b-Lactoglobulin Aggregates from Heating with Charged Cosolutes: Formation, Characterization and Foaming - Food Colloids: Self-Assembly and Material Science - page 184

aggregation pattern were similar in presence of the three cosolutes, but C cs * was
significantly lower for arginine HCl (70 mM) and GdnHCl (50 mM) than for
NaCl (150 mM).
The kinetics of denaturation and aggregation of the b-LG+cosolute mix-
tures has been investigated within 30 min at 801C by determination of the
remaining native soluble protein at pH 4.6 using RP-HPLC. Figure 2 shows the
calculation of the kinetic parameters for heat denaturation and aggregation of
the b-LG variants A and B in presence of arginine HCl. The application of the
general rate equation (1) demonstrates that b-LG aggregation follows a reac-
tion order of n ¼ 1.8-1.9 for variant A and n ¼ 2 for variant B. With increased
cosolute concentration the reaction order decreases to n ¼ 1.5 for both variants.
This is in agreement with results by Verheul and co-workers, 19 who explained
the decrease of the reaction order in terms of increased protein stability at
higher salt concentrations, and therefore reduced protein unfolding leading to
rate limitation. With further increase in cosolute concentration (100 mM
arginine HCl for both b-LG variants, 200 mM NaCl and 50 mM GdnHCl
for variant B), the reaction order increases slightly again to values of n 1.8.

Only for 400 mM of arginine HCl, the value of n increases to 2.2 and 2.0 for
b-LG variants A and B, respectively. Possible explanations for this latter
behaviour are more complex reactions due to arginine-induced protective
effects on the protein structure and/or suppressed aggregation. Overall, it
was found that the data for most of the conditions with the three cosolutes
fitted well with a reaction order of 1.5 (R 2 4 0.99) with a few exceptions for
b-LG variant B (R 2 ¼ 0.9736) and for cosolute concentrations higher than 200
mM (R 2

E

0.97-0.98). The reaction rate k was calculated from the HPLC
heating kinetics, with a reaction order of 1.5 for b-LG A and B (Figure 2). The
reaction rate was found to increase with increasing cosolute concentration up

B

5 7

1 1 1 1

. . . .

= = 0 0 k k t t + + 1 1 6 = = 0 0 k k t t + + 1 1

C C / / C C C C / / C C

4

t t 0 0 t t 0 0

5

3

4

3

2

2

1

1

A A B B

0 0

0 0 5 10 15 20 25 30 0 5 10 15 20 25 30

Time [min] Time [min]

Figure 2 Representation of the kinetics of heat denaturation and aggregation of (A)
b -LG A and (B) b -LG B in presence of arginine HCl (pH 7.0, 801C, 0-30 min,
protein concentration 10 g L 1 , cosolute concentration 0-400 mM): K , protein
alone; J , A20; ' , A70; m , A400. The lines indicate the decrease in protein
concentration for a given reaction order n, from which the reaction rate constant
k was derived