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to a maximum (k
max
) that corresponds to C
cs
* where the protein becomes
insolubilized. At higher concentrations than C
cs
*, k decreases significantly
(with exception of GdnHCl for b-LG A and B). This behaviour was found to be
similar for all three cosolutes. Normalization of all reaction rates by the fastest
one allowed calculation of the stability ratio W.
Looking closer at the stability ratio data reveals that, upon increased
arginine HCl concentration, W decreases and the sticking probability
a
in-
creases significantly, as shown in Figure 3; similar results were obtained for
NaCl and GdnHCl. This indicates that the proteins aggregate in the presence of
cosolutes, the likely reason being the surface charge neutralization of the
denatured proteins.
34
To understand the formation of soluble aggregates in
presence of cosolutes and the changes in the protein + solvent behaviour, the
second virial coefficient A
2
of the b-LG+cosolute mixtures was determined.
Even if overall positive values were found for A
2
, increasing cosolute concen-
tration led to a three to fourfold decrease in A
2
before precipitation occurred
(Figure 4). This indicates a lowered affinity of the protein for the solvent
compared to b-LG alone that might be explained by an increased surface
hydrophobicity, protein unfolding and aggregation.
35
In the tested conditions,
up to C
cs
*, the value of A
2
remained still positive after heating in presence of the
cosolute, indicating that the protein tends to aggregate but remains soluble
(A
2
4 0). Therefore sufficient repulsive forces are still present between the
protein molecules due to incomplete surface charge neutralization. At the
starting point of precipitation (C
cs
* at A70, N150 and G50), the stability ratio
reaches values close to unity, implying that the highest sticking probability for
10
1
0
100
200
300
400
Concentration in cosolute [mM]
Figure 3 Stability ratio W, as calculated from the reaction rate constant k, determined by
RP-HPLC (pH 7.0, 801C, 0-30 min, protein concentration 10 g L
1
, arginine
HCl concentrations 0-400 mM). Vertical error bars represent the standard
deviation
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