Structure of Self-Assembled Globular Proteins - Food Colloids: Self-Assembly and Material Science - page 44

added, or the pH is adjusted closer to pI, and as a consequence the amplitude of
the peak decreases.
When a protein solution is heated, the contribution of non-aggregated
protein decreases and that of the aggregates increases, until finally all protein
is transformed into aggregates - or, for C 4 C g , into a gel. The value of C g falls
from approximately 100 g L 1 in the absence of NaCl and far from pI,toaslow
as 10 g L 1 at high ionic strength. 29,32,43,44 In the latter case a precipitate may
be formed at even lower protein concentrations. An intriguing issue that has
not yet been resolved is why below a critical concentration the aggregates stop
growing. Apparently the aggregates cease to bind to each other in spite of the
fact that in some cases they can be highly interpenetrated. This effect cannot be
attributed to a dynamic equilibrium of bond formation and break-up, because
generally the aggregates are stable to dilution. The stability of the aggregates is
clearly related to electrostatic repulsion, because increasing the ionic strength,
or reducing the pH towards pI, induces further assembly of the clusters even at
room temperature. 10
At low ionic strength, and away from pI, the heated systems remain trans-
parent because the aggregate suspension and the gels are highly ordered and
still produce a peak in the static structure factor. 38,42,45,46 This is illustrated
in Figure 7, where the change of the structure factor is shown for b-LG heated
at pH ¼ 7 in the absence of added salt and at 100 mM NaCl. 45 Similar data
were obtained for OA. 45 Initially at 0 mM one observes the interaction peak
for native b-LG, and after extended heating one observes the interaction peak
for the gel. The upturn at the lowest q-values is an artefact caused by the
scattering cell. Surprisingly an iso-scattering point is observed during
the gelation process, which suggests separation between the aggregates and
the unaggregated protein during the process of self-assembly on the small
length-scales covered by SAXS.
For the heated systems, the value of q p increases with the square root of the
protein concentration, because the aggregates are locally rod-like. 42 The peak
height decreases with decreasing protein concentration and increasing salt
concentration because there is a reduction in the degree of order in the structure
of the gels. For the same reason, the apparent molar mass M a calculated from
I(0) increases with decreasing protein concentration and increasing salt con-
centration. Above 30 mM NaCl, a scattering peak is no longer observed and

the intensity at small q-values increases, as shown in Figure 7.
The ordered structure of concentrated aggregates of OA at low ionic strength
has been visualized by cryo-TEM as indicated in Figure 8. 42 The typical
distance between adjacent aggregate strands is close to the 2 p /q p obtained
from the SAXS measurements on the same system. Gels cannot be investigated
using cryo-TEM, and a highly perturbing treatment is necessary to observe
them with TEM or SEM. This is probably the reason why the TEM micro-
graphs of transparent gels do not clearly show the order that is implied by the
scattering experiments. However, as shown in Figure 8, TEM does indicate
cross-linked strands with diameters less than 10 nm. For this reason these
ordered transparent gels are often called as fine-stranded gels.

B