preformed microspheres containing the green fluorescent lysozyme molecules.
A change in colour from green to red was observed when following the
microspheres over time. It was further found that the protein molecules are able
to diffuse within the microspheres [91].
It was also observed that formation of the microspheres follows a aggregation–
reorganisation mechanism. First, a-lactalbumin–lysozyme heterodimers are
formed, followed by the formation of protein nanospheres. These nanospheres
form clusters that rearrange into microspheres. The total protein concentration
has an effect on the final size of the microspheres [91]. The formation kinetics of
calcium-depleted a-lactalbumin–lysozyme microspheres was investigated and it
was found that the kinetics were independent of temperature, but at 25
C the
clusters of nanospheres did not rearrange into microspheres and at 45
C they did.
A possible explanation for this temperature effect is that calcium-depleted
a-lactalbumin is in a molten globule state above 30
C. It was further observed
that the kinetics depend on the protein concentration and ionic strength [92].
Microsphere formation was not only observed for lysozyme and a-lactalbumin;
other oppositely charged protein couples (lysozyme/ovalbumin, ovalbumin/avidin
and lysozyme/BSA) showed a similar behaviour. All these systems showed
colocalisation of the protein molecules within the microspheres and were sensitive
to the ionic strength. An analogy to the isoprotic point (see Fig. 2) of two weakly
charged polyelectrolytes was found: the pH where optimal complexation occurred
was
1
2 ðpI protein þ þ pI protein À Þ. Charge compensation was not observed in all systems,
especially when there was a difference in protein size. In this case, the smaller
protein molecule was in excess within the microsphere [93].
How do the relaxation phenomena of protein–protein complexes compare to
polyelectrolyte complexes consisting of two polyelectrolytes? In Fig. 6, we
sketched, the three distances (d, r ij and h) that are important for the relaxation
behaviour of linear polyelectrolytes. The main difference between protein
molecules and polyelectrolyte complexes is the distance d because not only do
most protein molecules have a very low charge density, they are 3D nanoparticles.
The distance between opposite charges on different protein molecules is therefore
expected to be larger because optimal 3D packing is more complicated than for
linear polyelectrolytes. It is therefore not surprising that when two proteins are
different in size, it is difficult to obtain charge compensation [93]. Rearrangement
of the complexes was only found in systems where a-lactalbumin is in a molten
globule state. A molten globule is more like a linear weakly charged polyelectrolyte. It is well known that weakly charged polyelectrolytes and protein molecules
can form complex coacervate phases [8–12].
The ionic strength at which protein–protein complexes disintegrate is in general
lower than for polyelectrolyte complexes. Probably because of the low charge
density of the protein molecules and their less optimal packing within the complex.
In some systems, an increase in temperature results in disintegration of the
precipitates [82, 88]. This effect can be attributed to hydrophobic interactions and
hydrogen bond formation between the amino acids. These interactions are known to
be dependent on the temperature.
Relaxation Phenomena During Polyelectrolyte Complex Formation
169
A change in colour from green to red was observed when following the
microspheres over time. It was further found that the protein molecules are able
to diffuse within the microspheres [91].
It was also observed that formation of the microspheres follows a aggregation–
reorganisation mechanism. First, a-lactalbumin–lysozyme heterodimers are
formed, followed by the formation of protein nanospheres. These nanospheres
form clusters that rearrange into microspheres. The total protein concentration
has an effect on the final size of the microspheres [91]. The formation kinetics of
calcium-depleted a-lactalbumin–lysozyme microspheres was investigated and it
was found that the kinetics were independent of temperature, but at 25
C the
clusters of nanospheres did not rearrange into microspheres and at 45
C they did.
A possible explanation for this temperature effect is that calcium-depleted
a-lactalbumin is in a molten globule state above 30
C. It was further observed
that the kinetics depend on the protein concentration and ionic strength [92].
Microsphere formation was not only observed for lysozyme and a-lactalbumin;
other oppositely charged protein couples (lysozyme/ovalbumin, ovalbumin/avidin
and lysozyme/BSA) showed a similar behaviour. All these systems showed
colocalisation of the protein molecules within the microspheres and were sensitive
to the ionic strength. An analogy to the isoprotic point (see Fig. 2) of two weakly
charged polyelectrolytes was found: the pH where optimal complexation occurred
was
1
2 ðpI protein þ þ pI protein À Þ. Charge compensation was not observed in all systems,
especially when there was a difference in protein size. In this case, the smaller
protein molecule was in excess within the microsphere [93].
How do the relaxation phenomena of protein–protein complexes compare to
polyelectrolyte complexes consisting of two polyelectrolytes? In Fig. 6, we
sketched, the three distances (d, r ij and h) that are important for the relaxation
behaviour of linear polyelectrolytes. The main difference between protein
molecules and polyelectrolyte complexes is the distance d because not only do
most protein molecules have a very low charge density, they are 3D nanoparticles.
The distance between opposite charges on different protein molecules is therefore
expected to be larger because optimal 3D packing is more complicated than for
linear polyelectrolytes. It is therefore not surprising that when two proteins are
different in size, it is difficult to obtain charge compensation [93]. Rearrangement
of the complexes was only found in systems where a-lactalbumin is in a molten
globule state. A molten globule is more like a linear weakly charged polyelectrolyte. It is well known that weakly charged polyelectrolytes and protein molecules
can form complex coacervate phases [8–12].
The ionic strength at which protein–protein complexes disintegrate is in general
lower than for polyelectrolyte complexes. Probably because of the low charge
density of the protein molecules and their less optimal packing within the complex.
In some systems, an increase in temperature results in disintegration of the
precipitates [82, 88]. This effect can be attributed to hydrophobic interactions and
hydrogen bond formation between the amino acids. These interactions are known to
be dependent on the temperature.
Relaxation Phenomena During Polyelectrolyte Complex Formation
169