218
3.4
Conclusion
G. V ANDERHEEREN and J. HANSSENS
This study demonstrates that a more advanced research of the interaction of
hydrophobic probes offers information on the degeneration of the hydrophobic
domains during the protein denaturation.
In the molten globule state of a-lactalbumin, the two clusters with strong
hydrophobic character seem to be accessible. By heating the hydrophobic
domains degenerate, but the temperature of destabilization is different for the
two domains.
A similar evolution of the hydrophobic behavior is observed for apo- and
Ca 2 + - BLA, but the destabilization steps of Ca 2 + - BLA occur at higher temperatures
than those of apo-BLA. The results imply that Ca 2 + remains associated with BLA
after the thermal induced destabilization of its native tertiary structure.
4
The Perturbations Induced by the Hydrophobic Probe
in the Native State of Goat «-Lactalbumin
4.1
Aim
It has been shown that bis-ANS is helpful in following the behavior of the hydrophobic domains along the thermal unfolding pathway of an a-lactalbumin.
In this chapter we deal with the ability ofbis-ANS to penetrate into the hydrophobic core of native a-lactalbumin inducing perturbations in the native protein
structure. Again we distinguish the apo- and the Ca 2 + -bound protein. For this
study, goat a-lactalbumin is preferred to bovine a-lactalbumin because at low
temperatures a clear native state of the goat apo-protein is obtained with spectroscopic properties similar to those of the Ca2+ -bound protein.
4.2
Thermal Unfolding of GLA
The squares in Fig. 15.4 represent the ellipticity at 270 nm of GLA at different temperatures in the presence and absence of Ca 2 +. From the Fig. it can be deduced
that in a solution containing 10 mM Tris-HCl and 2 mM EGTA (filled squares) the
protein is in the fully native conformation only at temperatures below 10 °C. By
replacing EGTA for Ca 2 + (open squares), the native protein conformation remains
stable up to 55°C. The increased thermal stability resulting from Ca 2 + binding is
a general property of all a-lactalbumins (Acharya et al. 1989).
Next, we concentrate on the comparison of the effects of bis-ANS interaction
with native GLA at 4°C in 2 mM EGTA with those at 3]oC in 2mM Ca 2 +. In both
conditions the protein is in the native state close to the thermal transition and
shows the same spectroscopic properties. Therefore, although the temperatures
are different, the protein can be expected to be equally susceptible to conformational changes.
3.4
Conclusion
G. V ANDERHEEREN and J. HANSSENS
This study demonstrates that a more advanced research of the interaction of
hydrophobic probes offers information on the degeneration of the hydrophobic
domains during the protein denaturation.
In the molten globule state of a-lactalbumin, the two clusters with strong
hydrophobic character seem to be accessible. By heating the hydrophobic
domains degenerate, but the temperature of destabilization is different for the
two domains.
A similar evolution of the hydrophobic behavior is observed for apo- and
Ca 2 + - BLA, but the destabilization steps of Ca 2 + - BLA occur at higher temperatures
than those of apo-BLA. The results imply that Ca 2 + remains associated with BLA
after the thermal induced destabilization of its native tertiary structure.
4
The Perturbations Induced by the Hydrophobic Probe
in the Native State of Goat «-Lactalbumin
4.1
Aim
It has been shown that bis-ANS is helpful in following the behavior of the hydrophobic domains along the thermal unfolding pathway of an a-lactalbumin.
In this chapter we deal with the ability ofbis-ANS to penetrate into the hydrophobic core of native a-lactalbumin inducing perturbations in the native protein
structure. Again we distinguish the apo- and the Ca 2 + -bound protein. For this
study, goat a-lactalbumin is preferred to bovine a-lactalbumin because at low
temperatures a clear native state of the goat apo-protein is obtained with spectroscopic properties similar to those of the Ca2+ -bound protein.
4.2
Thermal Unfolding of GLA
The squares in Fig. 15.4 represent the ellipticity at 270 nm of GLA at different temperatures in the presence and absence of Ca 2 +. From the Fig. it can be deduced
that in a solution containing 10 mM Tris-HCl and 2 mM EGTA (filled squares) the
protein is in the fully native conformation only at temperatures below 10 °C. By
replacing EGTA for Ca 2 + (open squares), the native protein conformation remains
stable up to 55°C. The increased thermal stability resulting from Ca 2 + binding is
a general property of all a-lactalbumins (Acharya et al. 1989).
Next, we concentrate on the comparison of the effects of bis-ANS interaction
with native GLA at 4°C in 2 mM EGTA with those at 3]oC in 2mM Ca 2 +. In both
conditions the protein is in the native state close to the thermal transition and
shows the same spectroscopic properties. Therefore, although the temperatures
are different, the protein can be expected to be equally susceptible to conformational changes.