Different low molecular mass
ligands have been used to identify
amyloid deposits. Among these markers, the
dyes Thioflavin T and
Congo Red interact specifically with the beta-sheet structure arranged in a cross-beta conformation, which is characteristic of
amyloid. However, the molecular details of this interaction remain unknown. When labelled with technetium-99m, the
proteinase inhibitor
aprotinin has been shown to represent a very important
radiopharmaceutical agent for in vivo imaging of extra-abdominal deposition of
amyloid in
amyloidosis of the
immunoglobulin type. However, no information is available as to whether
aprotinin binds other types of
amyloid fibrils and on the nature and characteristics of the interaction. The present work shows
aprotinin binding to
insulin,
transthyretin,
beta-amyloid peptide and
immunoglobulin synthetic
amyloid fibrils by a specific dot-blot
ligand-binding assay.
Aprotinin did not bind amorphous precipitates and/or the soluble fibril precursors. A Ka of 2.9 microM-1 for the binding of
aprotinin to
insulin amyloid fibrils was determined by Scatchard analysis. In competition experiments, analogues such as an
aprotinin variant, a
spermadhesin and the soybean
trypsin inhibitor were tested and results suggest that both
aprotinin and the
spermadhesin interact with
amyloid fibrils through pairing of beta-sheets of the
ligands with exposed structures of the same type at the surface of
amyloid deposits. An electrostatic component may also be involved in the binding of
aprotinin to
amyloid fibrils because important differences in binding constants are observed when substitutions V15L17E52 are introduced in
aprotinin; on the other hand beta-sheet containing acidic
proteins, such as the soybean
trypsin inhibitor, are unable to bind
amyloid fibrils.