An improved method of preparing the amyloid beta-protein for fibrillogenesis and neurotoxicity experiments.
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 x 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 approximately 10.5 to neutral, avoiding the isoelectric point of A beta (pI approximately 5.5), at which A beta precipitation and aggregation propensity are maximal. Relative to trifluoroacetate (A beta x TFA) or hydrochloric acid (A beta x 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 x 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.
Fezoui
Y
Y
Center for Neurologic Diseases, Brigham and Women's Hospital, and Department of Neurology, Harvard Medical School, Boston, MA 02215, USA.
Hartley
D M
DM
Harper
J D
JD
Khurana
R
R
Walsh
D M
DM
Condron
M M
MM
Selkoe
D J
DJ
Lansbury
P T
PT
Fink
A L
AL
Teplow
D B
DB
D000595:Amino Acid Sequence; D016229:Amyloid beta-Peptides / Q000138:chemical synthesis* / Q000737:chemistry / Q000633:toxicity; D000818:Animals; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D018920:Coculture Techniques; D004396:Coloring Agents; D003224:Congo Red; D019281:Dimerization; D005374:Filtration; D005612:Freeze Drying; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D018625:Microscopy, Atomic Force; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009457:Neuroglia / Q000187:drug effects; D009474:Neurons / Q000187:drug effects; D010446:Peptide Fragments / Q000138:chemical synthesis* / Q000737:chemistry / Q000633:toxicity; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D051381:Rats; D012972:Sodium Hydroxide / Q000494:pharmacology; D012995:Solubility; D012997:Solvents / Q000494:pharmacology; D017550:Spectroscopy, Fourier Transform Infrared; D013329:Structure-Activity Relationship; D013997:Time Factors
D016229:Amyloid beta-Peptides; D004396:Coloring Agents; D010446:Peptide Fragments; D012997:Solvents; C078592:amyloid beta-protein (1-40); C075222:amyloid beta-protein (1-42); D003224:Congo Red; D012972:Sodium Hydroxide