Biomedical Engineering Reference
In-Depth Information
Little of the generic science depends on which of these two is studied. What does
matter, however, is the detail of the preparative conditions. This includes temperature
(and heating rate), pH, ionic strength (and speci
c concentration of cations), and even the
particulate batch number if the protein is obtained from a commercial source. This
explains why much of the work has been to try and deliver results in a
form so
that comparison within and between resultant gels can be made, without becoming
stalled by practical detail.
From all of the published work, the overall picture is that heating a suf
'
scaled
'
cient concen-
tration of protein
-
typically ~10% w/w
-
to just above the denaturation temperature
-
typically 60
-
80°C, depending upon other conditions
-
causes partial denaturation to take
place. This leads to the formation of inter-protein
-sheet, giving rise to a polymeric
network, the modulus of which is in turn accentuated by more prolonged heating, and/or
by cooling back to room temperature (Clark,
1998
). In this picture, the
β
'
strands
'
of the
network are typically a few protein diameters wide
-
around 5
-
10 nm
-
so these differ
substantially from the so-called
of gelatin and most other physi-
cally aggregated gel network strands, which are typically 1 nm in width (
Figure 1.1
).
Spectroscopic methods can be used to investigate the detailed structures of aggregates
formed during the heating and gelling of protein solutions. As mentioned in other
chapters, changes in structure at a molecular level (native-to-denatured) can be monitored
by optical techniques such as optical rotatory dispersion (ORD), circular dichroism (CD),
FTIR and Raman spectroscopy (Clark and Ross-Murphy,
1987
). The loss of the native
structure during aggregation and the development of new ordered structure (such as
β
'
molecular networks
'
cant, and CD and ORD have been used to calculate the relative
proportions of various secondary structures present in both native and denatured forms.
A limitation of the CD method is that suitable measurements can only be made on highly
dilute samples (concentrations of 0.01
-sheet) are usually signi
0.1% w/w).
Infrared and Raman spectroscopy, on the other hand, have been used to monitor
changes in protein solutions as they occur at higher concentrations. FTIR spectroscopy
is best for monitoring changes in
-
-sheet, although solutions have to be prepared in D
2
O,
while Raman and CD are better at following changes involving
β
-helix. For example,
Clark et al.(
1981a
,
1981b
) used scanning infrared and Raman spectroscopy to monitor
the aggregation of a range of globular proteins. Development of
α
β
-sheet during aggre-
(around 1620 cm
−
1
) in the Amide I
carbonyl stretching band. This is observed more or less generally, although the amount of
sheet seems to vary from system to system.
Traditional microscopy methods (i.e. using light or electrons) can be used to probe
aggregate and gel structures over longer distances. Since gelled and aggregated solution
samples contain large amounts of water, extensive sample preparation is required before an
image can be formed. However, one aspect which can be investigated in detail with
conventional (transmission) electron microscopy is the degree of network homogeneity
and the nature
gation is indicated by the development of a
'
shoulder
'
-
-
-
transparent or opaque
of the gel. Our archetype
ovalbumin, the protein
gel traditionally formed by boiling a hen
is of course opaque, but this is not an
intrinsic property. In principle, and indeed in practice, a transparent egg
'
segg
-
can be
prepared by dialyzing away some of the naturally occurring salt, or alternatively treating in
'
white
'
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