microspheres. For BSA/LYS mixture, microspheres are obtained between pH 7.4
and 9.2, with an optimal yield of cross-assembly at pH 8.9. The formed
microspheres contained 3 mol of LYS per mole of BSA. According to the considered dual protein mixtures, well-defined microspheres are observed for a narrow pH
window. Far from this window, either amorphous aggregates or no cross-assembly
are detected, according to the system. Comparing the conditions of formation of the
microspheres in the three binary systems reveals the following three similarities:
1. Existence of a pH value for maximum efficiency of cross-assembly
2. High sensitivity to the ionic strength, no assembly was detected above an ionic
strength of 30 mM
3. Microspheres are gently obtained at 25
C, a temperature significantly lower than
that required to form apo α-La/LYS microspheres, i.e. above 30
C [3]
The three dual systems also reveal some specificities with differences in:
1. The total protein concentration required to observe microspheres
2. The optimum pH for microsphere formation
3. The final stoichiometry in the formed microspheres
Whatever the considered binary system, a phenomenon of protein surface
charge compensation seems to prevail in the mechanism that governs microsphere
formation. This is consistent with the complexation theory between biopolymers
involving short- and long-range interactions [102]. However, this theory does not
completely explain the dual protein dependency of the final stoichiometry found in
microspheres. Fine examination of protein stoichiometries in various binary systems
shows that protein charge compensation by itself is necessary but not sufficient. The
results we obtained recently suggest that the difference in the surface area available
for interaction between two proteins is also an essential parameter that could explain
the change in the final stoichiometry. For each protein, this interacting surface is
assumed to be proportional to the molecular weight or size of the protein. Perfect
charge compensation is expected with a molar ratio of one between two proteins
of similar size, e.g. apo α-La/LYS or Ova/avidin. This is exactly what is seen
experimentally. The sizes of Ova and BSA are respectively two and three times
higher than that of LYS. This would explain the molar ratios of 2 and 3 observed in
the microspheres obtained with Ova/LYS and BSA/LYS, respectively [5]. These
results are in agreement with those reported for microspheres formed between gold
nanoparticles (grafted with positive charges) and negatively charged proteins of
different sizes [124]. When the nanoparticles are larger than the protein, several
protein molecules assemble with one gold nanoparticle and the inverse is true in the
case where the proteins are larger than the gold nanoparticles. In the case where the
dimensions of the nanoparticle and protein are in the same order of magnitude,
extended aggregates are observed [124]. Thus, the stoichiometry found in
microspheres of cross-assembled proteins depends, among other considerations, on
the extent of the relative size difference between mixed proteins. Consequently, as
well as charge compensation, size compensation is a key parameter that guides
protein assembly and their stoichiometry in microspheres in dual systems.
Spontaneous Assembly and Induced Aggregation of Food Proteins
91
and 9.2, with an optimal yield of cross-assembly at pH 8.9. The formed
microspheres contained 3 mol of LYS per mole of BSA. According to the considered dual protein mixtures, well-defined microspheres are observed for a narrow pH
window. Far from this window, either amorphous aggregates or no cross-assembly
are detected, according to the system. Comparing the conditions of formation of the
microspheres in the three binary systems reveals the following three similarities:
1. Existence of a pH value for maximum efficiency of cross-assembly
2. High sensitivity to the ionic strength, no assembly was detected above an ionic
strength of 30 mM
3. Microspheres are gently obtained at 25
C, a temperature significantly lower than
that required to form apo α-La/LYS microspheres, i.e. above 30
C [3]
The three dual systems also reveal some specificities with differences in:
1. The total protein concentration required to observe microspheres
2. The optimum pH for microsphere formation
3. The final stoichiometry in the formed microspheres
Whatever the considered binary system, a phenomenon of protein surface
charge compensation seems to prevail in the mechanism that governs microsphere
formation. This is consistent with the complexation theory between biopolymers
involving short- and long-range interactions [102]. However, this theory does not
completely explain the dual protein dependency of the final stoichiometry found in
microspheres. Fine examination of protein stoichiometries in various binary systems
shows that protein charge compensation by itself is necessary but not sufficient. The
results we obtained recently suggest that the difference in the surface area available
for interaction between two proteins is also an essential parameter that could explain
the change in the final stoichiometry. For each protein, this interacting surface is
assumed to be proportional to the molecular weight or size of the protein. Perfect
charge compensation is expected with a molar ratio of one between two proteins
of similar size, e.g. apo α-La/LYS or Ova/avidin. This is exactly what is seen
experimentally. The sizes of Ova and BSA are respectively two and three times
higher than that of LYS. This would explain the molar ratios of 2 and 3 observed in
the microspheres obtained with Ova/LYS and BSA/LYS, respectively [5]. These
results are in agreement with those reported for microspheres formed between gold
nanoparticles (grafted with positive charges) and negatively charged proteins of
different sizes [124]. When the nanoparticles are larger than the protein, several
protein molecules assemble with one gold nanoparticle and the inverse is true in the
case where the proteins are larger than the gold nanoparticles. In the case where the
dimensions of the nanoparticle and protein are in the same order of magnitude,
extended aggregates are observed [124]. Thus, the stoichiometry found in
microspheres of cross-assembled proteins depends, among other considerations, on
the extent of the relative size difference between mixed proteins. Consequently, as
well as charge compensation, size compensation is a key parameter that guides
protein assembly and their stoichiometry in microspheres in dual systems.
Spontaneous Assembly and Induced Aggregation of Food Proteins
91