Amyloid, The Amyloid B -Peptide and Alzheimer’s Disease: Structural Considerations - Molecules: Nucleation, Aggregation and Crystallization - page 295

sample buffer, dimers and trimers could be observed. One problem with
SDS-PAGE is that oligomers can be dissociated by SDS or during chro-
matography, and that SDS can induce oligomerization. In order to avoid
dissociation, cross-linking of A has been used. In this case it is impor-

tant to use short cross-linking times to avoid diffusion-dependent cross-
linking. Recently, it was suggested that photoinduced cross-linking of
unmodified proteins (PICUP) could be used for studying A

β

oligomers
(Bitan et al ., 2001). In this case, reagents that form radicals upon light
exposure activate A

β

molecules that are
closely associated in an oligomer can form. The duration of the light
pulse is usually one second or less, and the quenching of the reaction is
achieved in a few seconds by the addition of sample buffer containing a
reducing agent. After cross-linking, the oligomers are separated by PAGE
and visualized by silver staining of the gel. Alternatively, western blot-
ting can be used if high sensitivity is needed. By using urea in the sam-
ple buffer, artifactual interactions are minimized. One consideration
regarding PICUP is that the reactive amino acids (e.g. Tyr and Lys) must
be close in order to interact, and thus, it is likely that only a subset of
oligomers will be cross-linked.

β , and covalent bonds between A β

CD spectroscopy

As described above, CD spectroscopy can be used for studying the sec-
ondary structure of proteins. Since monomeric A in a buffer free from

organic solvents and detergents is in a mostly disordered conformation,
while fibrillar A

β

-sheet conformation, the polymerization can be
followed as an increased amplitude in the spectrum at the wavelengths
typical for

β is in β

-sheet (e.g. a minimum around 217 nm). Unfortunately, this
technique does not reveal minor changes in the sample, and it is not pos-
sible to follow the formation of oligomers since it is not clear what con-
formation they have. A comprehensive study including several different
A

β

β -variants showed that an α -helical conformation precedes the β -sheet

conformation (Kirkitadze et al ., 2001).