Ribbons result from the lateral stacking of fibrils. They are observed only after
a prolonged heating at acid pH of globular proteins as different as LYS or β-Lg.
The formation of multistranded ribbons occurs concomitantly with an extensive
hydrolysis of the proteins into low molecular weight peptides [23]. Such small
peptides are hypothesised to be responsible for the formation of multistranded
ribbons.
Spherulites result from the radial arrangement of unbranched protein fibrils and
are characterised by a semi-crystalline structure [90, 91]. Because of their inner
structure, spherulites exhibit a Maltese cross pattern when they are observed
between crossed polarizers in a light microscope [92]. The spherulites can reach
several hundred micrometres in diameter. In the larger spherulites, it is possible to
distinguish two regions: an amorphous nucleus and a semi-crystalline surrounding
corona [91, 93]. In contrast to the surrounding corona, the core progressively loses
its birefringence during the radial growth of the spherulite. Assuming that the
growth of the spherulite occurs through the periphery and that the radially ordered
fibrils do not convert into amorphous aggregates, it was hypothesised that a
distortion of fibrils in the core of the spherulite occurs during the growth, resulting
in a progressive loss of birefringence [8]. Although the mechanism triggering the
association of single fibrils into spherulites is not totally elucidated, it was
hypothesised that the balance between electrostatic, hydrophilic and hydrophobic
interactions is preponderant; heating temperature and pH are preponderant factors
in reducing the lag phase for spherulite observation [94]. In addition, the morphology of the spherulites is affected by the presence of salt, which favours larger
spherulites with a larger nucleus [74].
3.3 Particulate Aggregates
Globular proteins (BSA, β-Lg, LYS, etc.) are able to form irreversible, well-defined,
particulate aggregates of several tenths to hundreds of nanometres when heated close
to their pI or in the presence of salts (Fig. 2d) [95–97]. Heating induces the exposure
to the solvent of hydrophobic patches initially buried in the interior of the protein
structure. Hydrophobic interactions constitute the main driving force for protein
assembly, even if some authors underline the importance of electrostatic interactions
[10]. As for fibrils, proteins are held together by intermolecular β-sheets in the
particulate aggregates. However, intermolecular β-sheets are shorter in the particulate
aggregates and they have random orientations due to a faster aggregation step (low
electrostatic barriers for protein aggregation) [8]. Experiments on β-Lg indicate that
heat treatment of the unfolded proteins first causes aggregation into oligomers and
then into soluble aggregates [98, 99]. On prolonged heating, soluble aggregates
interact and form particulate aggregates of about one to several hundred nanometres
in diameter. These particulate aggregates self-assemble when the electrostatic
repulsions are too low for their stabilisation in solution [100]. The aggregation of
β-Lg slows down rapidly and even stops when the proportion of native proteins in
solution is lower than 10% [101].
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S. Bouhallab and T. Croguennec
a prolonged heating at acid pH of globular proteins as different as LYS or β-Lg.
The formation of multistranded ribbons occurs concomitantly with an extensive
hydrolysis of the proteins into low molecular weight peptides [23]. Such small
peptides are hypothesised to be responsible for the formation of multistranded
ribbons.
Spherulites result from the radial arrangement of unbranched protein fibrils and
are characterised by a semi-crystalline structure [90, 91]. Because of their inner
structure, spherulites exhibit a Maltese cross pattern when they are observed
between crossed polarizers in a light microscope [92]. The spherulites can reach
several hundred micrometres in diameter. In the larger spherulites, it is possible to
distinguish two regions: an amorphous nucleus and a semi-crystalline surrounding
corona [91, 93]. In contrast to the surrounding corona, the core progressively loses
its birefringence during the radial growth of the spherulite. Assuming that the
growth of the spherulite occurs through the periphery and that the radially ordered
fibrils do not convert into amorphous aggregates, it was hypothesised that a
distortion of fibrils in the core of the spherulite occurs during the growth, resulting
in a progressive loss of birefringence [8]. Although the mechanism triggering the
association of single fibrils into spherulites is not totally elucidated, it was
hypothesised that the balance between electrostatic, hydrophilic and hydrophobic
interactions is preponderant; heating temperature and pH are preponderant factors
in reducing the lag phase for spherulite observation [94]. In addition, the morphology of the spherulites is affected by the presence of salt, which favours larger
spherulites with a larger nucleus [74].
3.3 Particulate Aggregates
Globular proteins (BSA, β-Lg, LYS, etc.) are able to form irreversible, well-defined,
particulate aggregates of several tenths to hundreds of nanometres when heated close
to their pI or in the presence of salts (Fig. 2d) [95–97]. Heating induces the exposure
to the solvent of hydrophobic patches initially buried in the interior of the protein
structure. Hydrophobic interactions constitute the main driving force for protein
assembly, even if some authors underline the importance of electrostatic interactions
[10]. As for fibrils, proteins are held together by intermolecular β-sheets in the
particulate aggregates. However, intermolecular β-sheets are shorter in the particulate
aggregates and they have random orientations due to a faster aggregation step (low
electrostatic barriers for protein aggregation) [8]. Experiments on β-Lg indicate that
heat treatment of the unfolded proteins first causes aggregation into oligomers and
then into soluble aggregates [98, 99]. On prolonged heating, soluble aggregates
interact and form particulate aggregates of about one to several hundred nanometres
in diameter. These particulate aggregates self-assemble when the electrostatic
repulsions are too low for their stabilisation in solution [100]. The aggregation of
β-Lg slows down rapidly and even stops when the proportion of native proteins in
solution is lower than 10% [101].
78
S. Bouhallab and T. Croguennec