Biology Reference
In-Depth Information
18,19
for attachment (Fig. 8.2).
This adhesive film, of only 10 nm
thickness, supports a parasite up to 5 mm in length which will be
subjected to significant shear forces while attached to the host fish
in the ocean. This ratio of supported volume to adhesive volume
is extraordinary. As described later in this chapter, the functional
amyloid from the adhesives of the algae and parasite are surprisingly
similar in terms of their mechanical properties.
While the discovery of amyloid-based adhesives was initially
inferred from AFM mechanical responses attributed to the repetitive
protein elements,
14,15
the amyloid-specific dyes thioflavin-T and
Congo red were used as further evidence whenever possible.
14,16,17
Detection of amyloid is commonly based on the binding of these
dyes to the
β
-sheets of the protein,
20
and both could be used directly
on the adhesives
. It should be noted that neither is perfect
with regards to its specificity and usability. However, both are good
support for the presence of amyloid when used in combination.
Raman spectroscopy was used to provide additional evidence that
the structures characterized by AFM were amyloid,
in situ
16,17
and could
also be performed on an adhesive matrix
.
Definitive verification of the presence of amyloid is a major
issue particularly with secreted materials of complex composition.
Instead of a single method being used it is often only possible to
build up evidence based on a range of complementary techniques
(such as those detailed above) and when possible the additional
application of techniques, such as Fourier transform infrared (FTIR)
spectroscopy and far-UV circular dichroism (CD) spectroscopy. In
some cases, treatment with extremely strong organic acids may
release monomers that can be further purified for SDS-PAGE and
sequenced by mass spectrometry.
in situ
11
This, however, requires pure
cultures, ample expression of amyloid, and an efficient protocol
optimized for an individual organism, due to the robust and insoluble
structures involved.
11
For future studies, there exists a challenging
need to refine and develop procedures to identify amyloid more
clearly and readily in natural materials, such as biological adhesives
and cements.
In view of these significant difficulties, the potential presence of
amyloid in adhesives and cements in former studies may have been
overlooked simply because the detection methods were unsuitable,
difficult, or unavailable. This may have been the case for barnacle
cement. Barnacle cement is permanent, extremely insoluble, and
composed of proteinaceous nanofibrils.
1
It was noted as having some
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