Journal
AMYLOID-JOURNAL OF PROTEIN FOLDING DISORDERS
Volume 7, Issue 3, Pages 166-178Publisher
TAYLOR & FRANCIS LTD
DOI: 10.3109/13506120009146831
Keywords
Alzheimer's disease; amyloid beta-protein; amyloidogenesis; fibrillogenesis; neurotoxicity
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Funding
- NIA NIH HHS [1P01-AG14366, 1PO1-AG05134] Funding Source: Medline
- NINDS NIH HHS [1R01-NS38328] Funding Source: Medline
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Synthetic amyloid beta-protein (A beta) 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 A beta. To avoid these problems, we sought a simple, reliable means of preparing A beta for experimental use. We found that solvation of synthetic peptide with sodium hydroxide (A beta-NaOH) 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 similar to 10.5 to neutral, avoiding the isoelectric point of A beta (pI approximate to 5.5), at which A beta precipitation and aggregation propensity are maximal. Relative to trifluoroacetate (A beta-TFA) or hydrochloric acid (A beta-HCl) salts of A beta, yields of low molecular weight A beta (monomers and/or dimers) were improved significantly by NaOH pretreatment. Time-dependent changes in circular dichroism spectra and Congo red dye-binding showed that A beta-NaOH formed fibrils more readily than did the other A beta preparations and that these fibrils were equally neurotoxic. NaOH pretreatment thus offers advantages for the preparation of A beta for biophysical and physiologic studies.
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