b-Lactoglobulin Aggregates from Heating with Charged Cosolutes: Formation, Characterization and Foaming - Food Colloids: Self-Assembly and Material Science - page 187

0.0

-0.5

-1.0

-1.5

-2.0

-2.5

0 20 40 60 80 100

Concentration in cosolute [mM]

Figure 5 Electrophoretic mobility of b -LG aggregates heated in presence of cosolutes
(protein concentration 10 g L 1 , cosolute concentration 0-100 mM, pH 7.0,
heated at 801C for 10 min: K , arginine HCl; J , NaCl; . , GdnHCl. Vertical
error bars represent the standard deviation

Based on this set of results, a tentative mechanism of b-LG denaturation and
aggregation at pH 7.0 in presence of cosolute can be proposed. Considering
that b-LG unfolds at a temperature (T d B 751C) below the actual heating

temperature of 801C, the unfolding process should not be rate-limiting in those
conditions. However, due to an overall negative surface charge on the b-LG
molecules, repulsive forces certainly restrict immediate protein aggregation
upon cluster collision. Results in Figures 2-5 indicate that the overall negative
net charge of the protein is neutralized by interaction with positively charged
cations. In the presence of low concentrations of cosolute, negative protein
charges are partially screened by arginine, sodium and guanidinium cations.
Therefore the repulsive forces decrease, leading to an increased sticking prob-
ability (Figure 3) and fast particle aggregation (Figure 2). Consequently,
diffusion-limited aggregation gains an importance compared to rate-limited
aggregation, although DLCA and RLCA do coexist. 36 The higher the cosolute
concentration, the more particles are neutralized until a certain degree of
overall charge screening is reached, characterized by very heterogeneously
charged populations. In this case, the probability of interaction and the
aggregation rate increase. The reactivity between the particles becomes very
high, resulting in fast aggregation (W

1, see Figure 3). With increased
cosolute concentration the overall electrophoretic mobility of the particles
decreases for all three cosolutes to the same extent, until it levels off. This
indicates a certain degree of saturation with counter-ions (cosolutes) on the
particle surface, supporting the overcharging hypothesis. 37 At a certain degree
of surface charge neutralization, partial overcharging leads to higher repulsive
forces meaning that more particle contacts are necessary to induce aggregation.
We note that the effect of the guanidinium group on charge neutralization and
protein aggregation seems to be stronger than that of Na 1 . However, concern-
ing the overcharging effect, arginine HCl and NaCl lead to significantly higher

B