DPH fluorescence. The interaction of DPH with fibrillar Aβ was dependent on the incubation time of these two constituents. At fixed Aβ concentration and varying DPH concentration, there was a lag phase of DPH fluorescence from 1 to 4 μm DPH, that was followed by a linear increase in fluorescence from 4 to 8 μm DPH which then reached a plateau. Studies at varying pH showed that emission spectra of fAβ/DPH can be studied at a wide range of pH from 4 to 8. Aβ concentration studies showed that the fluorescence of DPH increases linearly with increase in the concentration of fibrillar Aβ 1‐40/Aβ 1‐42. Kinetic studies showed that one mole of fAβ binds to 15.5 mol of DPH. These results suggest that DPH fluorescence can be used as a standard assay method for the measurement of fibrillar Aβ.