An improved method of preparing the amyloid β-protein for fibrillogenesis and neurotoxicity experiments

Abstract

Synthetic amyloid β-protein (Aβ) is used widely to study fibril formation and the physiologic effects of low molecular weight and fibrillar forms of the peptide on cells in culture or in experimental animals. Not infrequently, conflicting results have arisen in these studies, in part due to variation in the starting conformation and assembly state of Aβ. To avoid these problems, we sought a simple, reliable means of preparing Aβ for experimental use. We found that solvation of synthetic peptide with sodium hydroxide (AβNaOH), followed by lyophilization, produced stocks with superior solubility and fibrillogenesis characteristics. Solubilization of the pretreated material with neutral buffers resulted in a pH transition from -10.5 to neutral, avoiding the isoelectric point of Aβ (pl=5.5), at which Aβ precipitation and aggregation propensity are maximal. Relative to trifluoroacetate (Aβ-TFA) or hydrochloric acid (Aβ-HCl) salts of Aβ, yields of “low molecular weight Aβ” (monomers and/or dimers) were improved significantly by NaOH pretreatment. Time-dependent changes in circular dichroism spectra and Congo red dye-binding showed that Aβ-NaOHformed fibrils more readily than did the other Aβ preparations and that these fibrils were equally neurotoxic. NaOH pretreatment thus offers advantages for the preparation of Aβ for biophysical and physiologic studies.